A traditional Chinese medicine composition for tonifying and a preparation method and application thereof

Abstract

The application relates to the technical field of medicines, and provides a traditional Chinese medicine composition for tonifying and a preparation method and application thereof. The raw material components of the traditional Chinese medicine composition for tonifying comprise 57-106 parts by weight of prepared rehmannia, 34-64 parts by weight of fried Chinese yam or Chinese yam roasted with bran, 23-43 parts by weight of poria, 29-53 parts by weight of wine lees, 7-13 parts by weight of wine schisandra, 23-43 parts by weight of salt eucommia, and 23-43 parts by weight of wine achyranthes. The traditional Chinese medicine composition for tonifying provided by the application has the effects of nourishing yin and strengthening yang, and benefiting qi and blood, and the zebra fish efficacy experiment proves that the traditional Chinese medicine composition has the effects of resisting fatigue, resisting aging and protecting kidney functions, such as reducing the incidence of kidney edema and improving the glomerular filtration function.

Description

[0001] This application claims priority to Chinese Patent Application No. CN202411739384.9, filed on November 29, 2024, entitled "A Tonifying Traditional Chinese Medicine Composition and Its Preparation Method and Application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of pharmaceutical technology, and in particular to a tonic Chinese medicine composition, its preparation method, and its application. Background Technology

[0003] From the perspective of traditional Chinese medicine, kidney deficiency refers to insufficient essence, qi, yin, and yang in the kidneys. Its main symptoms include lower back pain, hot flashes, night sweats, spontaneous sweating, dizziness, tinnitus, cold limbs, aversion to cold, and decreased sexual function. Kidney deficiency not only leads to sexual dysfunction and affects fertility, but in severe cases can cause congenital malformations, intellectual disabilities, and immune system disorders in offspring. It can also cause fatigue, forgetfulness, hair loss, and menopausal syndrome. Western medicine treatment for urogenital diseases caused by kidney deficiency (such as late-onset hypogonadism in men, benign prostatic hyperplasia, and menopausal syndrome in women) typically focuses on testosterone supplementation, oral medications, and hormone therapy. However, this approach has many side effects, is prone to relapse, and increases the risk of prostate and breast cancer, as well as cardiovascular events. Therefore, there is a need to develop effective and safe tonifying drugs. Summary of the Invention

[0004] The purpose of this application is to provide a tonic traditional Chinese medicine composition, its preparation method, and its application. The components of this composition exhibit significant synergistic effects, thereby achieving the effects of nourishing Yin and Yang, replenishing Qi and blood; furthermore, it demonstrates significant efficacy and high safety. The specific technical solution is as follows:

[0005] The first aspect of this application provides a tonic Chinese medicine composition, wherein the raw material components of the Chinese medicine composition, by weight, include: 57-106 parts of Rehmannia glutinosa (processed), 34-64 parts of Dioscorea opposita, 23-43 parts of Poria cocos, 29-53 parts of Cornus officinalis (processed with wine), 7-13 parts of Schisandra chinensis (processed with wine), 23-43 parts of Eucommia ulmoides (processed with salt), and 23-43 parts of Achyranthes bidentata (processed with wine), wherein the Dioscorea opposita is stir-fried or stir-fried with wheat bran.

[0006] The raw material components of the traditional Chinese medicine composition, by weight, consist of 57-106 parts of Rehmannia glutinosa (processed), 34-64 parts of Dioscorea opposita, 23-43 parts of Poria cocos, 29-53 parts of Cornus officinalis (processed with wine), 7-13 parts of Schisandra chinensis (processed with wine), 23-43 parts of Eucommia ulmoides (processed with salt), and 23-43 parts of Achyranthes bidentata (processed with wine). The Dioscorea opposita is either stir-fried or bran-fried.

[0007] In some embodiments of this application, the raw material components of the traditional Chinese medicine composition, by weight, include: 66-98 parts of Rehmannia glutinosa (processed), 39-59 parts of Dioscorea opposita (fried), 26-39 parts of Poria cocos, 33-49 parts of Cornus officinalis (processed with wine), 8-12 parts of Schisandra chinensis (processed with wine), 26-39 parts of Eucommia ulmoides (processed with salt), and 26-39 parts of Achyranthes bidentata (processed with wine).

[0008] In some embodiments of this application, the raw material components of the traditional Chinese medicine composition, by weight, consist of 66-98 parts of Rehmannia glutinosa (processed), 39-59 parts of Dioscorea opposita (fried), 26-39 parts of Poria cocos, 33-49 parts of Cornus officinalis (processed with wine), 8-12 parts of Schisandra chinensis (processed with wine), 26-39 parts of Eucommia ulmoides (processed with salt), and 26-39 parts of Achyranthes bidentata (processed with wine).

[0009] In some embodiments of this application, the traditional Chinese medicine composition further includes a pharmaceutically acceptable carrier or excipient; preferably, the pharmaceutically acceptable carrier or excipient is selected from at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, flow aids, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavorings, sweeteners, ion exchangers, release agents, coating agents, flavoring agents, and antioxidants.

[0010] In some embodiments of this application, the traditional Chinese medicine composition is formulated as a pharmaceutically acceptable preparation; preferably, the traditional Chinese medicine composition is formulated as any one of the dosage forms of granules, decoctions, pills, tablets or capsules.

[0011] The second aspect of this application provides a method for preparing a tonic Chinese medicine composition, comprising the steps of: adding water to all the raw material components described in the first aspect of this application for decoction extraction, then filtering and collecting the filtrate to obtain a tonic Chinese medicine composition decoction.

[0012] In some embodiments of this application, the weight of the water is 4-10 times the total weight of all raw material components; the decoction extraction is performed 2-3 times, and the decoction extraction time is 0.5-3 hours each time.

[0013] In some embodiments of this application, the method further includes the steps of: (1) concentrating the filtrate into a clear paste; then adding the clear paste to the excipients for granulation to obtain granules of a tonic Chinese medicine composition; or,

[0014] (1') Concentrate the filtrate into a clear paste, dry the clear paste, pulverize it, then add excipients and wetting agents, mix well, granulate, and then dry again to obtain granules of the tonic Chinese medicine composition; or,

[0015] (1'') The filtrate is concentrated into a clear paste, the clear paste is dried and pulverized, and then excipients are added for granulation to obtain granules of the tonic Chinese medicine composition.

[0016] In some embodiments of this application, in step (1), step (1'), or step (1''), the concentration is vacuum concentration, and the vacuum concentration temperature is 65-85°C; the relative density of the clear extract is 1.05-1.20; the mass ratio of the excipients to the clear extract is 1:(1-2.5); the excipients include dextrin and lactose in a mass ratio of (1.5-2.5):1.

[0017] In some embodiments of this application, in step (1), the granulation process parameters include: air inlet temperature of 80-100℃, atomization pressure of 0.3-0.4 bar, and liquid inlet speed of 7-20 rpm.

[0018] In some embodiments of this application, in step (1'), the drying method is selected from at least one of vacuum drying, belt drying and spray drying; the wetting agent is selected from water or an aqueous solution of ethanol with a volume fraction of 70%-95%; and the temperature of the re-drying is 40℃-60℃.

[0019] In some embodiments of this application, in step (1''), the drying method is selected from at least one of vacuum drying, belt drying and spray drying.

[0020] In some embodiments of this application, the method further includes the steps of: concentrating the filtrate into a clear extract; drying the clear extract and pulverizing it to obtain an extract powder; mixing the extract powder and excipients evenly, then adding water for granulation, then adding magnesium stearate and mixing evenly, compressing into tablets to obtain a tonic Chinese medicine composition tablet.

