Preparation process and application of dog kidney, desert cistanche, epimedium, arginine and zinc compound preparation for tonifying kidney

Abstract

The invention belongs to the field of health care products and biological medicines, and particularly discloses a preparation process and application of a dog kidney, desert cistanche, epimedium, arginine and zinc compound preparation for tonifying kidney. The compound preparation is prepared from 10-30 parts of dog kidney extract, 20-50 parts of desert cistanche extract, 15-40 parts of herba epimedii extract, 5-20 parts of arginine and 1-5 parts of zinc sulfate, all the components have a synergistic effect, the testosterone level is remarkably improved, spermatogenesis is promoted, and the kidney-yang deficiency symptom is comprehensively improved. Experiments show that the testosterone level of the compound preparation is increased by 128%, the sperm density is increased by 143%, and the compound preparation is obviously superior to a single preparation and a contrast without key components. After the preparation process is optimized, the bioavailability is improved by 1.5-2.3 times, the disintegration time limit of the tablet is less than or equal to 15 minutes, and the dissolution rate is as high as 93.5%. When the composition is combined with sildenafil to treat severe erectile dysfunction, the dosage of chemical drugs can be greatly reduced while the curative effect is reserved, and the adverse reaction rate is reduced by 71%. The invention provides an innovative solution for high efficiency, safety and stability of the kidney-tonifying medicine.

Description

Technical Field

[0001] The invention belongs to the field of health care products and biomedicine, and specifically discloses a preparation process and application of a compound prescription of dog kidney cistanche and epimedium arginine zinc for tonifying the kidney. Background Art

[0002] In the field of kidney-tonifying drug research and development, existing technical solutions have multi-dimensional limitations and are in urgent need of innovative breakthroughs. Traditional kidney-tonifying Chinese medicines mostly rely on compound prescriptions, such as Liuwei Dihuang Pills and Jinkui Shenqi Pills. Although they have the characteristics of "multi-component-multi-target" effects, they have significant defects:

[0003] Complex ingredients and vague mechanisms: Traditional compound prescriptions often contain dozens of medicinal materials (e.g. Liuwei Dihuang Wan contains 6 herbs, including Rehmannia glutinosa and Cornus officinalis), with unclear active ingredients and lack of modern molecular biological verification of the mechanism of action, resulting in unstable efficacy and difficulty in standardization. For example, although Lycium barbarum polysaccharides have been proven to improve symptoms of kidney deficiency, there is a lack of quantitative research on their synergistic effects in compound prescriptions, and clinical efficacy often requires continuous use for 4-6 weeks to manifest.

[0004] Risk of side effects of chemical drugs: Although chemical drugs represented by phosphodiesterase-5 inhibitors (such as sildenafil) can quickly improve erectile function, long-term use may cause adverse reactions such as cardiovascular events (incidence ≥ 12%), headaches and visual impairment, and cannot solve the fundamental problems such as low testosterone levels and insufficient sperm motility caused by kidney yang deficiency.

[0005] In summary, existing technologies are difficult to balance the triple demands of “efficiency-safety-stability”, and an innovative solution based on modern extraction technology, multi-component synergistic compatibility and precise formulation technology is urgently needed to break through the efficacy bottleneck of kidney-tonifying drugs. Summary of the invention

[0006] In view of the above problems, the present invention discloses a preparation process and application of a compound prescription of dog kidney cistanche and ephedra arginine zinc for tonifying the kidney, which is prepared by modern extraction technology and precise preparation technology. The components work synergistically to effectively activate the hypothalamus-pituitary-gonad axis, significantly increase testosterone levels, promote spermatogenesis, and comprehensively improve the symptoms of kidney yang deficiency.

[0007] The purpose of the present invention is achieved through the following technical solutions.

[0008] A compound prescription of dog kidney cistanche and ephedra arginine zinc for tonifying the kidney, comprising the following active ingredients by weight:

[0009] Dog kidney extract 10-30 parts

[0010] Desert Cistanche Extract 20-50 parts

[0011] Epimedium extract 15-40 parts

[0012] Arginine 5-20 parts

[0013] Zinc sulfate 1-5 parts.

[0014] Furthermore, the preparation method of the dog kidney extract of the above-mentioned dog kidney cistanche and ephedra arginine zinc compound for tonifying the kidney comprises the following steps:

[0015] a. Wash fresh dog kidneys and cut into 2-3 mm slices, and mix with pH 5.0 phosphate buffer at a mass ratio of 1:4;

[0016] b. Add 0.2% trypsin with an enzyme activity of ≥2500 U / mg, and perform enzymolysis at 55°C for 3 hours, followed by inactivation at 90°C for 15 minutes;

[0017] c. Centrifuge at 8000 rpm for 20 minutes, take the supernatant, and concentrate it to 1 / 5 of the original volume through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa;

[0018] d. Spray drying: inlet air temperature 160°C, outlet air temperature 85°C, to obtain dog kidney extract powder with a moisture content of ≤6%.

[0019] Furthermore, in the above-mentioned Cistanche deserticola extract and Echeveria arginine zinc compound for tonifying the kidney, the preparation method of the Cistanche deserticola extract comprises:

[0020] a. The dried Cistanche deserticola was crushed through a 60-mesh sieve and extracted with 70% ethanol at a solid-liquid ratio of 1:10 reflux twice, each for 2 hours;

[0021] b. The combined extracts were concentrated under reduced pressure until there was no alcohol taste, and loaded onto a D101 macroporous resin column, and eluted with a gradient of 20%, 30%, and 50% ethanol, and the 30% ethanol eluate was collected;

[0022] c. The eluate was pre-frozen at -40°C and freeze-dried at -50°C for 24 hours under a vacuum degree of 10 Pa to obtain an extract with a phenylethanoid glycoside content ≥ 8%.

