Sheep testis freeze-dried powder and preparation method thereof

Abstract

The invention belongs to the technical field of Tibetan medicine, and particularly relates to sheep testis freeze-dried powder and a preparation method thereof.In the preparation process, all active ingredients in dairy sheep testis pickled with salt can be reserved through vacuum freeze-drying, the water content of the freeze-dried sheep testis can reach 5% or below, and the freeze-dried sheep testis are loose in texture and free of toxic and side effects. The redissolution property is extremely high. The materials can be crushed in an environment of 0 DEG C or below through ultralow-temperature crushing, and the active ingredients of the freeze-dried sheep testicles can also be reserved to the maximum extent; and the tripeptide CWC is added before freeze-drying, so that the obtained sheep testis freeze-dried powder has the effects of tonifying yang, generating sperms, nourishing and strengthening the body. In addition, three medicinal materials, namely, gymnadenia conopsea, fructus piperis longi and freeze-dried emblic leafflower fruit which are featured by Tibetan medicines and homologous in medicine and food, have a synergistic effect, so that the efficacy of the sheep testis freeze-dried powder is further consolidated and enhanced

Description

Technical Field

[0001] The present invention belongs to the technical field of Tibetan medicine, and particularly relates to a freeze-dried powder of sheep testicles and a preparation method thereof. Background Art

[0002] The testicle is an important organ of the male animal's reproductive system, responsible for carrying on the family line and multiplying the offspring. In China, there has been a record of using animal testicles for medicinal purposes more than 2,000 years ago. The first pharmaceutical monograph, "Shennong Ben Cao Jing", and subsequent "Kaibao Bencao", "Yaoxing Lun", "Bencao Shiyi", "Haiyao Bencao", "Compendium of Materia Medica", etc. all have relevant records. The Tibetan Medicine Material Processing Specification of the Tibet Autonomous Region (2022) records that sheep testicles are important reproductive organs of male sheep and also important medicinal organs. They are regarded as "fleshy and emotional products", having the effects of strengthening yang, generating essence, and tonifying the body, and can treat impotence, premature ejaculation, etc.

[0003] There is no doubt that the medicinal efficacy of sheep testicles in these classic pharmaceutical monographs was summarized by the predecessors through countless observations and practices, and is the crystallization of the wisdom of countless medical experts. However, due to limitations in conditions, there are more superficial descriptions and less in-depth revelations, more perceptual knowledge and less rational understanding. There is even less knowledge about the effective components of the nature and flavor, their processing techniques, and quality inspection. In recent years, the varieties of Chinese patent medicines and tonic series put into production that mainly use animal testicles have been increasing, such as Sanbian Pills, Yangchun Pills, Yangshen Pills, Sanshen Pills, etc. Diet therapies and health-care dishes mainly based on medicinal porridges, medicinal diets, and medicinal wines have also become popular; there are also many successful cases of using single-component sheep testicles to treat difficult and complicated diseases.

[0004] As the core animal medicine material in the Tibetan medicine system, the application history of sheep testicles is closely intertwined with the nomadic civilization of the Qinghai-Tibet Plateau. In the handwritten copy of the "Four Medical Tantras" during the Tubo Dynasty, there is a clear record of "drying the testicles in the shade, grinding them, and mixing them with yak milk to treat spermatorrhea and infertility". Archaeologists detected specific proteins of sheep testicles through proteomic analysis in the residue of a medicine pestle found in the Ali region, confirming the unbroken medicinal tradition for thousands of years.

[0005] The adaptive evolution of plateau sheep (Ovis aries) in extreme environments endows its testicles with unique biological activities: the mitochondrial density in the testicle tissue of individuals above 4,000 meters above sea level is increased by 42% compared with the plain population, and the expression level of steroidogenic acute regulatory protein (StAR) in Leydig cells is increased by 3.1 times. This physiological characteristic makes it a high-quality medicine source. Modern breeding specifications require that medicinal sheep must be naturally grazed in alpine meadows at an altitude of 3,000 - 4,500 meters, and the use of synthetic feeds and growth hormones is prohibited. A 90-day drug withdrawal period is required before slaughter to ensure the clearance of metabolites.

[0006] As a classic animal medicinal material in Tibetan medicine, the processing technology of sheep testicles has been passed down for thousands of years, integrating the ecological wisdom of the plateau and the philosophical thoughts of Tibetan medicine. Traditional processing strictly follows the records in the Four Medical Tantras and the Crystal Bead Materia Medica. The core process includes four major stages: ecological collection, membrane removal and purification, wine immersion and fermentation, and nine steaming and nine sunning, aiming to stimulate the activity of the medicinal materials and reduce toxicity.

[0007] Ecological collection relies on the special ecological cycle of plateau pastoral areas. Medicinal sheep need to be naturally grazed in pastures above 3,000 meters above sea level for 3 - 5 years. The collection period is locked in 30 days after autumn breeding. At this time, the biological active substances in the testicles (such as testosterone, zinc and selenium elements) reach the annual peak. Traditional Tibetan medicine judges the harvesting time by observing the behavior of sheep and the morphological changes of the testicles. Modern research has confirmed that the fresh weight of the testicles can reach 120 - 150 grams at this stage, and the testosterone content is 2.3 times higher than that in daily life.

[0008] The primary processing stage needs to be completed within 2 hours after slaughter. Craftsmen manually peel off the testicular tunica vaginalis and epididymal tissues, remove blood vessels and lymph with yak bone knives, and then immerse them in highland barley wine and seal them for fermentation for seven days and nights. Yeasts in the koji (such as Saccharomyces cerevisiae) convert glycogen into active substances such as β-phenylethanol, and at the same time degrade immunogenic proteins. Metabolomics research in the 2020s showed that 23 new compounds are produced in this process, including phenylpropanoid glycosides with absorption-promoting effects.

[0009] Nine steaming and nine sunning is the essence of the process. The fermented testicles are wrapped with three alkalis of Tibetan medicine (natural sodium carbonate, plant ash and yak bone ash) and hung in a ventilated and dry place to undergo nine rounds of steaming and sunning cycles. Each round of steaming uses slow steaming with snow water over low heat for 4 hours, gradually hydrolyzing collagen into small molecule peptides; the sunning stage relies on the strong ultraviolet rays on the plateau (the average radiation dose reaches 8500 kJ / m 2 ) to promote the Maillard reaction and generate antioxidant substances such as melanoidins. Traditional experience believes that the complete cycle needs to span the waxing and waning of the moon phase. Modern analysis has confirmed that this operation increases the content of active peptides to 1.8 mg / g, and the proportion of free hormones is stable within the safe threshold.

[0010] The contemporary preparation method of sheep testicles is only recorded in the Tibetan Medicine Material Processing Specification of the Tibet Autonomous Region (2022) compiled by the Drug Administration of the Tibet Autonomous Region: Take the original medicinal materials, remove impurities, add sufficient goat milk and decoct until the milk juice is completely absorbed by the medicinal materials, dry, pulverize, and set aside.

[0011] With the progress of technology, the traditional process is being scientifically upgraded. The low-temperature freeze-drying technology shortens the drying time from 45 days to 72 hours, and the blockchain traceability system ensures the stable and controllable quality of each batch of medicinal materials.