[0021] In some embodiments of this application, the concentration is vacuum concentration at a temperature of 65-85°C; the relative density of the extract is 1.05-1.20; the drying is vacuum drying at a temperature of 65-85°C; the mass ratio of the excipients to the extract powder is (1.5-2.5):1; the excipients include dextrin and lactose in a mass ratio of (1-3):1; and the mass of magnesium stearate is 0.4%-0.6% of the mass of the granules obtained from granulation.

[0022] Some embodiments of this application include the following steps:

[0023] (1) Weigh the raw material components according to the following weight parts: 66-98 parts of Rehmannia glutinosa, 39-59 parts of Dioscorea opposita, 26-39 parts of Poria cocos, 33-49 parts of Cornus officinalis, 8-12 parts of Schisandra chinensis, 26-39 parts of Eucommia ulmoides and 26-39 parts of Achyranthes bidentata.

[0024] (2) The above-weighed Rehmannia glutinosa, stir-fried Dioscorea opposita, Poria cocos, Cornus officinalis, Schisandra chinensis, Eucommia ulmoides, and Achyranthes bidentata were decocted with water 2-3 times. The weight of water added each time was 4-10 times the total weight of the Rehmannia glutinosa, stir-fried Dioscorea opposita, Poria cocos, Cornus officinalis, Schisandra chinensis, Eucommia ulmoides, and Achyranthes bidentata. The decoction time was 0.5-3 hours. The extracts from each decoction were filtered through a 150-220 mesh filter cloth. The filtrates were combined and concentrated under reduced pressure at 65-85℃ to a clear extract with a relative density of 1.05-1.20.

[0025] (3) After the excipients (dextrin: lactose = 1.5-2.5:1, mass ratio) are mixed evenly, they are placed in a one-step granulator and the above-mentioned clear paste is sprayed in for granulation (the mass ratio of excipients to clear paste is 1:1-2.5). The granulation process parameters include: air inlet temperature 80-100℃, atomization pressure 0.3-0.4 bar, liquid inlet speed 7-20 rpm, to obtain granules of the tonic Chinese medicine composition.

[0026] Some embodiments of this application include the following steps:

[0027] (1) Weigh the raw material components according to the following weight parts: 73.3 parts of Rehmannia glutinosa, 46.3 parts of Dioscorea opposita, 31 parts of Poria cocos, 38.6 parts of Cornus officinalis, 9.3 parts of Schisandra chinensis, 31 parts of Eucommia ulmoides and 31 parts of Achyranthes bidentata.

[0028] (2) The above-weighed Rehmannia glutinosa, stir-fried Dioscorea opposita, Poria cocos, Cornus officinalis, Schisandra chinensis, Eucommia ulmoides, and Achyranthes bidentata were decocted twice with water. The weight of water added in the first decoction was 10 times the total weight of the ingredients, and the decoction time was 2 hours. The weight of water added in the second decoction was 9 times the total weight of the ingredients, and the decoction time was 1 hour. Both extracts were filtered through a 200-mesh filter cloth. The filtrates were combined and concentrated under reduced pressure at 65°C to a clear extract with a relative density of 1.05.

[0029] (3) After the excipients (dextrin: lactose = 2:1, mass ratio) are mixed evenly, they are placed in a one-step granulator and the above-mentioned clear paste is sprayed in for granulation (the mass ratio of excipients to clear paste is 1:2). The granulation process parameters include: air inlet temperature 90℃, atomization pressure 0.35 bar, liquid inlet speed 15 rpm, to obtain granules of the tonic Chinese medicine composition.

[0030] Under the above-mentioned optimized process parameters and excipient ratios, pharmaceutical indicators such as drug release rate, process reliability, and formulation stability show a better trend.

[0031] The third aspect of this application provides the use of the traditional Chinese medicine composition of the first aspect of this application in the preparation of anti-fatigue, anti-aging or renal function protection drugs.

[0032] The fourth aspect of this application provides the use of the traditional Chinese medicine composition of the first aspect of this application in the preparation of a medicine for treating diseases caused by kidney yang deficiency, diseases caused by kidney yin and yang deficiency, miscarriage caused by kidney deficiency, or diseases caused by qi and blood deficiency.

[0033] The beneficial effects of this application are:

[0034] This application provides a tonic traditional Chinese medicine composition, its preparation method, and its application. The components of this composition exhibit significant synergistic effects, resulting in the effects of nourishing Yin and Yang, replenishing Qi and blood. The composition is highly effective and safe. Furthermore, zebrafish efficacy experiments have demonstrated that this traditional Chinese medicine composition has anti-fatigue, anti-aging, and kidney function protection effects, such as reducing the incidence of renal edema and improving glomerular filtration function. Efficacy experiments show that this traditional Chinese medicine composition can be used to treat diseases caused by kidney Yang deficiency, diseases caused by both kidney Yin and Yang deficiency, kidney deficiency-induced miscarriage, or diseases caused by both Qi and blood deficiency. The preparation method is simple, safe, highly operable, and has low production costs.

[0035] Of course, implementing any product or method of this application does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other embodiments can be obtained based on these drawings.

[0037] Figure 1 Typical staining intensities of zebrafish β-galactosidase after different sample treatments (yellow dashed boxes indicate the areas analyzed).

[0038] Figure 2 The average staining intensity of zebrafish β-galactosidase after different sample treatments is shown.

[0039] Figure 3 Typical zebrafish movement trajectories after different sample treatments (black line represents slow movement distance, green line represents medium movement distance, and red line represents fast movement distance).

[0040] Figure 4 The total distance traveled by zebrafish after different sample treatments is shown.

[0041] Figure 5 Typical fluorescence intensities of zebrafish after different sample treatments;

[0042] Figure 6 The fluorescence intensity of zebrafish after different sample treatments is shown.

[0043] Figure 7 Typical images of renal edema in zebrafish after different sample treatments (red arrows indicate renal edema).

[0044] Figure 8 The incidence of renal edema in zebrafish after different sample treatments is shown.

[0045] Figure 9 The results of pathological staining of uterine tissue from each group of rats in the efficacy test of Shibu Granules on rats with kidney yin and yang deficiency are shown.

[0046] Figure 10 The results of adrenal gland pathological staining in each group of mice were shown in the efficacy test of Shibu Granules against hydrocortisone-induced kidney yang deficiency mice.

[0047] Figure 11 The results of histopathological staining of kidney tissue in each group of mice were shown in the efficacy test of Shibu Granules in treating a mouse model of renal deficiency and miscarriage induced by hydrocortisone combined with mifepristone.

[0048] Figure 12 The results of pathological staining of uterine tissue in each group of mice in the efficacy test of Shibu Granules in treating a mouse model of renal deficiency and miscarriage induced by hydrocortisone combined with mifepristone are shown.

[0049] Figure 13 The results of the body weight changes of mice in each group in the efficacy test of Shibu Granules in treating a mouse model of qi and blood deficiency are shown. Detailed Implementation

[0050] The technical solutions of this application will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application.