[0023] Furthermore, in the above-mentioned Herba Epimedii Cistanches and Herba Epimedii Arginine Zinc compound for tonifying the kidney, the preparation method of the Herba Epimedii extract comprises:

[0024] a. Grind the whole herb of Epimedium to 40 mesh, add 10 times the amount of pure water, and extract by ultrasonic at 50℃ and 40 kHz for 1.5 hours;

[0025] b. The extract was adsorbed on AB-8 macroporous resin at a dynamic adsorption flow rate of 2 BV / h and eluted with 70% ethanol at 1.5 BV / h;

[0026] c. The eluate is concentrated under reduced pressure to a relative density of 1.25 and dried under vacuum at 60°C, -0.08 MPa until the water content is ≤5%.

[0027] Furthermore, the above-mentioned compound prescription of Cistanche deserticola and Echeveria arginine zinc for tonifying the kidney is used in the preparation of tablets, capsules, pills or pills.

[0028] Furthermore, the method for preparing the above-mentioned compound into tablets or capsules comprises the following steps:

[0029] a. Dissolve the dog kidney extract and arginine in 5% citric acid solution and stir at 50°C until completely dissolved to obtain a mixed solution;

[0030] b. Mix Cistanche deserticola extract, Epimedium extract, zinc sulfate, microcrystalline cellulose and lactose and sieve through a 100-mesh sieve;

[0031] c. Using an efficient wet granulator, the mixture of step a and the powder of step b were mixed to control the water activity ≤ 0.25, using 8% hydroxypropyl methylcellulose ethanol solution as a binder;

[0032] d. The wet granules are dried in a fluidized bed at 45°C until the moisture content is ≤4%. After granulation, they are compressed into tablets or filled into capsules.

[0033] Furthermore, in the above preparation method, the disintegration time of the tablet is ≤15 minutes, and the particle size of the capsule content is ≤150 μm.

[0034] The present invention also discloses the use of the compound in combination with sildenafil in preparing a medicine for treating severe erectile dysfunction.

[0035] Furthermore, in the above application, in the medicine, the mass ratio of the compound to sildenafil is 10:1 to 50:1.

[0036] The present invention also discloses a method for preparing the compound into pills or granules, comprising the following steps:

[0037] Preparation method of pills:

[0038] 1) Raw material preparation:

[0039] Dissolve dog kidney extract and arginine in 5% citric acid solution, stir at 50°C until completely dissolved, to obtain a mixed solution.

[0040] The Cistanche deserticola extract, Epimedium extract, zinc sulfate, appropriate amounts of microcrystalline cellulose and lactose are mixed and passed through a 100-mesh sieve to obtain a uniform powder mixture.

[0041] 2) Mixing and pelleting:

[0042] The mixed solution obtained in step 1 and the powder mixture are thoroughly mixed.

[0043] As needed, you can add an appropriate amount of honey or refined honey as a binder to adjust the hardness to the appropriate level.

[0044] The mixture is made into pills using a pill making machine, and the size and shape of the pills can be adjusted as needed.

[0045] 3) Drying and packaging:

[0046] The prepared pills are placed in a ventilated and dry place for drying until the specified moisture content is reached;

[0047] Preparation method of granules:

[0048] 4) Wet granulation:

[0049] Add the mixed liquid obtained in step 1) and the powder mixture into a high-efficiency wet granulator, and control the water activity to ≤ 0.25;

[0050] Using 8% hydroxypropyl methylcellulose ethanol solution as a binder, start the granulator for wet granulation;

[0051] During the granulation process, the amount of binder and the parameters of the granulator can be adjusted in time according to the formation of the granules;

[0052] 5) Drying and granulation:

[0053] The wet granules were dried in a fluidized bed dryer at 45°C until the moisture content was ≤4%;

[0054] After drying, the granules are sized using a granulator to remove oversized or undersized particles to obtain uniform granules.

[0055] Compared with the existing technology, the present invention has the following advantages and beneficial effects:

[0056] The present invention achieves multiple breakthroughs in kidney-tonifying drugs by combining dog kidney extract, Cistanche deserticola, Epimedium, arginine and zinc sulfate in a scientific way, combined with modern extraction technology and precision preparation technology. The multi-component synergistic effect in the compound is significant, and it can restore endocrine function by activating the hypothalamus-pituitary-gonad axis, increase testosterone levels and promote sperm production, and comprehensively improve the symptoms of kidney yang deficiency. The enzymatic ultrafiltration, resin purification and arginine carrier technology are adopted to significantly improve the bioavailability of active ingredients and ensure the efficient absorption of fat-soluble components. When combined with sildenafil for treatment, the dosage of chemical drugs can be greatly reduced while retaining the efficacy, significantly reducing cardiovascular risks and the incidence of adverse reactions. The preparation process ensures the rapid disintegration and dissolution of tablets and capsules by accurately controlling water activity, low-temperature drying and optimizing adhesives, and shows excellent main component retention rate in accelerated stability tests. In addition, the compound can be flexibly prepared into various dosage forms such as tablets, capsules, pills or granules, with both long-term storage stability and clinical adaptability, providing innovative solutions for the efficiency, safety and stability of kidney-tonifying drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Safety evaluation of combined use of sildenafil: effective;

[0058] Figure 2 Safety evaluation of combined use of sildenafil: adverse reaction rate;

[0059] Figure 3 Safety evaluation of combined use of sildenafil: rate of electrocardiogram abnormalities;

[0060] Figure 4 Formulation process optimization verification: disintegration time (min);

[0061] Figure 5 Formulation process optimization and verification: dissolution (%);

[0062] Figure 6 Formulation process optimization verification: main component retention rate (%). DETAILED DESCRIPTION

[0063] To make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. All raw materials in the embodiments of the present invention can be obtained through commercial channels.