[0012] It is worth noting that the "Tibetan Medicine Processing Specifications" in 2025 requires the retention of the cultural link of "Tibetan medicine practitioners chanting scriptures for blessing". EEG monitoring shows that specific sound wave frequencies can increase the operator's concentration by 37%, indirectly ensuring process stability. After the sheep testicles are prepared into decoction pieces, they are called "Lutu Zhebu" in Tibetan.

[0013] Zangbianbao Bushen Pills is a classic compound preparation of Tibetan medicine, composed of nine animal medicines such as snow deer penis, yak testicles, Tibetan sheep testicles, wild horse testicles, etc., and compatible with plant medicines such as Gymnadenia conopsea, Polygonatum sibiricum, and Lycium barbarum, following the formula principle of the "Four Medical Canons". The core functions of this medicine are to tonify the kidney and strengthen yang, nourish yin and replenish essence, and it is mainly used to treat kidney deficiency impotence, sexual function decline, and lack of energy, and to improve symptoms such as mental fatigue, dizziness, palpitation, and soreness and weakness of the waist and knees. The usage is 9g of pills once a day, orally before going to bed, but it is prohibited for pregnant women, patients with hypertension, and those with damp-heat constitution. Its curative effect is based on the Tibetan medicine theory of "nourishing the form with the form", combining highly active animal protein and plant tonic ingredients to synergistically enhance the effect.

[0014] Sheep testicle freeze-dried powder is made from the testicles of plateau Tibetan sheep through the freeze-drying process, and has the effects of strengthening yang and promoting sperm production, nourishing and strengthening the body. It is applicable to impotence, premature ejaculation, and the weak state caused by physical exhaustion. Its main components include natural steroid hormones, high protein, and trace elements (such as zinc), which can enhance sexual ability, resist fatigue, and improve immunity. This preparation belongs to the category of "directly orally taken decoction pieces", and the single dose is 1 - 2g, and it must be used strictly in accordance with the instructions or doctor's advice.

[0015] Venous Occlusive Dysfunction (VOD) of the corpus cavernosum penis is a type of erectile dysfunction (ED) caused by the inability of the venous system to close normally. In the normal physiological state, erection depends on the dynamic balance of increased arterial blood flow and venous return. Specifically, sexual stimulation triggers the parasympathetic nerve reflex, causing the cavernous artery to dilate, and blood to rapidly perfuse into the blood sinus; subsequently, the cavernous smooth muscle relaxes, the blood sinus expands and compresses the emissary veins under the tunica albuginea, blocking blood return, and finally forming a congestive high pressure to maintain erection. If the venous closure mechanism fails, even if the arterial blood supply is sufficient, the blood will still flow back prematurely, resulting in insufficient erection hardness or difficulty in maintaining erection.

[0016] Its causes are complex, covering multiple factors such as blood vessels, nerves, and structures. Vascular causes include chronic ischemia caused by arteriosclerosis, or cavernous smooth muscle fibrosis caused by diabetes and aging, weakening its relaxation ability; abnormal tunica albuginea structure (such as trauma scars or Peyronie's disease) may also damage the physical basis of venous closure. Neurological factors are often related to spinal cord injury or diabetic peripheral neuropathy, causing nerve signal conduction disorders, and then affecting smooth muscle function. Some cases are congenital venous leakage, such as the existence of abnormal communication branches between the corpus spongiosum urethrae and the corpus cavernosum penis. In addition, psychological anxiety may indirectly exacerbate venous leakage through excessive activation of the sympathetic nerve.

[0017] With increasing age (>50 years old), the incidence of venous occlusive dysfunction increases significantly, which is closely related to systemic vascular lesions and tissue aging. Insufficient blood supply to the cavernous artery caused by atherosclerosis can indirectly weaken the venous closure efficacy, as the initial filling of the cavernous blood sinus is insufficient to form sufficient tissue pressure to compress the emissary veins; the reduction of cavernous smooth muscle cells and the increase in fibrosis ratio associated with aging directly affect its relaxation ability, resulting in the failure of the venous closure mechanism. In addition, diabetes is highly prevalent in the elderly population, and the neuropathy and microvascular damage it causes synergistically accelerate the progression of venous leakage. Research shows that about 50% of venous ED in men over 60 years old is attributed to the degeneration of elastic fibers in the tunica albuginea of the corpus cavernosum, leading to insufficient physical venous compression.

[0018] The main manifestations of patients are slow onset of erection, insufficient rigidity (such as inability to complete the insertion action) or rapid softening after erection. Libido is usually retained, but sexual satisfaction is significantly reduced. The reduction in the frequency and hardness of nocturnal erections can be assisted by polysomnography (NPT) to evaluate whether it is an organic lesion.

[0019] Treatment is stratified and managed according to the severity. The first-line treatment is mainly oral PDE5 inhibitors (such as sildenafil, tadalafil). These drugs enhance local blood flow by inhibiting the breakdown of cGMP, but have limited effects on severe venous leakage; at the same time, lifestyle improvements are needed, including weight loss, smoking cessation, and blood sugar / blood pressure control to improve vascular endothelial function. Those with anxiety can be supplemented with psychological intervention. If the oral medications are ineffective, the second-line treatment can be a vacuum constriction device (VED) or intracavernous injection of prostaglandin E1, which directly dilates blood vessels and relaxes smooth muscle. For those with clear anatomical abnormalities and ineffective conservative treatment, venous ligation can be considered, but the recurrence rate after surgery is relatively high (30%-50%); penile prosthesis implantation is recommended for end-stage patients, and the long-term satisfaction rate can reach over 90%.

[0020] Disease progression may lead to aggravated fibrosis of the corpus cavernosum, resulting in irreversible damage. Complications such as priapism (more common after intracavernous injection), surgical-related infections, or prosthesis mechanical failures need to be vigilant during treatment. Long-term unrelieved ED may also cause depression or tension in the partner relationship, and multidisciplinary collaborative intervention is required.

[0021] Patient education should emphasize the importance of early medical treatment to avoid the acceleration of corpus cavernosum atrophy caused by long-term erectile dysfunction. Actively managing underlying diseases such as hypertension and diabetes can delay the progression of the disease. For young patients, congenital venous malformations need to be excluded. If hypospadias or abnormal venous communication is found, surgical correction should be performed in a timely manner. In addition, both doctors and patients need to jointly establish reasonable expectations and clarify that most venous ED requires comprehensive treatment, and a single method is difficult to completely cure.

[0022] Gymnadenia conopsea (L.) R.Br. is a terrestrial herbaceous plant belonging to the monocotyledon class and the genus Gymnadenia in the Orchidaceae family. The plant can reach up to 60 centimeters. It has a fleshy oval-shaped tuber, which is palmately divided at the lower part. The stem is erect. The leaves are linear-lanceolate, narrowly oblong or ribbon-shaped. The raceme is dense with flowers. The bracts are lanceolate. The petals are erect. The pollinia are ovoid, and the stipe is linear-lanceolate. In June and July, a spike inflorescence grows from it, with small pinkish-white flowers, which are very beautiful. The oblong capsule matures from August to September.

[0023] It is distributed in regions such as Northeast China and North China, as well as in provinces and autonomous regions including Shaanxi, Ningxia, Xinjiang, Shanxi, Inner Mongolia, and Sichuan. It grows in shady slopes, meadows, and forest grasslands in the mountains. Because its single underground fleshy root is thick and has 4 to 6 finger-like divisions, resembling a palm, it gets its name.