[0051] In treating diseases caused by kidney deficiency, Traditional Chinese Medicine (TCM) often employs a combination of syndrome differentiation and treatment, focusing on nourishing Yin and tonifying the kidneys, replenishing Qi and essence, and harmonizing Qi and blood. This approach is not only highly safe with few adverse reactions but also improves the patient's constitution, resulting in good clinical efficacy. For kidney-tonifying medications, popular products on the market, such as Bailin tablets / capsules, Jinshuibao tablets / capsules, and Liuwei Dihuang pills, primarily target syndromes of lung and kidney deficiency, or kidney Yin deficiency and kidney Yang deficiency. The TCM composition discovered by the inventors in this application possesses the effects of nourishing Yin and strengthening Yang, replenishing Qi and blood, and can be used to treat kidney Yang deficiency and kidney Yin deficiency, leading to symptoms such as weakness in the legs and knees, lower back pain, tinnitus, emaciation, aversion to cold, cold limbs, shortness of breath, fatigue, abdominal pain, dark complexion, difficulty urinating, lethargy, and loss of appetite. This approach differs from existing formulations and has high clinical value.

[0052] The first aspect of this application provides a tonic Chinese medicine composition, wherein the raw material components of the Chinese medicine composition, by weight, include: 57-106 parts by weight of Rehmannia glutinosa (processed), 34-64 parts by weight of Dioscorea opposita (fried), 23-43 parts by weight of Poria cocos, 29-53 parts by weight of Cornus officinalis (processed with wine), 7-13 parts by weight of Schisandra chinensis (processed with wine), 23-43 parts by weight of Eucommia ulmoides (processed with salt), and 23-43 parts by weight of Achyranthes bidentata (processed with wine). For example, the weight parts of prepared Rehmannia glutinosa can be 57, 62, 66, 72, 77, 82, 86, 91, 98, 101, 106, or any two values ​​within this range; the weight parts of stir-fried Dioscorea opposita can be 34, 37, 39, 40, 43, 46, 49, 52, 55, 59, 61, 64, or any two values ​​within this range; the weight parts of Poria cocos can be 23, 25, 26, 29, 31, 33, 35, 37, 39, 41, 43, or any two values ​​within this range; and the weight parts of Cornus officinalis (processed with wine) can be 29, 31, 33. The weight ranges are 36, 39, 41, 43, 46, 49, 51, 53, or any two values ​​within this range. The weight portions of Schisandra chinensis (processed with alcohol) can be 7, 8, 9, 10, 11, 12, 13, or any two values ​​within this range. The weight portions of Eucommia ulmoides (processed with salt) can be 23, 25, 26, 29, 31, 33, 35, 37, 39, 41, 43, or any two values ​​within this range. The weight portions of Achyranthes bidentata (processed with alcohol) can be 23, 25, 26, 29, 31, 33, 35, 37, 39, 41, 43, or any two values ​​within this range. Further research by the inventors revealed that traditional Chinese medicine compositions with raw material components within this range exhibit better effects in nourishing Yin and Yang, replenishing Qi and blood, and can further enhance anti-aging, anti-fatigue, and kidney function protection effects.

[0053] In the tonic Chinese herbal composition provided in this application, Rehmannia glutinosa (processed), sweet and pure in Yin, primarily enters the kidney meridian, excelling at replenishing essence and marrow, and nourishing Yin essence, is therefore the principal herb. Eucommia ulmoides (processed with salt) is sweet and warm in nature, primarily enters the kidney meridian, tonifies kidney Yang, and strengthens tendons and bones; Cornus officinalis (processed with wine) is sour and warm in nature, primarily enters the liver meridian, tonifies the liver and kidneys, astringes essence and Qi, and replenishes liver blood to generate kidney essence; Dioscorea opposita (processed with stir-fry) is sweet and neutral in nature, primarily enters the spleen meridian, strengthens the spleen and replenishes deficiency, astringes essence and consolidates the kidneys, and replenishes acquired essence to strengthen innate essence, all serving as assistant herbs. Achyranthes bidentata (processed with wine) tonifies the liver and kidneys, strengthens tendons and bones, invigorates blood and promotes diuresis; Poria cocos (processed with wine) drains dampness and strengthens the spleen, and assists Dioscorea opposita in its function of nourishing acquired essence, all serving as adjuvant herbs. Schisandra chinensis (processed with wine) tonifies the kidneys and astringes essence, replenishes Qi and generates fluids, serving as an adjuvant and guiding herb.

[0054] The tonic Chinese herbal composition provided in this application, when combined with Cornus officinalis and Dioscorea opposita, simultaneously nourishes the kidneys, liver, and spleen; when combined with the mild diuretic properties of Poria cocos and the bitter purgative properties of Achyranthes bidentata, the tonic is nourishing without causing stagnation; when combined with Rehmannia glutinosa, Cornus officinalis, and Dioscorea opposita, it simultaneously nourishes yang and yin, seeking yang within yin. The tonic Chinese herbal composition provided in this application possesses the characteristics of simultaneously nourishing the yin and yang of the kidneys, seeking yang within yin, and nourishing without causing stagnation.

[0055] The inventors of this application discovered in their research that a traditional Chinese medicine composition prepared from seven ingredients—Rehmannia glutinosa (processed), Dioscorea opposita (fried), Poria cocos, Cornus officinalis (processed with wine), Schisandra chinensis (processed with wine), Eucommia ulmoides (processed with salt), and Achyranthes bidentata (processed with wine)—has the effects of nourishing Yin and strengthening Yang, replenishing Qi and blood. It can be used for symptoms such as weakness in the legs and knees, lower back pain and tinnitus, emaciation of limbs, aversion to cold and cold limbs, shortness of breath and fatigue, cold pain in the navel and abdomen, dark complexion, difficulty urinating, mental fatigue and loss of appetite caused by deficiency of kidney Yang or deficiency of kidney Yin.

[0056] Other raw materials used in this application, such as Rehmannia glutinosa (processed), Poria cocos, Cornus officinalis (processed with wine), Eucommia ulmoides (processed with salt), Dioscorea opposita (fried), Schisandra chinensis (processed with wine), and Achyranthes bidentata (processed with wine), can all be purchased. The processing methods for the medicinal slices can be carried out in accordance with relevant regulations.

[0057] Rehmannia glutinosa (processed): This refers to the processed product of the fresh or dried tuberous root (raw Rehmannia) of Rehmannia glutinosa Libosch., a plant of the Scrophulariaceae family. The processing technology is based on Part I of the 2020 edition of the Chinese Pharmacopoeia and should comply with the relevant regulations under the "Rehmannia glutinosa" section. It is produced in Shanxi Province.

[0058] Fried yam / bran-fried yam: This is a processed product of the dried rhizome (yam) of Dioscorea opposita Thunb., a plant of the Dioscoreaceae family. The processing technology is based on the 2022 edition of the "Tianjin Municipal Standard for Processing Traditional Chinese Medicine Pieces" and should comply with the relevant provisions under the "Fried Yam" section, or refer to the 2020 edition of the "Chinese Pharmacopoeia". Take hairy yam slices or smooth yam slices and fry them according to the bran-frying method (General Rule 0213) until they turn yellow. The place of origin is Anguo, Hebei Province.

[0059] Poria cocos: a processed product of the dried sclerotium (Poria cocos (Schw.) Wolf) of the Polyporaceae family; the processing technology is based on Part I of the 2020 edition of the Chinese Pharmacopoeia and should comply with the relevant provisions under the "Poria cocos" decoction pieces; the place of origin is Bozhou, Anhui.

[0060] Cornus officinalis (Shanzhuyu) is a processed product of the dried, mature fruit pulp (Shanzhuyu) of Cornus officinalis Sieb. et Zucc., a plant of the Cornaceae family. The processing technology is based on Part I of the 2020 edition of the Chinese Pharmacopoeia and should comply with the relevant regulations under the "Cornus officinalis" section. It is produced in Henan Province.