[0064] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0065] The following are the sources of the raw materials used in the examples of the present invention.

[0066] 1. Dog kidney

[0067] Source: Artificially bred dogs (beagle breed), in compliance with the "Regulations on the Management of Experimental Animals" and the "Quality Management Standards for the Production of Animal-derived Medicinal Raw Materials".

[0068] Quality Standards:

[0069] Freshly collected, free of lesions and parasites

[0070] Meet the requirements of "Animal Organ Raw Materials" in the 2020 edition of the Chinese Pharmacopoeia

[0071] Microbiological limit: Total aerobic bacteria ≤1000 CFU / g, Salmonella shall not be detected.

[0072] 2. Cistanche deserticola

[0073] Source: Artificially cultivated product, produced in Alxa League, Inner Mongolia (Chinese Geographical Indication Product), moisture content of dried product ≤10%.

[0074] Extract Standard:

[0075] Phenylethanol glycoside content ≥ 8% (HPLC detection, refer to the method under Cistanche deserticola in the 2020 edition of the Chinese Pharmacopoeia)

[0076] Heavy metals: lead ≤5 ppm, arsenic ≤2 ppm (in compliance with GB 2762-2017 national food safety standard).

[0077] 3. Epimedium brevicornu

[0078] Source: Authentic production areas such as Shaanxi and Gansu, whole herb dried product, in compliance with the provisions of the Epimedium brevifolia 2020 edition of the Chinese Pharmacopoeia.

[0079] Extract Standard:

[0080] Icariin ≥5% (UV detection)

[0081] Total flavonoids ≥ 30%

[0082] The remaining chemical raw materials were of chemical or analytical grade and purchased from the market.

[0083] Macroporous adsorption resin

[0084] D101 type resin:

[0085] Pore ​​diameter: 13-14 nm, surface area ≥500 m² / g

[0086] Supplier: Cangzhou Baoen Chemical

[0087] AB-8 type resin:

[0088] Polarity: Weak polarity, suitable for flavonoid adsorption

[0089] Supplier: Xi'an Lanxiao Technology.

[0090] Example 1

[0091] A compound prescription of dog kidney cistanche and ephedra arginine zinc for tonifying the kidney, comprising the following active ingredients by weight:

[0092] Dog kidney extract 10 parts

[0093] Desert Cistanche Extract 20 parts

[0094] Epimedium Extract 15 parts

[0095] Arginine 5 parts

[0096] 1 part zinc sulfate.

[0097] The preparation method of the dog kidney extract comprises the following steps:

[0098] a. Wash fresh dog kidneys and cut into 2-3 mm slices, and mix with pH 5.0 phosphate buffer at a mass ratio of 1:4;

[0099] b. Add 0.2% trypsin with an enzyme activity of ≥2500 U / mg, and perform enzymolysis at 55°C for 3 hours, followed by inactivation at 90°C for 15 minutes;

[0100] c. Centrifuge at 8000 rpm for 20 minutes, take the supernatant, and concentrate it to 1 / 5 of the original volume through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa;

[0101] d. Spray drying: inlet air temperature 160°C, outlet air temperature 85°C, to obtain dog kidney extract powder with a moisture content of ≤6%.

[0102] The preparation method of the desert cistanche extract comprises:

[0103] a. The dried Cistanche deserticola was crushed through a 60-mesh sieve and extracted with 70% ethanol at a solid-liquid ratio of 1:10 reflux twice, each for 2 hours;

[0104] b. The combined extracts were concentrated under reduced pressure until there was no alcohol taste, and loaded onto a D101 macroporous resin column, and eluted with a gradient of 20%, 30%, and 50% ethanol, and the 30% ethanol eluate was collected;

[0105] c. The eluate was pre-frozen at -40°C and freeze-dried at -50°C for 24 hours under a vacuum degree of 10 Pa to obtain an extract with a phenylethanoid glycoside content ≥ 8%.

[0106] The preparation method of the epimedium extract comprises:

[0107] a. Grind the whole herb of Epimedium to 40 mesh, add 10 times the amount of pure water, and extract at 50℃ and 40 kHz ultrasonic extraction for 1.5 hours;

[0108] b. The extract was adsorbed on AB-8 macroporous resin at a dynamic adsorption flow rate of 2 BV / h and eluted with 70% ethanol at 1.5 BV / h;

[0109] c. The eluate is concentrated under reduced pressure to a relative density of 1.25 and dried under vacuum at 60°C, -0.08 MPa until the water content is ≤5%.

[0110] The dog kidney extract, desert cistanche extract, epimedium extract, arginine and zinc sulfate are mixed, further ground and crushed, and passed through a 200-mesh sieve to obtain a compound prescription.

[0111] Example 2

[0112] A compound prescription of dog kidney cistanche and ephedra arginine zinc for tonifying the kidney, comprising the following active ingredients by weight:

[0113] Dog kidney extract 20 parts

[0114] Desert Cistanche Extract 35 parts

[0115] Epimedium Extract 30 servings

[0116] Arginine 10 parts

[0117] 3 parts of zinc sulfate.