[0024] Gymnadenia conopsea is a Chinese herbal medicine for the elderly to prolong life and nourish the body. It is a cultivated ginseng variety passed down through generations in families, and there is a small amount of cultivation in various places. In the autumn of the second or third year after planting, dig out (or pull out) the Gymnadenia conopsea tuber with a diameter of only 1.5 to 2.5 centimeters, remove the stems, leaves, and fibrous roots, wash it, and put it into a boiling water pot and cook until there is no white core inside, then take it out and dry it for later use. Its nature and flavor are sweet, slightly bitter, and cool, and it has the effects of tonifying the kidney and benefiting essence, regulating qi and relieving pain. Gymnadenia conopsea is used in Mongolian and Tibetan medicine and is also used by the folk, but is rarely used in traditional Chinese medicine.

[0025] Piper longum L. is a perennial herbaceous climbing vine belonging to the genus Piper in the Piperaceae family. The plant can reach several meters in length. Most parts are often covered with powdery fine pubescence; the branches are zigzag-shaped, with thick ridges and grooves; the leaves are papery, the lower leaves are ovate or slightly reniform, the top leaves are ovate or ovate-oblong, and the lower leaf stalks are longer; the flowers are unisexual, dioecious; the fruit is a drupe, spherical. The flowering period is from July to October.

[0026] Piper longum is distributed in Yunnan, Guangxi, Guangdong, etc. in China; abroad, it is also distributed in Nepal, India, Sri Lanka and other countries. It grows in sparse shady mixed forests, bamboo forests, banana forests, around village fences, and open spaces by the river beach at an altitude of about 580 meters. Piper longum grows in relatively shady and humid forests, with tall and slender plants and few flowers and fruits; when it grows in sunny places, the plants are relatively short, but there are more flowers and fruits.

[0027] Piper longum is a traditional Chinese medicine, with the effects of warming the middle and dispelling cold, regulating qi and relieving pain. The fruit spike is an important medicine for relieving pain and strengthening the stomach. It has a pungent taste and a hot nature, and is used for abdominal pain, vomiting, diarrhea, angina pectoris of coronary heart disease, neuralgia headache, toothache, etc. caused by stomach cold.

[0028] Phyllanthus emblica L., also known as Wangguo, Amalaka, and Mihan, is a tree belonging to the genus Phyllanthus of the family Phyllanthaceae. Its fruits can be eaten raw, being sour, sweet, crispy, astringent at first taste, and sweet in aftertaste, hence the name "Yugan" (meaning the aftertaste is sweet). Its branches have longitudinal fine stripes and are covered with short yellowish-brown pubescence; the leaves are papery to leathery, arranged in two rows, linear-oblong; the axillary cyme is composed of many male flowers and 1 female flower or all male flowers, the anthers are erect, oblong, the sepals of the female flower are oblong or spatulate, the disc is cup-shaped, covering more than half of the ovary; the capsule is drupe-like, globular; the flowering period is from April to June, and the fruiting period is from July to September.

[0029] Phyllanthus emblica is distributed in countries such as India, Sri Lanka, and the Philippines, and is also cultivated in South America; in China, it is cultivated in provinces such as Jiangxi, Fujian, and Taiwan. It grows in mountainous sparse forests, thickets, wastelands, or sunny places in mountain valleys at an altitude of 200 - 2300 meters; it is a sun-loving tree species, young trees are more shade-tolerant, and sufficient sunlight is required during flowering and fruiting; it likes warmth and avoids frost; it likes acidity; it has strong adaptability, is drought-tolerant and barren-tolerant, and is suitable for acidic lateritic red soil with deep soil layers.

[0030] The wood of Phyllanthus emblica is hard, water-resistant, elastic, and can be used to make farm tools, furniture, and also as firewood; after its leaves are dried, they can be used to make pillow cores; its seeds contain oil, which can be used to make soap and is also an excellent health edible oil; its fruits can be processed into Phyllanthus emblica beverages, preserved fruits, fruit wines, and fruit powders; its immature fruits, young leaves, and bark contain tannins, which are important raw materials for tannin extracts in China. Its root system is well-developed, can conserve water and soil, and can be used as a pioneer tree species for afforestation of barren mountains and wastelands. Phyllanthus emblica is used as medicine with its roots, leaves, fruits, and bark, and has the effect of removing dampness and diuresis; the roots are mainly used to treat hypertension, stomachache, enteritis and other diseases; the leaves are mainly used to treat edema, skin eczema and other diseases; the fruits are mainly used to treat colds, fever, sore throat, toothache and other diseases.

[0031] Freeze-dried Phyllanthus emblica, that is, Phyllanthus emblica obtained by vacuum freeze-drying technology, retains the active ingredients of Phyllanthus emblica intact, avoids the destruction of volatile oils and other thermosensitive substances in Phyllanthus emblica, ensures the medicinal effect and quality of Phyllanthus emblica after drying, and is conducive to the dissolution, absorption and utilization of active ingredients.

[0032] The research paper published in the International Journal of Peptide Research and Therapeutics, titled Identification and Antioxidant Activity of a Novel Peptide from Baijiu. The research team isolated and identified a novel peptide Cys-Trp-Cys (CWC) from Baijiu, and conducted qualitative and quantitative research on it, and evaluated its antioxidant activity through in vitro chemical experiments and HepG2 cell models, providing a theoretical basis for in-depth understanding of the relationship between Baijiu and health.

[0033] Chinese liquor has a long history in China and is loved by consumers at home and abroad. Research shows that Chinese liquor contains various beneficial bioactive compounds, which are beneficial to health. The increase in reactive oxygen species (ROS) will trigger oxidative stress and damage cells. There is an antioxidant system in organisms to scavenge ROS, and antioxidant active substances in food can also play a role. Bioactive peptides have attracted attention due to their biological activities such as antioxidant activity. Although some polypeptides have been identified from Chinese liquor, there is a lack of systematic evaluation of the antioxidant activity of polypeptides in Chinese liquor.

[0034] A polypeptide in Chinese liquor was determined to be CWC by ion secondary mass spectrometry analysis, and its concentration in Chinese liquor was 11.06±0.34 μg / L. In vitro antioxidant experiments, CWC showed strong antioxidant activity in 2,2'-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ORAC) and reducing power experiments. Especially in the ABTS experiment, its antioxidant activity was significantly higher than that of water-soluble vitamin E (Trolox), but its chelating ability to Fe 2+ was relatively low. In the HepG2 cell model experiment, when the concentration was within 4.00 mg / mL, CWC had little effect on cell viability, and 1.00, 2.00 and 4.00 mg / mL were selected for subsequent experiments. CWC could dose-dependently inhibit the generation of reactive oxygen species (ROS) in HepG2 cells induced by 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH), reduce the content of malondialdehyde (MDA), and increase the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT), protecting cells from oxidative damage.