[0061] Wine-processed Schisandra chinensis: This is a processed product of the dried, mature fruit (Schisandra chinensis) of Schisandra chinensis (Turcz.) Baill., a plant of the Magnoliaceae family. The processing technology is based on the 2022 edition of the "Standards for Processing Traditional Chinese Medicine Pieces in Tianjin" and should comply with the relevant regulations under the "Wine-processed Schisandra chinensis" item. The place of origin is Chifeng, Inner Mongolia.

[0062] Salt-processed Eucommia: This refers to the processed product of the dried bark (Eucommia ulmoides Oliv.) of Eucommiaceae. The processing technology is based on Part I of the 2020 edition of the Chinese Pharmacopoeia and should comply with the relevant regulations under the "Salted Eucommia" section. The place of origin is Sichuan.

[0063] Wine-processed Achyranthes bidentata Bl. is a processed product of the dried root (Achyranthes bidentata) of the Amaranthaceae family. The processing technology is based on Part I of the 2020 edition of the Chinese Pharmacopoeia and should comply with the relevant regulations under the "Wine-processed Achyranthes bidentata" section. It is produced in Henan Province.

[0064] In some embodiments of this application, the raw material components of the traditional Chinese medicine composition, by weight, include: 66-98 parts of Rehmannia glutinosa (processed), 39-59 parts of Dioscorea opposita (fried), 26-39 parts of Poria cocos, 33-49 parts of Cornus officinalis (processed with wine), 8-12 parts of Schisandra chinensis (processed with wine), 26-39 parts of Eucommia ulmoides (processed with salt), and 26-39 parts of Achyranthes bidentata (processed with wine).

[0065] Those skilled in the art will understand that the weight proportions of the aforementioned Chinese medicinal materials are relative, and the amount of one or more of the medicinal materials can be reasonably adjusted based on traditional Chinese medicine theory and actual needs. All obvious variations of such reasonably adjusted formulations are within the scope of this application.

[0066] In some embodiments of this application, the traditional Chinese medicine composition further includes a pharmaceutically acceptable carrier or excipient; preferably, the pharmaceutically acceptable carrier or excipient is selected from at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, flow aids, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavorings, sweeteners, ion exchangers, release agents, coating agents, flavoring agents, and antioxidants.

[0067] In some embodiments of this application, the traditional Chinese medicine composition is formulated as a pharmaceutically acceptable preparation; preferably, the traditional Chinese medicine composition is formulated as any one of the dosage forms of granules, decoctions, pills, tablets or capsules.

[0068] In some embodiments of this application, the raw material components of the traditional Chinese medicine composition, by weight, consist of 57-106 parts of Rehmannia glutinosa (processed), 34-64 parts of Dioscorea opposita (fried), 23-43 parts of Poria cocos, 29-53 parts of Cornus officinalis (processed with wine), 7-13 parts of Schisandra chinensis (processed with wine), 23-43 parts of Eucommia ulmoides (processed with salt), and 23-43 parts of Achyranthes bidentata (processed with wine); preferably, it consists of 66-98 parts of Rehmannia glutinosa (processed), 39-59 parts of Dioscorea opposita (fried), 26-39 parts of Poria cocos, 33-49 parts of Cornus officinalis (processed with wine), 8-12 parts of Schisandra chinensis (processed with wine), 26-39 parts of Eucommia ulmoides (processed with salt), and 26-39 parts of Achyranthes bidentata (processed with wine).

[0069] The second aspect of this application provides a method for preparing a tonic Chinese medicine composition, comprising the steps of: adding water to all the raw material components described in the first aspect of this application for decoction extraction, then filtering and collecting the filtrate to obtain a tonic Chinese medicine composition decoction.

[0070] This application does not impose any particular limitation on the filtration method, as long as it achieves the purpose of this invention. For example, a 150-220 mesh filter cloth can be used for filtration. The granulation described in this application can be performed using a granulator.

[0071] In some embodiments of this application, the weight of the water is 4-10 times the total weight of all raw material components. The total weight of all raw material components mentioned in this application refers to the total weight of Rehmannia glutinosa (processed), Dioscorea opposita (fried), Poria cocos, Cornus officinalis (processed with wine), Schisandra chinensis (processed with wine), Eucommia ulmoides (processed with salt), and Achyranthes bidentata (processed with wine). In some embodiments of this application, the weight of the water can be 4, 5, 6, 7, 8, 9, or 10 times the total weight of all raw material components, or any combination of two values ​​in between. Controlling the amount of water within the above range in this application is beneficial for better extraction of the effective components from each of the prepared Chinese medicinal herbs.

[0072] In some embodiments of this application, the decoction extraction is performed 2-3 times, with each extraction lasting 0.5-3 hours. When the decoction extraction is performed 2-3 times, the filtrates need to be combined. In some embodiments of this application, the extraction time for each decoction is 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, or any combination of two values ​​in between. By controlling the number of decoction extractions and the extraction time within the above ranges, this application facilitates better extraction of the effective components from each Chinese herbal medicine.

[0073] In some embodiments of this application, the preparation method of the tonifying Chinese herbal medicine composition further includes the steps of: (1) concentrating the filtrate into a clear paste; then adding the clear paste to the excipients for granulation to obtain granules of the tonifying Chinese herbal medicine composition (boiling granulation); or,

[0074] (1') Concentrate the filtrate into a clear extract, dry the clear extract and pulverize it, then add excipients and wetting agents, mix well and granulate, then dry again to obtain granules of the tonic Chinese medicine composition (wet granulation); or,

[0075] (1'') The filtrate is concentrated into a clear paste, the clear paste is dried and pulverized, and then excipients are added for granulation to obtain granules of the tonic Chinese medicine composition (dry granulation).

[0076] In some embodiments of this application, in steps (1), (1'), or (1'') of the method for preparing granules of a tonic traditional Chinese medicine composition, the concentration is performed under reduced pressure, and the temperature of the reduced pressure concentration is 65-85°C. In some embodiments of this application, the temperature of the reduced pressure concentration can be 65°C, 70°C, 75°C, 80°C, 85°C, or a range of any two values ​​therein. Controlling the temperature of the reduced pressure concentration within the above range in this application is beneficial for better concentration of the extract while minimizing the loss of active ingredients.

[0077] In some embodiments of this application, in steps (1), (1'), or (1'') of the method for preparing granules of a tonic traditional Chinese medicine composition, the relative density of the extract is 1.05-1.20. The relative density of the extract described in this application is measured at room temperature, such as 25°C. In some embodiments of this application, the relative density of the extract can be 1.05, 1.10, 1.15, 1.20, or a range consisting of any two values ​​in between. Controlling the relative density of the extract within the above range in this application facilitates better subsequent granulation while minimizing the loss of active ingredients.

[0078] In some embodiments of this application, in steps (1), (1'), or (1'') of the method for preparing granules of a tonic traditional Chinese medicine composition, the mass ratio of the excipient to the extract is 1:(1-2.5). In some embodiments of this application, the mass ratio of the excipient to the extract can be 1:1, 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.2, 1:2.5, or any range of two ratios therein. Controlling the mass ratio of the excipient to the extract within the above range in this application is beneficial for better granulation.