[0118] The preparation method of the dog kidney extract comprises the following steps:

[0119] a. Wash fresh dog kidneys and cut into 2-3 mm slices, and mix with pH 5.0 phosphate buffer at a mass ratio of 1:4;

[0120] b. Add 0.2% trypsin with an enzyme activity of ≥2500 U / mg, and perform enzymolysis at 55°C for 3 hours, followed by inactivation at 90°C for 15 minutes;

[0121] c. Centrifuge at 8000 rpm for 20 minutes, take the supernatant, and concentrate it to 1 / 5 of the original volume through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa;

[0122] d. Spray drying: inlet air temperature 160°C, outlet air temperature 85°C, to obtain dog kidney extract powder with a moisture content of ≤6%.

[0123] The preparation method of the desert cistanche extract comprises:

[0124] a. The dried Cistanche deserticola was crushed through a 60-mesh sieve and extracted with 70% ethanol at a solid-liquid ratio of 1:10 reflux twice, each for 2 hours;

[0125] b. The combined extracts were concentrated under reduced pressure until there was no alcohol taste, and loaded onto a D101 macroporous resin column, and eluted with a gradient of 20%, 30%, and 50% ethanol, and the 30% ethanol eluate was collected;

[0126] c. The eluate was pre-frozen at -40°C and freeze-dried at -50°C for 24 hours under a vacuum degree of 10 Pa to obtain an extract with a phenylethanoid glycoside content ≥ 8%.

[0127] The preparation method of the epimedium extract comprises:

[0128] a. Grind the whole herb of Epimedium to 40 mesh, add 10 times the amount of pure water, and extract by ultrasonic at 50℃ and 40 kHz for 1.5 hours;

[0129] b. The extract was adsorbed on AB-8 macroporous resin at a dynamic adsorption flow rate of 2 BV / h and eluted with 70% ethanol at 1.5 BV / h;

[0130] c. The eluate is concentrated under reduced pressure to a relative density of 1.25 and dried under vacuum at 60°C, -0.08 MPa until the water content is ≤5%.

[0131] The dog kidney extract, desert cistanche extract, epimedium extract, arginine and zinc sulfate are mixed, further ground and crushed, and passed through a 200-mesh sieve to obtain a compound prescription.

[0132] Example 3

[0133] A compound prescription of dog kidney cistanche and ephedra arginine zinc for tonifying the kidney, comprising the following active ingredients by weight:

[0134] Dog kidney extract 30 parts

[0135] Desert Cistanche Extract 50 parts

[0136] Epimedium Extract 40 parts

[0137] Arginine 20 parts

[0138] 5 parts of zinc sulfate.

[0139] The preparation method of the dog kidney extract comprises the following steps:

[0140] a. Wash fresh dog kidneys and cut into 2-3 mm slices, and mix with pH 5.0 phosphate buffer at a mass ratio of 1:4;

[0141] b. Add 0.2% trypsin with an enzyme activity of ≥2500 U / mg, and perform enzymolysis at 55°C for 3 hours, followed by inactivation at 90°C for 15 minutes;

[0142] c. Centrifuge at 8000 rpm for 20 minutes, take the supernatant, and concentrate it to 1 / 5 of the original volume through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa;

[0143] d. Spray drying: inlet air temperature 160°C, outlet air temperature 85°C, to obtain dog kidney extract powder with a moisture content of ≤6%.

[0144] The preparation method of the desert cistanche extract comprises:

[0145] a. The dried Cistanche deserticola was crushed through a 60-mesh sieve and extracted with 70% ethanol at a solid-liquid ratio of 1:10 reflux twice, each for 2 hours;

[0146] b. The combined extracts were concentrated under reduced pressure until there was no alcohol taste, and loaded onto a D101 macroporous resin column, and eluted with a gradient of 20%, 30%, and 50% ethanol, and the 30% ethanol eluate was collected;

[0147] c. The eluate was pre-frozen at -40°C and freeze-dried at -50°C for 24 hours under a vacuum degree of 10 Pa to obtain an extract with a phenylethanoid glycoside content ≥ 8%.

[0148] The preparation method of the epimedium extract comprises:

[0149] a. Grind the whole herb of Epimedium to 40 mesh, add 10 times the amount of pure water, and extract at 50℃ and 40 kHz ultrasonic extraction for 1.5 hours;

[0150] b. The extract was adsorbed on AB-8 macroporous resin at a dynamic adsorption flow rate of 2 BV / h and eluted with 70% ethanol at 1.5 BV / h;

[0151] c. The eluate is concentrated under reduced pressure to a relative density of 1.25 and dried under vacuum at 60°C, -0.08 MPa until the water content is ≤5%.

[0152] The dog kidney extract, desert cistanche extract, epimedium extract, arginine and zinc sulfate are mixed, further ground and crushed, and passed through a 200-mesh sieve to obtain a compound prescription.

[0153] Comparative Example 1

[0154] Missing dog kidney extract

[0155] formula:

[0156] 35 parts of Cistanche deserticola extract, 30 parts of Epimedium extract, 10 parts of arginine, and 3 parts of zinc sulfate.

[0157] No dog kidney extract added.

[0158] Preparation process: Same as Example 2.

[0159] Comparative Example 2

[0160] Missing dog kidney extract and desert cistanche extract

[0161] formula:

[0162] 30 parts of epimedium extract, 10 parts of arginine, and 3 parts of zinc sulfate.

[0163] No dog kidney extract or desert cistanche extract is added.

[0164] Preparation process: Same as Example 2.

[0165] Comparative Example 3

[0166] Epimedium Extract

[0167] Formula: Contains only 30 parts of epimedium extract, no other ingredients are added.

[0168] Preparation process: the same as the preparation method of the epimedium extract in Example 2.

[0169] Comparative Example 4

[0170] Zinc sulfate deficiency

[0171] Formula: 20 parts of dog kidney extract, 35 parts of Cistanche extract, 30 parts of Epimedium extract, and 10 parts of arginine.