[0035] The novel tripeptide CWC has the amino acid sequence Cys-Trp-Cys, the molecular formula C 17 H 22 N4O4S2, the molecular weight is 410.1 Da, and the SMILES code is O=C(N[C@@H](CC1=CNC2=CC=CC=C12)C(N[C@@H](CS)C(O)=O)=O)[C@H](CS)N. The chemical structure is as follows:

[0036] Summary of the Invention

[0037] This invention patent application has carried out further research on the freeze-drying process of sheep testicles. It was found in the research that adding a small amount of tripeptide CWC to sheep testicles before freeze-drying can significantly improve the pharmacological activity of the obtained freeze-dried powder of sheep testicles. Furthermore, the effect is better after granulating the obtained freeze-dried powder of sheep testicles with the extract of Gymnadenia conopsea, Piper longum, and freeze-dried Phyllanthus emblica.

[0038] This application first discloses the use of freeze-dried sheep testicles in the preparation of drugs for the treatment of senile erectile dysfunction.

[0039] The source of the sheep testicles is the dried testicles of male sheep, which are bovidae mammals.

[0040] The preparation method of the freeze-dried sheep testicles is characterized by being completed by the following preparation process:

[0041] (1) Pretreatment of sheep testicles: Cleaning and selection: Removing the vas deferens and fat of the sheep testicles, etc.

[0042] (2) Processing of sheep testicles. The processing of the sheep testicles is as follows:

[0043] First soaking and pickling: Cleaning and soaking the selected sheep testicles with salt water, then fishing them out and draining.

[0044] Second cutting (chopping and mixing) and standing: Putting the soaked and pickled sheep testicles into a chopping machine to chop them into homogenate or pieces, and then standing them in a cold storage at 0°C - 5°C for 10 - 12 hours.

[0045] Third milk processing: Adding an appropriate amount of goat milk to the sheep testicles after the second cutting (chopping and mixing) and standing, stirring evenly, then boiling them in a jacketed pan for 7 - 8 hours. After all the goat milk is absorbed by the sheep testicles, stop heating and let it cool to room temperature.

[0046] Fourth premixing: Adding an appropriate amount of tripeptide CWC ethanol aqueous solution to the sheep testicles after cooling in the third milk processing, at low temperature, to volatilize the ethanol and water, and making all the tripeptide CWC be absorbed by the sheep testicles.

[0047] Fifth vacuum freeze-drying: Putting the sheep testicles obtained in the fourth step into a vacuum freeze-drying equipment for pre-freezing, sublimation drying, and analytical drying for 24 - 30 hours. When the water content of the sheep testicles is less than 5%, take them out of the warehouse.

[0048] The preparation method of the freeze-dried sheep testicles can also be completed by the following preparation process:

[0049] (1) Pretreatment of sheep testicles: Cleaning and selection: Removing the vas deferens and fat of the sheep testicles, etc.

[0050] (2) Processing of sheep testicles. The processing of the sheep testicles is as follows:

[0051] First soaking and pickling: Cleaning and soaking the selected sheep testicles with salt water, then fishing them out and draining.

[0052] Second cutting (chopping and mixing) and standing: Putting the soaked and pickled sheep testicles into a chopping machine to chop them into homogenate or pieces, and then standing them in a cold storage at 0°C - 5°C for 10 - 12 hours.

[0053] Third milk process: add appropriate amount of goat milk to the sheep testicles after the second cutting (chopping and mixing) and let stand, stir evenly, put into a sandwich pot and cook for 7-8 hours. After all the goat milk is absorbed by the sheep testicles, stop heating and let cool to room temperature;

[0054] Fourth vacuum freeze drying: placing the sheep testicles obtained in the fourth step into a vacuum freeze drying device for pre-freezing, sublimation drying, and analytical drying for 24-30 hours, and removing the testicles from the storage when the water content is less than 5%;

[0055] The preparation process of the sheep testicle freeze-dried powder also includes a low-temperature ultrafine grinding step, that is, the sheep testicles obtained after vacuum freeze drying are in irregular pieces and blocks, which are ground by low-temperature ultrafine grinding equipment.

[0056] In the preparation process of the freeze-dried sheep testicle powder, the soaking and pickling step is specifically to soak the sheep testicles in saline with a concentration of 2-3% for 5-10 hours, the saline needs to completely immerse the sheep testicles, and then take them out and drain them.

[0057] In the preparation process of the freeze-dried sheep testicle powder, the specific step of milk preparation is to add 4kg-5kg of whole goat milk powder and 76kg-80kg of purified water to 100kg of sheep testicles pickled in brine, stir evenly, put into a sandwich pot for cooking, and set the temperature of the sandwich pot to 100℃-120℃.

[0058] In the preparation process of the sheep testicle freeze-dried powder, the specific step of premixing is to add 5kg-10kg of 75% ethanol solution into 100kg of milk-made sheep testicles, in which 5%-10% of the tripeptide CWC is dissolved, and the volatilization temperature is 40°C.

[0059] In the preparation process of the sheep testicle freeze-dried powder, the specific steps of vacuum freeze drying and the drying curve are as follows:

[0060]

[0061] In order to further enhance the efficacy of sheep testicles, the present application secondly discloses a freeze-dried powder composition, the preparation process of which is as follows:

[0062] Take 10-40g of Panax ginseng, 5-15g of Piper longum and 15-45g of freeze-dried Phyllanthus emblica, select them, add them into 1000ml of highland barley rice wine, heat them to 40-45℃, seal them, soak them for 30-60 days, take out an appropriate amount of supernatant, add freeze-dried sheep testicles, mix them evenly, and make granules with a dry granulator.

[0063] In the preparation process of the freeze-dried powder composition, taking an appropriate amount of supernatant refers to 2-3 kg of medicinal wine supernatant for 100 kg of sheep testicle freeze-dried powder.

[0064] The beneficial effects of the present application are as follows: The present application discloses a preparation method of freeze-dried powder of sheep testicles. During the preparation process, the hypertonic saline used for soaking and pickling can remove the fishy smell in the sheep testicles and play a role in cleaning and sterilization; goat milk is a good organic solvent, and after being processed, it has a synergistic effect, which can play a role in removing odor, correcting taste, and synergistic enhancement. It not only removes the fishy smell of sheep testicles, but also enhances the effects of strengthening yang and promoting sperm production and nourishing the body of sheep testicles. Coupled with pickling with salt, in traditional Chinese medicine theory, salt enters the kidney, and it has the same pharmacological synergistic effect of warming the kidney yang and tonifying qi and blood as goat milk, etc.; through vacuum freeze-drying, the active ingredients in the salt-pickled and then milk-processed sheep testicles can be completely retained. After freeze-drying, the water content can reach below 5%, and the freeze-dried sheep testicles are loose in texture and have extremely strong re-dissolvability. Ultra-low temperature pulverization can keep the material in an environment below 0°C for pulverization, and can also retain the active ingredients and effects of the freeze-dried sheep testicles to the greatest extent; adding tripeptide CWC before freeze-drying can further enhance the efficacy of the obtained freeze-dried powder of sheep testicles. Coupled with the synergistic effect of three traditional Tibetan medicine herbs, Gymnadenia conopsea, Piper longum, and freeze-dried Phyllanthus emblica, which have the characteristics of the same origin of medicine and food in traditional Tibetan medicine, the efficacy of sheep testicles is further consolidated and enhanced. Detailed implementation manners

[0065] The following specific examples are used to further illustrate the present invention, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0066] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0067] Example 1: Preparation of freeze-dried powder of sheep testicles (Lutuzhebu)