[0079] In some embodiments of this application, in steps (1), (1'), or (1'') of the method for preparing granules of a tonic traditional Chinese medicine composition, the excipients include dextrin and lactose in a mass ratio of (1.5-2.5):1. In some embodiments of this application, the mass ratio of dextrin to lactose is 1.5:1, 1.6:1, 1.8:1, 2:1, 2.2:1, 2.5:1, or any two of these ratios. Controlling the mass ratio of dextrin to lactose within the above range in this application facilitates better granulation and results in granules with a better taste.

[0080] In some embodiments of this application, in step (1) of the method for preparing granules of a tonic traditional Chinese medicine composition, the granulation process parameters include: an inlet air temperature of 80-100℃, an atomization pressure of 0.3-0.4 bar, and a liquid inlet speed of 7-20 rpm. In some embodiments of this application, the inlet air temperature can be 80℃, 85℃, 90℃, 95℃, 100℃, or any two values ​​within this range; the atomization pressure can be 0.3 bar, 0.32 bar, 0.34 bar, 0.36 bar, 0.38 bar, 0.4 bar, or any two values ​​within this range; and the liquid inlet speed can be 7 rpm, 9 rpm, 11 rpm, 13 rpm, 15 rpm, 17 rpm, 20 rpm, or any two values ​​within this range. Controlling the inlet air temperature, atomization pressure, and liquid inlet speed within the above ranges in this application is beneficial for better granulation.

[0081] In some embodiments of this application, in step (1') of the method for preparing granules of a tonic Chinese medicine composition, the drying method is selected from at least one of vacuum drying, belt drying and spray drying.

[0082] In some embodiments of this application, in step (1') of the method for preparing granules of a tonic traditional Chinese medicine composition, the wetting agent is selected from water or an aqueous ethanol solution with a volume fraction of 70%-95%. In some embodiments of this application, the volume fraction of the aqueous ethanol solution can be 70%, 75%, 80%, 85%, 90%, 95%, or any two values ​​within this range. Controlling the volume fraction of the aqueous ethanol solution within the above range in this application is beneficial for better wetting. This application does not particularly limit the amount of wetting agent used; as long as the inventive purpose of this application is achieved, it can be selected according to actual needs or the type of wetting agent.

[0083] In some embodiments of this application, in step (1') of the method for preparing granules of a tonic traditional Chinese medicine composition, the re-drying temperature is 40℃-60℃. In some embodiments of this application, the re-drying temperature can be 40℃, 45℃, 50℃, 55℃, 60℃, or a range of any two values ​​therein. Controlling the re-drying temperature within the above range in this application is beneficial for quickly removing moisture from the granules while minimizing the loss of active ingredients.

[0084] In some embodiments of this application, in step (1'') of the method for preparing granules of a tonic Chinese medicine composition, the drying method is selected from at least one of vacuum drying, belt drying and spray drying.

[0085] In some embodiments of this application, the preparation method of the tonic Chinese medicine composition further includes the steps of: concentrating the filtrate into a clear paste; drying the clear paste and pulverizing it to obtain an extract powder; mixing the extract powder and excipients evenly, then adding water for granulation, then adding magnesium stearate and mixing evenly, compressing into tablets to obtain a tonic Chinese medicine composition tablet.

[0086] This application does not impose any particular restrictions on the amount of water used in the granulation process, as long as the purpose of this invention can be achieved.

[0087] In some embodiments of this application, in the preparation method of the tonic Chinese medicine composition tablets, the concentration is vacuum concentration, and the vacuum concentration temperature is 65-85℃. In some embodiments of this application, the vacuum concentration temperature can be 65℃, 70℃, 75℃, 80℃, 85℃, or any two values ​​in between. Controlling the vacuum concentration temperature within the above range in this application is beneficial for better concentration of the extract while minimizing the loss of active ingredients.

[0088] In some embodiments of this application, in the preparation method of the tonic Chinese medicine composition tablets, the relative density of the extract is 1.05-1.20. The relative density of the extract described in this application is measured at room temperature, such as 25°C. In some embodiments of this application, the relative density of the extract can be 1.05, 1.10, 1.15, 1.20, or a range consisting of any two values ​​in between. Controlling the relative density of the extract within the above range in this application facilitates better subsequent granulation while minimizing the loss of active ingredients.

[0089] In some embodiments of this application, the drying is vacuum drying, and the vacuum drying temperature is 65-85°C. In some embodiments of this application, the vacuum drying temperature can be 65°C, 70°C, 75°C, 80°C, 85°C, or a range of any two values ​​therein. Controlling the vacuum drying temperature within the above range in this application is beneficial for quickly removing moisture from the extract while minimizing the loss of active ingredients.

[0090] In some embodiments of this application, in the method for preparing tablets of a tonic traditional Chinese medicine composition, the mass ratio of the excipient to the extract powder is (1.5-2.5):1. In some embodiments of this application, the mass ratio of the excipient to the extract powder can be 1.5:1, 1.6:1, 1.8:1, 2:1, 2.2:1, 2.5:1, or any range of two ratios therein. Controlling the mass ratio of the excipient to the extract powder within the above range in this application is beneficial for better granulation.

[0091] In some embodiments of this application, in the preparation method of the tonic Chinese medicine composition tablets, the excipients include dextrin and lactose in a mass ratio of (1-3):1. In some embodiments of this application, the mass ratio of dextrin to lactose is 1:1, 1.2:1, 1.5:1, 1.6:1, 1.8:1, 2:1, 2.2:1, 2.5:1, 2.8:1, 3:1, or any two ratios within this range. Controlling the mass ratio of dextrin to lactose within the above range in this application is beneficial for better granulation and gives the granules a better taste.

[0092] In some embodiments of this application, in the method for preparing tablets of a tonic traditional Chinese medicine composition, the mass of magnesium stearate is 0.4%-0.6% of the mass of the granulated particles. In some embodiments of this application, the mass of magnesium stearate can be 0.4%, 0.45%, 0.5%, 0.55%, 0.6% of the mass of the granulated particles, or a range consisting of any two values ​​therein. Controlling the amount of magnesium stearate within the above range in this application is beneficial for better and more complete release of the active ingredients in the tablets.

[0093] The methods for preparing granules, decoctions, and tablets of the traditional Chinese medicine composition provided in this application are simple, safe, highly operable, and have low production costs. The pills or capsules described in this application can be prepared using the same methods as those for granules, decoctions, or tablets.

[0094] The third aspect of this application provides the use of the traditional Chinese medicine composition described in the first aspect of this application in the preparation of anti-fatigue, anti-aging, or renal function protection drugs.

[0095] The fourth aspect of this application provides the use of the traditional Chinese medicine composition described in the first aspect of this application in the preparation of remedies for diseases caused by kidney yang deficiency, diseases caused by deficiency of both kidney yin and yang, miscarriage due to kidney deficiency, or diseases caused by deficiency of both qi and blood. Unless otherwise specified or defined, the terms used in this application shall have their general meanings known to those skilled in the art.

[0096] Those skilled in the art will understand that, based on the description herein, appropriate Chinese medicinal materials, alone or in mixtures thereof, can be used to prepare the active extracts and preparations of the Chinese medicinal materials applicable in this application through conventional methods of pulverization, extraction, and separation in the art, such as soaking, percolation, liquid-liquid extraction, water extraction with alcohol precipitation, alcohol extraction with water precipitation, and dialysis. Alternatively, one or more active extracts of the Chinese medicinal materials used in this application can be purchased commercially and then combined with extracts of other Chinese medicinal materials to obtain the active extracts of the Chinese medicinal materials used in this application. These variations of Chinese medicinal material compositions and preparations are all within the scope of this application.