[0172] No zinc sulfate added.

[0173] Preparation process: Same as Example 2.

[0174] Comparative Example 5

[0175] Arginine and zinc sulfate deficiency

[0176] formula:

[0177] 20 parts of dog kidney extract, 35 parts of Cistanche deserticola extract, 30 parts of Epimedium extract;

[0178] No added arginine or zinc sulfate.

[0179] Preparation process: Same as Example 2.

[0180] Test Example 1

[0181] Verification of multi-target synergistic effects

[0182] Objective: To verify the comprehensive improvement effect of multiple components in the compound prescription on the kidney yang deficiency model

[0183] method:

[0184] Animal model: SPF SD rats (n=60) were given hydrocortisone (25 mg / kg / d) by intramuscular injection for 15 days to establish the kidney yang deficiency model.

[0185] Grouping (n=10 / group):

[0186] Blank control group (normal saline)

[0187] Model group (untreated)

[0188] Example 2 Group (200 mg / kg / d)

[0189] Comparative Example 1 (same dose, lack of dog kidney extract)

[0190] Comparative Example 2 (equal dose, lacking dog kidney extract and desert Cistanche extract)

[0191] Comparative Example 3 (Equal dose, single prescription Epimedium extract)

[0192] Comparative Example 4 (Equal dose, lack of zinc sulfate)

[0193] Comparative Example 5 (Equal dose, lack of arginine and zinc sulfate)

[0194] positive control group (Jinkui Shenqi Pills, 1.5 g / kg / d);

[0195] Detection indicators:

[0196] On the 28th day, serum testosterone (ELISA) and sperm density (blood cell counting plate) were measured.

[0197] Testicular histopathological score (HE staining) and expression of genes related to the hypothalamus-pituitary-gonad axis (GnRH, LHβ) (qRT-PCR).

[0198] The results are shown in Table 1

[0199]

[0200] Data analysis was performed according to Table 1:

[0201] 1. Multi-target synergistic effect:

[0202] In the kidney yang deficiency model rats in Example 2, the serum testosterone level recovered to 4.1 ng / mL (1.8 ng / mL in the model group and 5.2 ng / mL in the blank group), an increase of 128%; the sperm density reached 54.3×10^6 / mL (22.4×10^6 / mL in the model group and 58.7×10^6 / mL in the blank group), an increase of 143%.

[0203] When the dog kidney extract and desert Cistanche extract (Comparative Example 2) or the single Epimedium extract (Comparative Example 3) were missing, the effect was significantly reduced, indicating the importance of the synergistic effect of multiple components.

[0204] 2. Endocrine axis regulation:

[0205] The GnRH mRNA expression in Example 2 group recovered to 0.89 (1.00 in the blank group and 0.32 in the model group), indicating that the compound achieved multi-target regulation by activating the hypothalamus-pituitary-gonad axis.

[0206] 3. Verification of key ingredients:

[0207] The synergistic effect of dog kidney extract, desert Cistanche extract, arginine and zinc sulfate in the compound prescription is significantly better than that of a single prescription or a partially missing prescription, verifying the indispensability of each ingredient in testosterone production, sperm density improvement and endocrine axis regulation.

[0208] Test Example 2

[0209] Comparative bioavailability studies

[0210] Evaluating the role of modern extraction processes in enhancing the absorption of active ingredients

[0211] method:

[0212] Test substance:

[0213] Test group: Extract from Example 2 (containing arginine)

[0214] Control group: traditional decoction extract (same raw materials, without arginine, using traditional decoction, not purified)

[0215] Experimental design: Beagle dogs (n=6), crossover administration (500 mg / kg), LC-MS / MS determination:

[0216] Cistanche deserticola marker: echinacoside

[0217] Epimedium Marker: Icariin

[0218] Zinc ion concentration (ICP-MS)

[0219] The pharmacokinetic parameters are shown in Table 2

[0220]

[0221] Data analysis for Table 2:

[0222] 1. Echinacoside and icariin:

[0223] The AUCs of the experimental groups were 352.4 ng·h / mL and 487.5 ng·h / mL, respectively, which were 131% higher than those of the control group (152.6 and 210.8), indicating that modern extraction technology (resin purification) significantly improved the dissolution and absorption of fat-soluble components.

[0224] Arginine as a carrier may promote absorption by enhancing intestinal permeability.

[0225] 2. Zinc ion:

[0226] The zinc AUC in the experimental group (18.6 μg / mL) was 51% higher than that in the control group (12.3 μg / mL). The chelation of zinc sulfate and arginine may reduce the gastric acid degradation of zinc and improve its bioavailability.

[0227] Conclusion: Modern extraction technology combined with arginine carrier can increase the bioavailability of key ingredients by 1.5-2.3 times.

[0228] Test Example 3

[0229] Safety evaluation of combined use of sildenafil

[0230] Objective: To verify the safety advantage of compound medicine in reducing the dosage of chemical drugs

[0231] method:

[0232] Clinical design: 180 patients with severe ED (IIEF-5≤11) were included

[0233] Grouping:

[0234] Group A: sildenafil 50 mg alone

[0235] Group B: Preparation of Example 2 (containing 5 mg of sildenafil) + compound medicine 500 mg

[0236] Group C: 500 mg of the compound alone

[0237] Observation indicators:

[0238] 4-week effective rate (IIEF-5 increased by ≥5 points)

[0239] Incidence of adverse reactions (headache, flushing, abnormal vision)

[0240] The abnormal rate of dynamic electrocardiogram, the above results are shown in Figure 1 , Figure 2 and Figure 3 , and Table 3.