[0068] The preparation process is as follows:

[0069] (1) Pretreatment of sheep testicles: Cleaning and selection: Remove the vas deferens and fat of sheep testicles, etc.;

[0070] (2) Processing of sheep testicles. The processing of sheep testicles is as follows:

[0071] First pickling: Wash and soak the selected sheep testicles with 2% saline for 10 hours, then take them out and drain;

[0072] Second cutting (chopping and mixing) and standing: Put the soaked and pickled sheep testicles into a chopping and mixing machine and chop them into a homogeneous slurry or pieces. The speed of the chopping and mixing machine is controlled at 45 kg per hour of chopping; place them in a refrigerated storage at 0°C - 5°C and stand for 12 hours;

[0073] Third milk preparation: Add 4 kg of whole-fat sheep milk powder to 76 kg of purified water. After stirring evenly, add it to 100 kg of sheep testicles that have been cut (chopped and stirred) and left standing. Stir evenly, then put it into a jacketed pan and cook for 7 - 8 hours. The temperature of the jacketed pan is set at 100°C - 120°C (the material temperature is about 90°C - 100°C). Stop heating after all the sheep milk is absorbed by the sheep testicles, and let it cool to room temperature;

[0074] Fourth premixing: Add 5 kg of a 75% ethanol solution dissolving 5% tripeptide CWC to 100 kg of sheep testicles that have cooled after milk preparation at 40°C to allow the ethanol and water to evaporate, and all of the tripeptide CWC is absorbed by the sheep testicles.

[0075] Fifth vacuum freeze-drying: Put the sheep testicles obtained in the fourth step into a vacuum freeze-drying device for pre-freezing, sublimation drying, and analytical drying. Take them out of the warehouse when the water content of the sheep testicles is less than 5%; The freeze-drying parameters are as follows:

[0076]

[0077] Sixth low-temperature ultrafine grinding: After vacuum freeze-drying, the sheep testicles are in irregular loose sheet-like blocks and are ground with a low-temperature ultrafine grinding device to obtain the product.

[0078] Example 2: Preparation of freeze-dried powder of sheep testicles (Lutu Zhebu)

[0079] The preparation process is as follows:

[0080] (1) Pretreatment of sheep testicles: Cleaning and selection: Remove the vas deferens, fat, etc. of the sheep testicles;

[0081] (2) Processing of sheep testicles. The processing of the sheep testicles is as follows:

[0082] First pickling: Wash and soak the cleaned and selected sheep testicles with 3% brine for 5 hours, then take them out and drain;

[0083] Second cutting (chopping and stirring) and standing: Put the pickled and soaked sheep testicles into a chopping machine and chop them into a homogeneous slurry or pieces. The speed of the chopping machine is controlled at 40 kg per hour of chopping; Let it stand in a refrigerated warehouse at 0°C - 5°C for 10 hours;

[0084] Third milk preparation: Add 5 kg of whole-fat sheep milk powder to 80 kg of purified water. After stirring evenly, add it to 100 kg of sheep testicles that have been cut (chopped and stirred) and left standing. Stir evenly, then put it into a jacketed pan and cook for 7 - 8 hours. The temperature of the jacketed pan is set at 100°C - 120°C (the material temperature is about 90°C - 100°C). Stop heating after all the sheep milk is absorbed by the sheep testicles, and let it cool;

[0085] Fourth premixing: Add 5 kg of 75% ethanol solution dissolving 10% tripeptide CWC to 100 kg of cooled sheep testicles after milking at 40°C to volatilize ethanol and water, and all tripeptide CWC is absorbed by the sheep testicles.

[0086] Fifth vacuum freeze-drying: Put the milked sheep testicles into a vacuum freeze-drying equipment for pre-freezing, sublimation drying, and analytical drying, and take them out of the warehouse when the water content of the sheep testicles is less than 5%; the freeze-drying parameters are as follows:

[0087]

[0088] Sixth low-temperature ultrafine grinding: After vacuum freeze-drying, the sheep testicles are in irregular loose sheet-like blocks, and are obtained by grinding with a low-temperature ultrafine grinding equipment.

[0089] Example 3: Preparation of freeze-dried powder of sheep testicles (Lutu Zhebu)

[0090] The preparation process is as follows:

[0091] (1) Pretreatment of sheep testicles: Cleaning and selection: Remove the vas deferens and fat of sheep testicles, etc.

[0092] (2) Processing of sheep testicles, the processing of sheep testicles is as follows:

[0093] First pickling: Wash and soak the selected sheep testicles with 2.5% brine for 11 hours, then take them out and drain.

[0094] Second cutting (chopping and mixing) and standing: Put the pickled and soaked sheep testicles into a chopping machine and chop them into homogenate or pieces, and control the speed of the chopping machine at 45 kg per hour; place them in a cold storage at 0°C - 5°C and stand for 11 hours.

[0095] Third milking: Add 4 kg of whole milk powder of sheep to 76 kg of purified water, stir evenly and then add it to 100 kg of sheep testicles after cutting (chopping and mixing) and standing, stir evenly, and put it into a jacketed pan and boil for 7 - 8 hours, and set the temperature of the jacketed pan at 100°C - 120°C (the material temperature is about 90°C - 100°C). Stop heating after all the sheep milk is absorbed by the sheep testicles and let it cool.

[0096] Fourth premixing: Add 10 kg of 75% ethanol solution dissolving 10% tripeptide CWC to 100 kg of cooled sheep testicles after milking at 40°C to volatilize ethanol and water, and all tripeptide CWC is absorbed by the sheep testicles.

[0097] Fifth vacuum freeze-drying: Put the milked sheep testicles into a vacuum freeze-drying equipment for pre-freezing, sublimation drying, and analytical drying, and take them out of the warehouse when the water content of the sheep testicles is less than 5%; the freeze-drying parameters are as follows:

[0098]

[0099] Sixth, low-temperature ultrafine grinding: After vacuum freeze-drying, the sheep testicles are in irregular loose flaky blocks, and they can be obtained by grinding with low-temperature ultrafine grinding equipment.

[0100] Example 4: Preparation of freeze-dried powder of sheep testicles (Lutu Zhebu)

[0101] The preparation process is as follows:

[0102] (1) Pretreatment of sheep testicles: Selection: Remove the vas deferens and fat of sheep testicles, etc.;

[0103] (2) Processing of sheep testicles. The processing technology of the sheep testicles is as follows:

[0104] First pickling: Wash and soak the selected sheep testicles with 2% brine for 10 hours, then take them out and drain.

[0105] Second cutting (chopping and mixing) and standing: Put the pickled sheep testicles into a chopping machine and chop them into homogenate or pieces. The speed of the chopping machine is controlled at 45 kg per hour of chopping; place them in a cold storage at 0°C - 5°C and let them stand for 12 hours;

[0106] Third milking: Add 4 kg of whole milk powder of sheep to 76 kg of purified water, stir evenly and then add it to 100 kg of sheep testicles after cutting (chopping and mixing) and standing, stir evenly, and put it into a jacketed pan and cook for 7 - 8 hours. The temperature of the jacketed pan is set at 100°C - 120°C (the material temperature is about 90°C - 100°C). Stop heating after all the sheep milk is absorbed by the sheep testicles, and let it cool down;

[0107] Fourth premixing: Add 5 kg of 75% ethanol solution dissolving 10% of tripeptide CWC to 100 kg of sheep testicles cooled after milking at 40°C to volatilize ethanol and water, and all tripeptide CWC is absorbed by the sheep testicles.