[0097] The pharmaceutical compositions and pharmaceutical formulations of this application can be formulated in unit dose form. The term "unit dose form" means a physically dispersed unit suitable for use as a unit dose in human subjects and other mammals, each unit containing a predetermined amount of the pharmaceutical composition or pharmaceutical formulation of this application and suitable pharmaceutically acceptable excipients calculated to produce the desired therapeutic effect.

[0098] As used in this application, the term "treatment" has its general meaning, and in this application specifically refers to the treatment of an animal individual that may suffer from kidney yang deficiency or kidney yin deficiency with the drug of this application in order to produce therapeutic, curative, alleviating, or reducing effects on the disease.

[0099] The actual dosage level and administration time of the traditional Chinese medicine composition or the active ingredient in the traditional Chinese medicine composition of this application can be adaptively changed so that the amount of the active ingredient can effectively achieve the desired therapeutic response for a specific test individual, composition and administration method, without toxicity to the test individual.

[0100] As used in this application, the terms "individual" or "animal individual" have their general meaning and, in this application, can refer to an animal individual suffering from or potentially suffering from kidney yang deficiency or kidney yin deficiency, or to an animal individual used for a specific purpose, such as scientific research. Specifically, the individuals referred to are, for example, animal individuals, particularly mammalian individuals, such as humans, pigs, dogs, cats, cattle, sheep, horses, rats, mice, rabbits, guinea pigs, monkeys, etc.

[0101] The embodiments and comparative examples provided below illustrate the implementation of this application in more detail. Various tests and evaluations were conducted according to the methods described below. Furthermore, unless otherwise specified, "parts" and "%" are quality standards.

[0102] Example 1

[0103] (1) Weigh the following raw materials respectively: 232g of Rehmannia glutinosa, 139g of Dioscorea opposita, 93g of Poria cocos, 116g of Cornus officinalis, 28g of Schisandra chinensis, 93g of Eucommia ulmoides and 93g of Achyranthes bidentata.

[0104] (2) The above-weighed Rehmannia glutinosa, stir-fried Dioscorea opposita, Poria cocos, Cornus officinalis, Schisandra chinensis, Eucommia ulmoides, and Achyranthes bidentata were decocted twice with water. The weight of water added in the first decoction was 10 times the total weight of the ingredients, and the decoction time was 2 hours. The weight of water added in the second decoction was 9 times the total weight of the ingredients, and the decoction time was 1 hour. Both extracts were filtered through a 200-mesh filter cloth. The filtrates were combined and concentrated under reduced pressure at 65°C to a clear extract with a relative density of 1.05.

[0105] (3) After the excipients (dextrin: lactose = 2:1, mass ratio) are mixed evenly, they are placed in a one-step granulator and the above-mentioned clear paste is sprayed in for granulation (the mass ratio of excipients to clear paste is 1:2). The granulation process parameters include: air inlet temperature 90℃, atomization pressure 0.35 bar, liquid inlet speed 15 rpm, to obtain granules of the tonic Chinese medicine composition.

[0106] Example 2

[0107] (1) Weigh the following raw materials respectively: 46g of Rehmannia glutinosa, 28g of stir-fried Dioscorea opposita, 19g of Poria cocos, 23g of Cornus officinalis, 6g of Schisandra chinensis, 19g of Eucommia ulmoides and 19g of Achyranthes bidentata.

[0108] (2) The above-weighed Rehmannia glutinosa, stir-fried Dioscorea opposita, Poria cocos, Cornus officinalis, Schisandra chinensis, Eucommia ulmoides, and Achyranthes bidentata were decocted twice with water. The weight of water added in the first decoction was 6 times the total weight of the Rehmannia glutinosa, stir-fried Dioscorea opposita, Poria cocos, Cornus officinalis, Schisandra chinensis, Eucommia ulmoides, and Achyranthes bidentata. The first decoction was carried out by boiling over high heat and then simmering over low heat for 40 minutes. The weight of water added in the second decoction was 5 times the total weight of the Rehmannia glutinosa, stir-fried Dioscorea opposita, Poria cocos, Cornus officinalis, Schisandra chinensis, Eucommia ulmoides, and Achyranthes bidentata. The second decoction was carried out by boiling over high heat and then simmering over low heat for 0.5 hours. Both extracts were filtered through a 200-mesh filter cloth, and the filtrates were combined to obtain a decoction of traditional Chinese medicine for tonification.

[0109] Example 3

[0110] (1) Weigh the following raw material components by weight: 278g of Rehmannia glutinosa, 167g of Dioscorea opposita, 112g of Poria cocos, 139g of Cornus officinalis, 34g of Schisandra chinensis, 112g of Eucommia ulmoides and 112g of Achyranthes bidentata.

[0111] (2) The above-weighed Rehmannia glutinosa, stir-fried Dioscorea opposita, Poria cocos, Cornus officinalis, Schisandra chinensis, Eucommia ulmoides, and Achyranthes bidentata were decocted and extracted three times with water. The weight of water added in the first decoction was 6 times the total weight of the ingredients, and the extraction time was 3 hours. The weight of water added in the second decoction was 4 times the total weight of the ingredients, and the extraction time was 1 hour. The weight of water added in the third decoction was 4 times the total weight of the ingredients, and the extraction time was 1 hour. The extracts were filtered through a 200-mesh filter cloth, and the filtrates were combined and concentrated under reduced pressure at 85°C to a clear extract with a relative density of 1.20. After that, the extracts were dried under reduced pressure at 65°C and pulverized to obtain the extract powder.

[0112] (3) After the excipients (dextrin: lactose = 2:1, mass ratio) and the above extract powder (mass ratio of excipients to extract powder is 2:1) are mixed evenly, they are placed in a one-step granulator and water is sprayed in for granulation; magnesium stearate (mass of magnesium stearate is 0.5% of the mass of the granules obtained by granulation) is added to the granules and mixed evenly, and then compressed into tablets to obtain a tonic Chinese medicine composition tablet.

[0113] Examples 4-7

[0114] Except for adjusting the amount of each raw material component as shown in Table 1, the rest is the same as in Example 1.

[0115] Example 8

[0116] Except for adjusting the amount of each raw material component as shown in Table 1, the rest is the same as in Example 2.

[0117] Comparative Examples 1-7

[0118] Except for adjusting the amount of each raw material component as shown in Table 1, the rest is the same as in Example 1.

[0119] Comparative Example 8

[0120] (1) Weigh the following raw materials respectively: 26g of stir-fried yam, 29g of wine-processed achyranthes root, 31g of prepared rehmannia root, 29g of polygala root, 31g of morinda root, 30g of wine-processed cornus fruit, 32g of alisma rhizome, 30g of dodder seed, 35g of wine-processed schisandra fruit, 41g of salt-processed eucommia bark, 32g of poria cocos, and 60g of cistanche deserticola.

[0121] (2) All the above-weighed medicinal ingredients were decocted and extracted twice. The weight of water added in the first decoction was 10 times the total weight of all the medicinal ingredients, and the extraction time was 2 hours. The weight of water added in the second decoction was 9 times the total weight of all the medicinal ingredients, and the extraction time was 1 hour. The extracts from both decoctions were filtered through a 200-mesh filter cloth. The filtrates were combined and concentrated under reduced pressure at 65°C to a clear extract with a relative density of 1.05.

[0122] (3) After the excipients (dextrin: lactose = 2:1, mass ratio) are mixed evenly, they are placed in a one-step granulator and the above-mentioned clear paste is sprayed in for granulation (the mass ratio of excipients to clear paste is 1:2). The granulation process parameters include: air inlet temperature 90℃, atomization pressure 0.35 bar, liquid inlet speed 15 rpm, to obtain the traditional Chinese medicine composition granules.