[0241]

[0242] Data analysis for Table 3:

[0243] 1. Effectiveness:

[0244] There was no statistical difference between group B (78%) and group A (82%) (P>0.05), but the dosage of chemical drugs was reduced by 90% (5 mg vs 50 mg), verifying the conclusion of "retaining 85% efficacy".

[0245] 2. Adverse reaction rate:

[0246] The adverse reaction rate in group B (9.8%) was significantly lower than that in group A (26.7%) (*P<0.05), indicating that the compound medicine can reduce the side effects of sildenafil.

[0247] Group C (pure natural compound) had the lowest adverse reaction rate (3.3%), but the effective rate was low (65%), suggesting the need to balance efficacy and safety.

[0248] Conclusion: The combination therapy significantly reduces the side effects associated with chemical drugs while maintaining 85% of the efficacy.

[0249] Example 4

[0250] Tablet preparation

[0251] formula:

[0252] 20 parts of dog kidney extract, 35 parts of Cistanche deserticola extract, 30 parts of Epimedium extract, 10 parts of arginine, and 3 parts of zinc sulfate.

[0253] Excipients: microcrystalline cellulose (20 parts), lactose (15 parts), hypromellose (HPMC, 8% ethanol solution)

[0254] Preparation steps:

[0255] Preparation of mixed solution: Dissolve dog kidney extract and arginine in 5% citric acid solution (stir at 50°C until dissolved).

[0256] Powder mixing: Cistanche deserticola extract, Epimedium extract, zinc sulfate, microcrystalline cellulose and lactose are sieved through a 100-mesh sieve.

[0257] Wet granulation:

[0258] The mixed liquid and powder are added into a high-efficiency wet granulator at a mass ratio of 1:3.

[0259] The water activity Aw was controlled at 0.22, and the amount of binder (8% HPMC ethanol solution) was 12% of the total material.

[0260] Drying and tableting:

[0261] The wet granules were dried in a fluid bed at 45°C to a moisture content of 3.8%.

[0262] After whole granules, they are compressed into tablets with a tablet weight of 500 mg and a hardness of 8-10 kp.

[0263] result:

[0264] Disintegration time: 12 minutes (meets the requirement of ≤15 minutes).

[0265] Dissolution (45 minutes): 93.5% (pH 6.8 medium).

[0266] Example 5

[0267] Capsule preparation

[0268] Formulation: Same as Example 4, filled into gelatin capsules (specification: 500 mg / capsule).

[0269] Preparation steps:

[0270] Wet granulation: adjust the HPMC dosage to 10% and the water activity Aw = 0.20.

[0271] Fluidized bed drying: Dry at 45°C to a moisture content of 3.5%.

[0272] Filled capsules: particle size ≤150 μm, and the fluidity of the capsule contents after filling is good (angle of repose ≤30°).

[0273] result:

[0274] Dissolution (45 minutes): 95.2%

[0275] Accelerated stability (40°C / 75% RH, 3 months): Main component retention rate 96.8%

[0276] Comparative Example 6

[0277] Water activity exceeds the standard (Aw=0.30)

[0278] Preparation method:

[0279] In step c, the water activity (Aw=0.30) was not controlled, and the other steps were the same as in Example 4.

[0280] result:

[0281] Disintegration time: 28 minutes (much higher than 15 minutes)

[0282] Accelerated stability (3 months): Main component retention rate 78.5% (degradation due to excessive moisture).

[0283] Comparative Example 7

[0284] High temperature drying (60℃ fluidized bed)

[0285] In step d, the drying temperature is increased to 60° C., and the rest is the same as in Example 4.

[0286] result:

[0287] Active ingredient loss: Icariin content decreased by 15% (due to degradation of heat-sensitive ingredients)

[0288] Dissolution rate (45 minutes): 82.1% (hardening of particle surface).

[0289] Comparative Example 8

[0290] No HPMC binder used

[0291] Preparation method:

[0292] In step c, starch slurry is used to replace HPMC ethanol solution, and the rest is the same as in Example 4.

[0293] result:

[0294] Poor particle adhesion, tablets are fragile after compression (hardness ≤ 5 kp)

[0295] Dissolution (45 minutes): 71.3% (due to slow disintegration)

[0296] Test Example 4

[0297] Formulation process optimization and validation

[0298] Grouping:

[0299] Example 4 (Aw=0.22, dried at 45°C, HPMC adhesive)

[0300] Comparative Example 6 (Aw=0.30)

[0301] Comparative Example 7 (60°C Drying)

[0302] Comparative Example 8 (Starch Adhesive)

[0303] Detection indicators:

[0304] Disintegration time (Chinese Pharmacopoeia 0931 method)

[0305] Dissolution (paddle method, pH 6.8 medium, 45 minutes)

[0306] Accelerated stability (40℃ / 75% RH, 3-month main component retention rate)

[0307] Results Figure 4 , Figure 5 and Figure 6 And Table 4:

[0308]

[0309] Data Analysis:

[0310] Water activity (Aw):

[0311] The disintegration time of Comparative Example 6 (Aw=0.30) was 28 minutes (far exceeding 15 minutes), and the main component retention rate was only 78.5%, indicating that high moisture content accelerated the degradation of components.

[0312] Example 4 (Aw=0.22) had a disintegration time of 12 minutes and a dissolution rate of 93.5%, which verified the necessity of controlling the water activity.

[0313] Drying temperature:

[0314] In comparative example 7 (dried at 60°C), the icariin loss was 15%, and the solubility decreased to 82.1%, indicating the protective effect of low-temperature drying (≤45°C) on heat-sensitive components.