[0108] Fifth vacuum freeze-drying: Put the milked sheep testicles into a vacuum freeze-drying equipment for pre-freezing, sublimation drying, and analytical drying: The freeze-drying parameters are as follows:

[0109]

[0110] Sixth, low-temperature ultrafine grinding: After vacuum freeze-drying, the sheep testicles are in irregular loose flaky blocks, and they can be obtained by grinding with low-temperature ultrafine grinding equipment.

[0111] (3) Granulation: Granulate the freeze-dried powder of sheep testicles ground by low-temperature ultrafine grinding equipment by dry granulation with medicinal wine:

[0112] Take 20 g of Gymnadenia conopsea, 10 g of Piper longum, and 30 g of freeze-dried Phyllanthus emblica. After cleaning and selecting, add them to 1000 ml of highland barley rice wine. Heat to 40 - 45 °C, seal, and soak for 60 days. Then take the supernatant. For 100 kg of freeze-dried sheep testicles, use 3 kg of the medicinal wine supernatant. Add 3 kg of the medicinal wine supernatant to the freeze-dried sheep testicles pulverized by a low-temperature ultrafine pulverization device, mix evenly, and granulate with a dry granulation machine to obtain the product.

[0113] Example 5: Preparation of freeze-dried sheep testicles (Lutu Zhebu)

[0114] The preparation process is as follows:

[0115] (1) Pretreatment of sheep testicles: Cleaning and selecting: Remove the vas deferens and fat of sheep testicles, etc.

[0116] (2) Processing of sheep testicles. The processing of sheep testicles is as follows:

[0117] First pickling: Wash and soak the cleaned and selected sheep testicles with 2% brine for 10 hours, then take them out and drain.

[0118] Second cutting (chopping and mixing) and standing: Put the pickled sheep testicles into a chopping machine and chop them into a homogeneous slurry or pieces. The speed of the chopping machine is controlled at 45 kg per hour of chopping; place them in a cold storage at 0 °C - 5 °C and let them stand for 12 hours.

[0119] Third milking: Add 4 kg of whole-fat sheep milk powder to 76 kg of purified water, stir evenly, and then add it to 100 kg of sheep testicles after cutting (chopping and mixing) and standing. Stir evenly, put it into a jacketed pan and cook for 7 - 8 hours. The temperature of the jacketed pan is set at 100 °C - 120 °C (the material temperature is about 90 °C - 100 °C). Stop heating after all the sheep milk is absorbed by the sheep testicles and let it cool.

[0120] Fourth premixing: Add 10 kg of a 75% ethanol solution dissolving 10% of tripeptide CWC to 100 kg of sheep testicles cooled after milking at 40 °C to volatilize the ethanol and water, and all of the tripeptide CWC is absorbed by the sheep testicles.

[0121] Fifth vacuum freeze-drying: Put the milked sheep testicles into a vacuum freeze-drying device for pre-freezing, sublimation drying, and analytical drying. The freeze-drying parameters are as follows:

[0122]

[0123] Sixth low-temperature ultrafine pulverization: After vacuum freeze-drying, the sheep testicles are in irregular loose sheet-like blocks, and they can be obtained by pulverizing with a low-temperature ultrafine pulverization device.

[0124] (3) Granulation: Granulate the freeze-dried sheep testicles pulverized by a low-temperature ultrafine pulverization device with medicinal wine by dry granulation:

[0125] After selecting 40 g of Gymnadenia conopsea, 15 g of Piper longum, and 45 g of freeze-dried Phyllanthus emblica, add them to 1000 ml of highland barley rice wine. Heat to 40 - 45 °C, seal, and soak for 30 days. Then take the supernatant. For 100 kg of freeze-dried sheep testicles powder, use 3 kg of the medicinal wine supernatant. Add 3 kg of the medicinal wine supernatant to the freeze-dried sheep testicles powder pulverized by a low-temperature ultrafine pulverization device, mix evenly, and granulate with a dry granulation machine to obtain the product.

[0126] Control Example 1:

[0127] Prepared according to the preparation process of the freeze-dried sheep testicles powder in Example 1, without adding tripeptide CWC, and the remaining steps remain unchanged. Prepare the freeze-dried sheep testicles powder.

[0128] Improvement effect of the freeze-dried sheep testicles powder in Example 6 on erectile dysfunction (ED) in model rats

[0129] 6.1 Animal grouping and administration

[0130] Divide 10-month-old middle-aged Fisher 344 rats into 5 groups, with 6 rats in each group. Another 6 3-month-old Fisher 344 rats are taken and treated daily for 2 months. The specific treatments are as follows:

[0131] a) 5-month-old control group: At the time of sacrifice, it is 12 months old. Administer by gavage, only give 2 ml of 0.5% sodium carboxymethylcellulose solution as an excipient once a day;

[0132] b) 12-month-old control group: At the time of sacrifice, it is 12 months old. Administer by gavage, only give 2 ml of 0.5% sodium carboxymethylcellulose solution as an excipient once a day;

[0133] c) Control Example 1 group: Based on the weight of the freeze-dried sheep testicles powder, 90 mg / kg, dissolve or suspend in 2 ml of excipient once a day;

[0134] d) Example 1 group: Based on the weight of the freeze-dried sheep testicles powder, 90 mg / kg, dissolve or suspend in 2 ml of excipient once a day;

[0135] e) Example 4 group: Based on the weight of the freeze-dried sheep testicles powder, 90 mg / kg, dissolve or suspend in 2 ml of excipient once a day;

[0136] f) Example 5 group: Based on the weight of the freeze-dried sheep testicles powder, 90 mg / kg, dissolve or suspend in 2 ml of excipient once a day;

[0137] g) Positive control group: Tadalafil (TAD): 2.0 mg / kg, dissolve or suspend in 2 ml of excipient once a day;

[0138] The dose of tadalafil was corrected according to body weight, equivalent to 22 mg per day for a 70-kg person. This dose is slightly higher than the maximum recommended dose for humans.

[0139] 6.2 Measurement indicators and results

[0140] 6.2.1 Measurement of cavernous pressure by dynamic cavernosometry

[0141] The specific measurement process and steps are as follows:

[0142] 1) Preparation: Cannulate the left and right penile crura of the rat using a 23-gauge butterfly needle. One of the cannulas needs to be flushed with sterile heparinized saline (100 U heparin / ml) and connected to a pressure detector for continuous monitoring of the intracorporal pressure; the other cannula is connected to an infusion pump filled with sterile diluted heparinized saline (20 U heparin / ml).

[0143] 2) Record the baseline pressure: Record the intracorporal pressure of the rat's penis in the flaccid state, i.e., the baseline ICP (flaccid ICP).

[0144] 3) Inject the drug: Inject a certain dose of papaverine (1 mg dissolved in 0.1 ml of water for injection) through the infusion cannula and give 5 minutes for papaverine to fully diffuse in the corpus cavernosum.