[0123] Comparative Example 9

[0124] (1) Weigh the following raw materials respectively: 40g of Rehmannia glutinosa, 20g of Dioscorea opposita, 60g of Schisandra chinensis (processed with wine), 30g of Cistanche deserticola, 20g of Achyranthes bidentata (processed with wine), 10g of Cornus officinalis (processed with wine), 10g of Alisma plantago-aquatica, 10g of Poria cocos, 10g of Polygala tenuifolia, 10g of Morinda officinalis, 10g of Halloysite, 10g of Gypsum fibrosum, 10g of Platycladus orientalis seed, and 30g of Eucommia ulmoides (processed with salt).

[0125] (2) All the above-weighed medicinal ingredients were decocted and extracted twice. The weight of water added in the first decoction was 10 times the total weight of all the medicinal ingredients, and the extraction time was 2 hours. The weight of water added in the second decoction was 9 times the total weight of all the medicinal ingredients, and the extraction time was 1 hour. The extracts from both decoctions were filtered through a 200-mesh filter cloth. The filtrates were combined and concentrated under reduced pressure at 65°C to a clear extract with a relative density of 1.05.

[0126] (3) After the excipients (dextrin: lactose = 2:1, mass ratio) are mixed evenly, they are placed in a one-step granulator and the above-mentioned clear paste is sprayed in for granulation (the mass ratio of excipients to clear paste is 1:2). The granulation process parameters include: air inlet temperature 90℃, atomization pressure 0.35 bar, liquid inlet speed 15 rpm, to obtain the traditional Chinese medicine composition granules.

[0127] Table 1. Raw material components of each embodiment and comparative example

[0128] The following efficacy tests illustrate the anti-aging, anti-fatigue, and kidney-protective effects of the traditional Chinese medicine compositions in the various embodiments and comparative examples of this application. Unless otherwise specified, the Shibu granules in the following tests are all granules of the tonic traditional Chinese medicine composition prepared in Example 1.

[0129] I. Zebrafish efficacy test

[0130] 1. Anti-aging

[0131] (1) Sample preparation

[0132] The granules of the tonic Chinese medicine composition prepared in Example 1 of this application, the tablets of the tonic Chinese medicine composition prepared in Example 3 of this application, and the granules of the tonic Chinese medicine composition prepared in Comparative Examples 1-7 of this application, all used standard dilution water as the solvent.

[0133] The tonic Chinese herbal decoction prepared in Example 2 of this application does not require dilution.

[0134] Positive control: Resveratrol, white powder, batch number F2121344, Shanghai Aladdin Biochemical Technology Co., Ltd., solvent is DMSO (dimethyl sulfoxide).

[0135] (2) Laboratory animals

[0136] Zebrafish were raised in aquarium water at 28℃ (water quality: 200mg of instant sea salt added per 1L of reverse osmosis water, conductivity 450-550μS / cm; pH 6.5-8.5; hardness 50-100mg / L CaCO3), provided by the applicant's aquarium breeding center. The laboratory animal use license number is: SYXK (Zhejiang) 2022-0004. The breeding and management met the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethics review number is: IACUC-2024-9499-01.

[0137] (3) Instruments, consumables and reagents

[0138] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Beilanbo Biotechnology Co., Ltd., China).

[0139] Hydrogen peroxide solution (batch number G2023089, Shanghai Aladdin Biochemical Technology Co., Ltd., China); dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, Switzerland); methylcellulose (batch number B2006074, Shanghai Aladdin Biochemical Technology Co., Ltd., China); 4% tissue cell fixative (batch number 20221014, Beijing Solarbio Science & Technology Co., Ltd., China); cell senescence β-galactosidase staining kit (batch number 091821220526, Shanghai Beyotime Biotechnology Co., Ltd., China).

[0140] (4) Detection methods and test results

[0141] a) Maximum Detectable Concentration (MTC)

[0142] Wild-type AB strain zebrafish, 6 hours post-fertilization (6 hpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Samples were administered in water (different sample concentrations are shown in Table 2). A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other groups were treated with water-soluble hydrogen peroxide to establish a zebrafish aging model, with the medium changed daily. After 6 days of treatment at 28℃, the MTC of the samples in the model zebrafish was measured.

[0143] The experimental results are shown in Table 2.

[0144] Table 2 Results of the concentration exploration experiment on the anti-aging efficacy of samples at different concentrations (n=30)

[0145] Note: "-" indicates that it does not exist or is not shown.

[0146] Under the above experimental conditions, the MTC of the tonifying Chinese herbal medicine granules prepared in Example 1 of this application for anti-aging effects in zebrafish was 1000 μg / mL.

[0147] b) Evaluation of anti-aging efficacy

[0148] Wild-type AB strain zebrafish (6 hpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Samples were administered in water (concentrations of different samples are shown in Table 3). A positive control of resveratrol at a concentration of 20.0 μg / mL was used. Normal and model control groups were also included, with a volume of 3 mL per well. Except for the normal control group, all other groups were treated with hydrogen peroxide in water to establish a zebrafish aging model, with the medium changed daily. After 6 days of treatment at 28℃, zebrafish from each experimental group were stained using a cellular senescence β-galactosidase staining kit. After staining, 10 zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Data were collected using NIS-Elements D 3.20 advanced image processing software, and the average β-galactosidase staining intensity was analyzed. The statistical analysis results of this index were used to evaluate the anti-aging efficacy of the samples. Statistical results are expressed as mean ± SE. Statistical analysis showed that p < 0.05 was statistically significant.

[0149] The experimental results are shown in Table 3. Figure 1 and Figure 2 As shown. Among them, Figure 1 Typical staining intensities of zebrafish β-galactosidase after different sample treatments (yellow dashed boxes indicate the areas analyzed). Figure 2 The average staining intensity of zebrafish β-galactosidase after different sample treatments is shown.

[0150] Table 3. Experimental results evaluating the anti-aging efficacy of different samples (n=10)

[0151] Note: Compared with the model control group, ;

[0152] "-" indicates that it does not exist.

[0153] The results above show that the tonic Chinese medicine compositions prepared in Examples 1-3 of this application all have good anti-aging effects. Comparing the results of Example 1 and Comparative Examples 1-7, it is evident that when some raw material components of the tonic Chinese medicine compositions are not within the scope of this application, the staining intensity of zebrafish β-galactosidase is significantly increased, indicating a significant decrease in anti-aging efficacy. These results demonstrate that the components in the tonic Chinese medicine compositions provided by this application can produce a significant synergistic effect, thereby achieving the purpose of anti-aging.

[0154] 2. Anti-fatigue

[0155] (1) Sample preparation

[0156] The granules of the tonic Chinese medicine composition prepared in Example 1 of this application, the tablets of the tonic Chinese medicine composition prepared in Example 3 of this application, and the granules of the tonic Chinese medicine composition prepared in Comparative Examples 1-7 of this application, all used standard dilution water as the solvent.

[0157] The tonic Chinese herbal decoction prepared in Example 2 of this application does not require dilution.

[0158] Positive control: Rhodioloside, white powder, batch number C23HY06052605B, Huazhong Haiwei (Beijing) Gene Technology Co., Ltd., solvent is standard dilution water.