[0315] Adhesive Selection:

[0316] The dissolution rate of comparative example 8 (starch adhesive) is only 71.3%. HPMC has better film-forming properties, ensuring uniform dispersion and rapid disintegration of particles.

[0317] Conclusion: The present invention successfully optimized the formulation by precisely controlling water activity, low-temperature drying and HPMC adhesive.

[0318] Test Example 5

[0319] Comparison of dosage form adaptability

[0320] Objective: To verify the dissolution behavior and storage stability of tablets and capsules in different media.

[0321] method:

[0322] Samples: Example 4 (tablets), Example 5 (capsules)

[0323] Dissolution medium:

[0324] Simulated gastric fluid (0.1N HCl, pH 1.2)

[0325] Simulated intestinal fluid (pH 6.8 phosphate buffer)

[0326] Detection:

[0327] Dissolution curve (sampling at 0, 15, 30, and 45 minutes, main component determination by HPLC)

[0328] Long-term stability (25°C / 60% RH, 12 months)

[0329] result:

[0330] The dissolution comparison is shown in Table 5, and the long-term stability is shown in Table 6.

[0331]

[0332]

[0333] Data analysis of Table 5 and Table 6:

[0334] 1. Dissolution behavior:

[0335] The dissolution of capsules in gastric juice (72.8% in 15 minutes) and intestinal juice (95.2% in 45 minutes) was better than that of tablets (65.2% and 93.5%), which may be related to the capsule's sealing property that reduces the damage of gastric acid to the active ingredients.

[0336] 2. Long-term stability:

[0337] The main ingredient retention rate of capsules (96.3%) is higher than that of tablets (94.1%) because the capsule shell isolates moisture and oxygen, delaying oxidation reactions.

[0338] in conclusion:

[0339] Capsules are slightly better in dissolution rate and stability, but tablets still meet clinical needs, and dosage form selection can be based on patient preference. Both dosage forms meet the requirements of rapid release (gastric juice > 60% in 15 minutes) and complete dissolution (intestinal juice > 90% in 45 minutes).

[0340] Summary of embodiments:

[0341] The following conclusions can be drawn from the above embodiments and test examples, and the present invention has achieved the following progress:

[0342] 1. Multi-target synergistic effect

[0343] Testosterone levels and sperm density increased significantly:

[0344] In the kidney yang deficiency model rats in Example 2, the serum testosterone level was restored to 4.1 ng / mL (1.8 ng / mL in the model group and 5.2 ng / mL in the blank group), an increase of 128%; the sperm density reached 54.3×10^6 / mL (22.4×10^6 / mL in the model group and 58.7×10^6 / mL in the blank group), an increase of 143%, which was significantly better than the lack of dog kidney extract (Comparative Example 1: 2.9 ng / mL, 38.2×10^6 / mL) or the lack of zinc (Comparative Example 4: 3.2 ng / mL, 41.5×10^6 / mL) (P<0.01).

[0345] 2. Endocrine axis regulation:

[0346] The expression of GnRH mRNA in Example 2 group recovered to 0.89 (1.00 in the blank group and 0.32 in the model group), indicating that the compound achieved multi-target regulation by activating the hypothalamus-pituitary-gonad axis.

[0347] 3. Bioavailability is significantly improved

[0348] Optimized absorption of active ingredients:

[0349] Modern extraction technology (enzymatic hydrolysis-ultrafiltration, resin purification) combined with arginine carrier has improved the AUC of echinacoside and icariin. 0-24h They reached 352.4 ng·h / mL and 487.5 ng·h / mL respectively, an increase of 231% compared with traditional water decoction (152.6 and 210.8); the zinc ion AUC increased by 51% (18.6 vs 12.3 μg / mL).

[0350] 4. Safety optimization and advantages of combined medication

[0351] Reduce the amount of chemical drugs:

[0352] The effective rate of combined use with sildenafil (Group B: 500 mg of the compound + 5 mg of sildenafil) was 78%, which was not significantly different from that of 50 mg of sildenafil alone (82% in Group A), but the dosage of chemical drugs was reduced by 90%.

[0353] Significantly reduced side effects:

[0354] The adverse reaction rate (9.8%) and electrocardiogram abnormality rate (1.6%) in group B were significantly lower than those in group A (26.7%, 8.3%) (P<0.05), and the adverse reaction rate of the pure compound (group C) was only 3.3%.

[0355] 5. Preparation stability and process innovation

[0356] Rapid dissolution and disintegration:

[0357] The disintegration time of the tablets of Example 4 is ≤12 minutes (28 minutes in Comparative Example 6 when the water activity is not controlled), and the dissolution rate is ≥93.5% in 45 minutes (pH 6.8 medium).

[0358] Stability breakthrough:

[0359] The main component retention rate of the accelerated test (40℃ / 75% RH, 3 months) was ≥95.7%, far exceeding that of the traditional physical mixed preparation (the retention rate of Comparative Example 6 was 78.5%).

[0360] Key process parameters:

[0361] Control water activity (Aw≤0.25), low temperature drying (≤45℃) and HPMC binder (8% ethanol solution) to avoid degradation of heat-sensitive ingredients (15% loss of icariin in Comparative Example 7) and delayed disintegration (dissolution rate of Comparative Example 8 is 71.3%).

[0362] 6. Dosage form flexibility and adaptability

[0363] Multiple dosage forms available:

[0364] Both the tablet (Example 4) and the capsule (Example 5) satisfy the requirements of rapid release (dissolution in gastric juice > 65% in 15 minutes) and complete dissolution (dissolution in intestinal juice > 93% in 45 minutes).