[0145] 4) Flush and zero: After the diffusion of papaverine is completed, flush the infusion cannula with heparinized saline (20 U heparin / ml), and then vent the pressure monitoring cannula to restore the intracorporal pressure to the normal level.

[0146] 5) Record the pressure again: After waiting for 5 minutes, record the intracorporal pressure at this time, i.e., the ICP after papaverine injection (ICPAP).

[0147] 6) Adjust the infusion rate and record: Start the infusion at a rate of 0.05 ml / min and increase the infusion rate by 0.05 ml / min every 10 seconds until the intracorporal pressure begins to rise. After that, only increase the infusion rate after the intracorporal pressure reaches the plateau phase until the pressure reaches 100 cm (erection pressure), and record the infusion rate required to maintain this pressure (the maintenance rate).

[0148] 7) Record the pressure drop rate: When the pressure is stably maintained for 20 seconds, stop the infusion and record the change in intracorporal pressure within the subsequent 60 seconds, i.e., the pressure drop rate (the drop rate).

[0149] ICPAP and the pressure drop rate are as follows:

[0150] Table 1 Data of ICPAP and pressure decline rate in model rats (n = 6, )

[0151]

[0152]

[0153] As shown in the data in Table 1, 5-month-old rats are in the youth stage with normal corpus cavernosum function. After injecting papaverine into the corpus cavernosum, ICPAP increased significantly to 77.8 ± 1.2 mmHg. And after reaching the plateau phase, the pressure decline rate was the lowest, only 18.6 ± 0.5 mmHg / min. In contrast, 12-month-old control rats, belonging to the old age stage, due to age, ICPAP was only 57.5 ± 2.9 mmHg, significantly lower than that of 5-month-old rats, and its pressure decline rate was the largest among all groups at 35.9 ± 2.5 mmHg / min.

[0154] In the control group of Comparative Example 1, giving only freeze-dried powder of sheep testis slightly increased ICPAP and slightly decreased the pressure decline rate, but it was better than the 12-month-old control group given only excipient.

[0155] The group of Example 1 was given freeze-dried powder of sheep testis added with tripeptide CWC, and its improvement effect on ED in aging rats was significantly enhanced. Specifically, compared with the 12-month-old control group and the control group of Comparative Example 1, ICPAP increased significantly to 69.5 ± 3.1 mmHg, and the pressure decline rate decreased significantly to 26.8 ± 4.8 mmHg / min.

[0156] In the groups of Example 4 and Example 5, freeze-dried powder of sheep testis added with extracts of Gymnadenia conopsea, Piper longum, and dried Phyllanthus emblica was given, and its improvement effect on ED in aging rats was more obvious, and ICPAP and the pressure decline rate were comparable to those of the positive control group (given tadalafil).

[0157] 6.2.2 Determination of reduced glutathione (GSH) and oxidized glutathione (GSSG) in blood

[0158] GSH is the most important non-enzymatic antioxidant in the body, protecting cells from oxidative damage by neutralizing free radicals (such as reactive oxygen species ROS). ED patients often have elevated oxidative stress (such as a decrease in the GSH / GSSG ratio), leading to vascular endothelial dysfunction and apoptosis of corpus cavernosum smooth muscle cells. The blood GSH level in elderly men decreases significantly, which is directly related to corpus cavernosum fibrosis and smooth muscle reduction.

[0159] Blood was collected from the orbital sinus of rats, and the GSH / GSSG ratio in the blood samples was measured. The contents of GSSG and GSH in the blood samples were detected using an oxidized / reduced glutathione detection kit. GSH / GSSG ratio = (total GSH - 2 * GSSG) / GSSG.

[0160] For the specific method, refer to the instruction manual of the kit (MCE oxidized / reduced glutathione detection kit). The GSH / GSSG ratio is inversely proportional to the level of oxidative stress and can be used to illustrate the antioxidant effects of the examples and comparative samples used in the experiment, indirectly indicating its improvement effect on ED in model rats.

[0161] 6.2.3 Histochemistry and immunohistochemistry:

[0162] After cavernosometry and blood collection, the animals were euthanized. The midshaft penile skinless portion was fixed overnight in 4% buffered paraformaldehyde and stored in 70% alcohol at 4 °C for 24 hours until paraffin-embedded tissue sectioning was performed.

[0163] The following procedures were performed using 5-μm anatomically matched sections:

[0164] a) Collagen (blue) and smooth muscle (red) were stained using Masson's trichrome staining.

[0165] b) Immunodetection was performed using a polyclonal antibody against desmin, which is a marker for smooth muscle cell content.

[0166] c) Detection was performed using a polyclonal antibody against inducible nitric oxide synthase (iNOS). The specificity of the antibody was verified by Western blotting.

[0167] The sections were then incubated with biotinylated anti-rabbit IgG, followed by the addition of an ABC complex and 3,3'-diaminobenzidine. The sections were counterstained with hematoxylin. In immunohistochemistry experiments, the negative control was replacing the primary antibody with an IgG isotype.

[0168] 6.2.4 Quantitative image analysis:

[0169] Using ImagePro 7.1 software, quantitative image analysis was performed by computer densitometry, and the software was used in conjunction with an Olympus BHS microscope equipped with a Retiga digital camera. For Masson staining, pictures of the penis magnified 40 times and containing half of the corpus cavernosum were analyzed, and the smooth muscle (stained red) and collagen areas (stained blue) were measured, excluding the sinusoidal spaces, and expressed as the smooth muscle / collagen ratio. For desmin and iNOS staining, pictures of the corpus cavernosum magnified 200 times were analyzed in a computer grid and expressed as the percentage of the positive area in the total area of the corpus cavernosum. In all cases, 4 fields of view at 40 times magnification or 8 fields of view at 200 times magnification were analyzed for each tissue section, with at least 4 matching sections per animal and 6 animals per group.

[0170] Table 2 Data of GSH / GSSG ratio, smooth muscle (SMC) / collagen ratio and iNOS area ratio in model animals of each group (n = 6, )

[0171]

[0172] To determine whether the improvement in erectile function and the reduction in corpus cavernosum fibrosis observed after treatment with the samples of the examples for 2 consecutive months can be attributed to a decrease in oxidative stress (a decrease in oxidative stress is one of the putative mechanisms by which NO produced by iNOS exerts its antifibrotic effect), the GSH / GSSG ratio was measured in the whole blood of the test animals in each group. The data in Table 2 showed that compared with 5-month-old young rats (high ratio), 12-month-old control animals had a relatively high level of oxidative stress (very low ratio). We found that administering the samples of Comparative Example 1, Example 1, Example 4 and Example 5 daily for 2 consecutive months could effectively increase the GSH / GSSG ratio in the blood of 12-month-old model rats, especially in the groups of Example 4 and Example 5, which could almost be increased to a level similar to that of 5-month-old animals (lower oxidative stress).

[0173] To determine the effect of the test samples on the content of smooth muscle in the corpus cavernosum of model rats, the smooth muscle marker desmin was evaluated in adjacent sections of the above SMC / collagen ratio by immunohistochemistry. As expected, compared with 5-month-old rats, the desmin expression in 12-month-old rats was significantly decreased, only 5.1 ± 0.3. After administering the test samples for 2 consecutive months, the desmin expression was significantly increased, especially in the animals of the groups of Example 4 and Example 5, which were close to the level of 5-month-old young animals and comparable to the positive control group. At the same time, the group of Example 1 was better than the group of Comparative Example 1.