[0365] Long-term stability:

[0366] Due to its better sealing properties, the 12-month main ingredient retention rate of capsules is 96.3%, which is better than the 94.1% of tablets.

[0367] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Therefore, based on the innovative concept of the present invention, changes and modifications to the embodiments described herein, or equivalent structures or equivalent process transformations made using the contents of the present invention specification, directly or indirectly applying the above technical solutions to other related technical fields are all included in the protection scope of the patent of the present invention.

Claims

1. A compound prescription of Cistanche deserticola and Ephedra arginine zinc for tonifying the kidney, characterized in that: The active ingredients are as follows: Dog kidney extract 10-30 parts Desert Cistanche Extract 20-50 parts Epimedium extract 15-40 parts Arginine 5-20 parts Zinc sulfate 1-5 parts.

2. The compound according to claim 1, characterized in that The preparation method of the dog kidney extract comprises the following steps: a. Wash fresh dog kidneys and cut into 2-3 mm slices, and mix with pH 5.0 phosphate buffer at a mass ratio of 1:4; b. Add 0.2% trypsin with an enzyme activity of ≥2500 U / mg, and perform enzymolysis at 55°C for 3 hours, followed by inactivation at 90°C for 15 minutes; c. Centrifuge at 8000 rpm for 20 minutes, take the supernatant, and concentrate it to 1 / 5 of the original volume through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa; d. Spray drying: inlet air temperature 160°C, outlet air temperature 85°C, to obtain dog kidney extract powder with a moisture content of ≤6%.

3. The compound according to claim 1, characterized in that The preparation method of the desert cistanche extract comprises: a. The dried Cistanche deserticola was crushed through a 60-mesh sieve and extracted with 70% ethanol at a solid-liquid ratio of 1:10 reflux twice, each for 2 hours; b. The combined extracts were concentrated under reduced pressure until there was no alcohol taste, and loaded onto a D101 macroporous resin column, and eluted with a gradient of 20%, 30%, and 50% ethanol, and the 30% ethanol eluate was collected; c. The eluate was pre-frozen at -40°C and freeze-dried at -50°C for 24 hours under a vacuum degree of 10 Pa to obtain an extract with a phenylethanoid glycoside content ≥ 8%.

4. The compound according to claim 1, characterized in that The preparation method of the epimedium extract comprises: a. Grind the whole herb of Epimedium to 40 mesh, add 10 times the amount of pure water, and extract at 50℃ and 40 kHz ultrasonic extraction for 1.5 hours; b. The extract was adsorbed on AB-8 macroporous resin at a dynamic adsorption flow rate of 2 BV / h and eluted with 70% ethanol at 1.5 BV / h; c. The eluate is concentrated under reduced pressure to a relative density of 1.25 and dried under vacuum at 60°C, -0.08 MPa until the water content is ≤5%.

5. Use of the compound according to any one of claims 1 to 4 in the preparation of tablets, capsules, pills or pills.

6. A method for preparing tablets or capsules using the compound according to any one of claims 1 to 4, characterized in that: The following steps are involved: a. Dissolve the dog kidney extract and arginine in 5% citric acid solution and stir at 50°C until completely dissolved to obtain a mixed solution; b. Mix Cistanche deserticola extract, Epimedium extract, zinc sulfate, microcrystalline cellulose and lactose and sieve through a 100-mesh sieve; c. Using an efficient wet granulator, the mixture of step a and the powder of step b were mixed to control the water activity ≤ 0.25, using 8% hydroxypropyl methylcellulose ethanol solution as a binder; d. The wet granules are dried in a fluidized bed at 45°C until the moisture content is ≤4%. After granulation, they are compressed into tablets or filled into capsules.

7. The preparation method according to claim 6, characterized in that: The disintegration time of the tablet is ≤15 minutes, and the particle size of the capsule content is ≤150 μm.

8. Use of the compound according to any one of claims 1 to 4 in combination with sildenafil in the preparation of a medicament for treating severe erectile dysfunction.

9. The use according to claim 8, characterized in that: In the medicine, the mass ratio of the compound to sildenafil is 10:1 to 50:

1.

10. A method for preparing a pill or granule using the compound according to any one of claims 1 to 4, characterized in that: The following steps are involved: Preparation method of pills: 1) Raw material preparation: Dissolve the dog kidney extract and arginine in 5% citric acid solution, and stir at 50°C until completely dissolved to obtain a mixed solution; Mix the Cistanche deserticola extract, Epimedium herb extract, zinc sulfate, appropriate amounts of microcrystalline cellulose and lactose, and pass through a 100-mesh sieve to obtain a uniform powder mixture; 2) Mixing and pelleting: The mixed solution obtained in step 1 is thoroughly mixed with the powder mixture; According to the needs, you can add an appropriate amount of honey or refined honey as a binder to adjust the hardness to the appropriate softness; The mixture is made into pills using a pill making machine, and the size and shape of the pills can be adjusted as needed; 3) Drying and packaging: The prepared pills are placed in a ventilated and dry place for drying until the specified moisture content is reached; Preparation method of granules: 4) Wet granulation: Add the mixed liquid obtained in step 1) and the powder mixture into a high-efficiency wet granulator, and control the water activity to ≤ 0.25; Using 8% hydroxypropyl methylcellulose ethanol solution as a binder, start the granulator for wet granulation; During the granulation process, the amount of binder and the parameters of the granulator can be adjusted in time according to the formation of the granules; 5) Drying and granulation: The wet granules were dried in a fluidized bed dryer at 45°C until the moisture content was ≤4%; After drying, the granules are sized using a granulator to remove oversized or undersized particles to obtain uniform granules.

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