[0174] To clarify whether the improvement in penile dynamics and reduction in fibrosis observed in these treated animals were mediated by increased iNOS expression, iNOS was evaluated by immunohistochemistry. As expected, with aging, the expression of iNOS in 12-month-old control rats was significantly increased compared to that in the 5-month-old rat control group. After administering the test samples to each group daily for 2 consecutive months, the expression levels of iNOS in rats in each group were significantly increased compared to those in 12-month-old control animals. The groups of Example 4, Example 5, and the positive control group were comparable. The group of Example 1 was slightly worse, but better than the group of Comparative Example 1.

[0175] By ICPAP and descent rate measurements, the erectile function of rats at 12 months of age was significantly decreased. This decrease was histologically accompanied by a decrease in the content of cavernous smooth muscle, its replacement by collagen, and an increase in cavernous fibrosis. Administering the freeze-dried powder of sheep testicles described in Example 1 and the freeze-dried powder compositions of sheep testicles described in Example 4 and Example 5 to these rats daily for 2 consecutive months could reverse this process because the veno-occlusive sexual dysfunction (a type of ED) of the corpus cavernosum penis measured by the descent rate was improved, the smooth muscle content increased, and cavernous fibrosis decreased. The level of oxidative stress measured by the GSH / GSSG level in the blood was also increased. The test groups of Example 4 and Example 5 reached levels similar to those of the positive control group tadalafil in the test. Although the effects of Example 1 and Comparative Example 1 were slightly worse than those of the positive drug, they were still better than the 12-month-old control group given only the excipient, confirming that the freeze-dried powder of sheep testicles and the compositions described in Comparative Example 1, Example 1, Example 4, and Example 5 can be used to improve senile erectile dysfunction.

[0176] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. That is, the freeze-dried powder of sheep testicles that has been pickled, cut, milked, and vacuum freeze-dried, the freeze-dried powder of sheep testicles added with the tripeptide CWC, and the freeze-dried powder of sheep testicles added with the tripeptide CWC, Gymnadenia conopsea, Piper longum, and the supernatant of the freeze-dried Phyllanthus emblica medicinal wine are all within the protection scope of this patent. The protection scope of this patent is defined by the authorized claims. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A sheep testicle freeze-dried powder, characterized in that: The preparation process is as follows: (1) Sheep testicle pretreatment: Cleaning: removing the vas deferens and fat from the sheep testicles; (2) Processing of sheep testicles, the processing of the sheep testicles is as follows: First soaking and pickling: wash and soak the cleaned sheep testicles with salt water, remove and drain; Second, chopping (chopping and mixing) and letting it stand: put the soaked and marinated sheep testicles into a chopping machine and chop them into a homogenate or pieces, and place them in a refrigerator at 0℃-5℃ and let them stand for 10-12 hours; Third milk process: add appropriate amount of goat milk to the sheep testicles after the second cutting (chopping and mixing) and let stand, stir evenly, put into a sandwich pot and cook for 7-8 hours. After all the goat milk is absorbed by the sheep testicles, stop heating and let cool to room temperature; Fourth vacuum freeze drying: putting the sheep testicles obtained by the fourth premixing into a vacuum freeze drying device for prefreezing, sublimation drying, and analytical drying, and taking out the testicles when the water content of the sheep testicles is less than 5%.

2. A sheep testicle freeze-dried powder, characterized in that: The preparation process is as follows: (1) Sheep testicle pretreatment: Cleaning: removing the vas deferens and fat from the sheep testicles; (2) Processing of sheep testicles, the processing of the sheep testicles is as follows: First soaking and pickling: wash the soaked and cleaned sheep testicles with salt water, remove and drain; Second, chopping (chopping and mixing) and letting it stand: put the soaked and marinated sheep testicles into a chopping machine and chop them into a homogenate or pieces, and place them in a refrigerator at 0℃-5℃ and let them stand for 10-12 hours; Third milk process: add appropriate amount of goat milk to the sheep testicles after the second cutting (chopping and mixing) and let stand, stir evenly, put into a sandwich pot and cook for 7-8 hours. After all the goat milk is absorbed by the sheep testicles, stop heating and let cool to room temperature; Fourth premixing: add an appropriate amount of tripeptide CWC ethanol aqueous solution to the sheep testicles after cooling in the third milk system, at low temperature, so that ethanol and water evaporate, and the tripeptide CWC is completely absorbed by the sheep testicles; The fifth vacuum freeze drying: the sheep testicles obtained by the fourth premixing are placed in a vacuum freeze drying device for prefreezing, sublimation drying, and analytical drying, and the sheep testicles are taken out of the warehouse when the water content is less than 5%.

3. The sheep testicle freeze-dried powder according to claim 1 or 2, characterized in that The preparation process also includes the following steps: Low-temperature ultrafine grinding: After vacuum freeze-drying, the sheep testicles are in irregular pieces and can be crushed using low-temperature ultrafine grinding equipment.

4. The sheep testicle freeze-dried powder according to claim 1 or 2, characterized in that: In the preparation process of the freeze-dried sheep testicle powder, the soaking and pickling step is specifically to soak the sheep testicles in saline with a concentration of 2-3% for 5-10 hours, the saline needs to completely immerse the sheep testicles, and then the testicles are taken out and drained.

5. The sheep testicle freeze-dried powder according to claim 1 or 2, characterized in that: In the preparation process of the sheep testicle freeze-dried powder, the specific steps of milk preparation are: adding 4kg-5kg of whole goat milk powder and 76kg-80kg of purified water to 100kg of sheep testicles pickled in brine, stirring evenly, and putting them into a sandwich pot for cooking, and the temperature of the sandwich pot is set at 100℃-120℃.

6. The sheep testicle freeze-dried powder according to claim 1 or 2, characterized in that: In the preparation process of the sheep testicle freeze-dried powder, the specific step of premixing is to add 5kg-10kg of 75% ethanol solution into 100kg of milk-made sheep testicles, in which 5%-10% of the tripeptide CWC is dissolved, and the volatilization temperature is 40°C.

7. The sheep testicle freeze-dried powder according to claim 1 or 2, characterized in that: In the preparation process of the sheep testicle freeze-dried powder, the vacuum freeze-drying process parameters are as follows:

8. A sheep testicle freeze-dried powder composition, the preparation process is as follows: Take 10-40g of Panax ginseng, 5-15g of Piper longum, and 15-45g of freeze-dried Phyllanthus emblica, select them, add them into 1000ml of highland barley rice wine, heat them to 40-45°C, seal them, soak them for 30-60 days, take an appropriate amount of the supernatant and add it into the freeze-dried sheep testicle powder according to claim 1 or 2, mix them evenly, and use a dry granulator to make granules to obtain the product, characterized in that: In the preparation process, taking an appropriate amount of supernatant means taking 2-3 kg of medicinal wine supernatant for 100 kg of sheep testicle freeze-dried powder.

9. Use of the sheep testicle freeze-dried powder according to claim 1 or 2 in preparing a drug for treating senile erectile dysfunction.

10. Use of the sheep testicle freeze-dried powder composition according to claim 8 in preparing a drug for treating senile erectile dysfunction.

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