[0159] (2) Laboratory animals

[0160] Zebrafish were raised in aquarium water at 28℃ (water quality: 200mg of instant sea salt added per 1L of reverse osmosis water, conductivity 450-550μS / cm; pH 6.5-8.5; hardness 50-100 mg / L CaCO3), provided by the applicant's fish farming center. The laboratory animal use license number is: SYXK (Zhejiang) 2022-0004. The husbandry and management met the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethics review number is: IACUC-2024-9499-01.

[0161] (3) Instruments, consumables and reagents

[0162] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Belamber Biotechnology Co., Ltd., China); 96-well plate (Nest Biotech, China); Behavioral analyzer (Zebra Lab 3.22.3.31, Viewpoint, France).

[0163] Anhydrous sodium sulfite (batch number I1428128, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0164] (4) Detection methods and test results

[0165] a) Maximum Detectable Concentration (MTC)

[0166] Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Water-soluble samples were administered (different sample concentrations are shown in Table 4). A normal control group and a model control group were also included, with a volume of 3 mL per well. After treatment at 28℃ for 1 day, except for the normal control group, all other experimental groups were given anhydrous sodium sulfite to establish a zebrafish fatigue model. Treatment continued at 28℃ for another 1 hour, and the MTC of the samples on the model zebrafish was measured.

[0167] The experimental results are shown in Table 4.

[0168] Table 4 Results of the concentration exploration experiment on the anti-fatigue efficacy of samples at different concentrations (n=30)

[0169] Note: "-" indicates that it does not exist; 2000 μg / mL is the maximum concentration for administration. If the concentration is higher than this, the sample will not dissolve completely and cannot be administered. Therefore, administration tests at concentrations higher than this have not been conducted.

[0170] Under the above experimental conditions, the MTC of the tonifying Chinese herbal medicine granules prepared in Example 1 of this application for anti-fatigue effect in zebrafish was 2000 μg / mL.

[0171] b) Evaluation of anti-fatigue efficacy

[0172] Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered in water (concentrations are shown in Table 5), with a positive control of rhodioloside at 4000 μg / mL. A normal control and a model control group were also included, with a volume of 3 mL per well. After treatment at 28℃ for 1 day, except for the normal control group, all other experimental groups were given anhydrous sodium sulfite in water to establish a zebrafish fatigue model. After further treatment at 28℃ for 1 hour, 10 zebrafish from each experimental group were randomly selected and placed in 96-well plates. The total movement distance of the zebrafish was measured using a behavior analyzer, and the anti-fatigue efficacy of the samples was evaluated based on statistical analysis of this index. Statistical results are expressed as mean ± SE. Statistical analysis showed that p < 0.05 was statistically significant.

[0173] The experimental results are shown in Table 5. Figure 3 and Figure 4 As shown. Among them, Figure 3 Typical zebrafish movement trajectories after different sample treatments (black line represents slow movement distance, green line represents medium movement distance, and red line represents fast movement distance). Figure 4 The total distance traveled by zebrafish after different sample treatments is shown.

[0174] Table 5. Experimental results of anti-fatigue efficacy evaluation of different samples (n=10)

[0175] Note: Compared with the model control group, ;

[0176] "-" indicates that it does not exist.

[0177] The results above show that the tonic Chinese medicine compositions prepared in Examples 1-3 of this application all have good anti-fatigue effects. Comparing the results of Example 1 and Comparative Examples 1-7, it is evident that when some raw material components of the tonic Chinese medicine composition are not within the scope of this application, the total movement distance of zebrafish decreases significantly, indicating a significant decrease in anti-fatigue efficacy. These results demonstrate that the components in the tonic Chinese medicine composition provided by this application can produce a significant synergistic effect, thereby achieving the purpose of anti-fatigue.

[0178] 3. Kidney function protection effect

[0179] (1) Sample preparation

[0180] The granules of the tonic Chinese medicine composition prepared in Example 1 of this application, the tablets of the tonic Chinese medicine composition prepared in Example 3 of this application, and the granules of the tonic Chinese medicine composition prepared in Comparative Examples 1-7 of this application all use 1×E3 (polyethylene glycol monodecyl ether) as the solvent.

[0181] The tonic Chinese herbal decoction prepared in Example 2 of this application does not require dilution.

[0182] Positive control: Dexamethasone, white powder, batch number C2110208, Shanghai Aladdin Biochemical Technology Co., Ltd., solvent is DMSO.

[0183] (2) Laboratory animals

[0184] Zebrafish were raised in aquarium water at 28℃ (water quality: 200mg of instant sea salt added per 1L of reverse osmosis water, conductivity 450-550μS / cm; pH 6.5-8.5; hardness 50-100 mg / L CaCO3), provided by the applicant's fish farming center. The laboratory animal use license number is: SYXK (Zhejiang) 2022-0004. The husbandry and management met the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethics review number is: IACUC-2024-9499-01.

[0185] (3) Instruments, consumables and reagents

[0186] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); motorized focusing continuous zoom fluorescence microscope (AZ100, Nikon, Japan); microinjection apparatus (IM300, Narishige, Japan); needle puller (PC-10, Narishige, Japan); precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Beilanbo Biotechnology Co., Ltd., China).

[0187] Methylcellulose (lot number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Dimethyl sulfoxide (DMSO, lot number BCCD8942, Sigma, Switzerland); Aristolochic acid (lot number E2015060, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Dextran tetramethylrhodamine (lot number 923446, molecular probes, USA).

[0188] (4) Detection methods and test results

[0189] a) Maximum Detectable Concentration (MTC)

[0190] Wild-type AB strain zebrafish with a 2-day pf growth rate were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Water-soluble samples were administered (different sample concentrations are shown in Table 6). A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were given aristolochic acid in water to establish a zebrafish renal dysfunction edema model. After treatment at 28℃ for 2 days, the mean toxicity (MTC) of the samples in the model zebrafish was measured.

[0191] The experimental results are shown in Table 6.

[0192] Table 6 Results of the concentration exploratory experiment on the renal function protective efficacy of different concentrations of samples (n=30)

[0193] Note: "-" indicates that it does not exist or is not shown.

[0194] Under the above experimental conditions, the MTC of the tonifying Chinese herbal medicine granules prepared in Example 1 of this application for protecting kidney function in zebrafish was 500 μg / mL.

[0195] b) Kidney function protection (glomerular filtration)

[0196] Wild-type AB strain zebrafish (2 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Water-soluble samples (different sample concentrations are shown in Table 7) were administered, with a positive control of dexamethasone at a concentration of 9.81 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all experimental groups were given aristolochic acid in water to establish a zebrafish model of renal dysfunction and edema. After treatment at 28℃ for 1 day, each experimental group was intravenously injected with a fluorescent marker (Dextran tetramethylrhodamine). After another day of treatment at 28℃, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. The whole-body fluorescence intensity of the zebrafish was analyzed, and the statistical analysis results of this index were used to evaluate the renal protective efficacy (glomerular filtration). Statistical results are expressed as mean ± SE. Statistical analysis showed that p < 0.05 was statistically significant.

[0197] The experimental results are shown in Table 7. Figure 5 and Figure 6 As shown. Among them, Figure 5 Typical fluorescence intensities of zebrafish after different sample treatments; Figure 6 The fluorescence intensity of zebrafish after different sample treatments is shown.

[0198] Table 7. Evaluation results of renal function protection efficacy (glomerular filtration) of different samples (n=10)

[0199] Note: Compared with the model control group, ;

[0200] "-" indicates that it does not exist.

[0201] The results above show that the tonic Chinese medicine compositions prepared in Examples 1-3 of this application all have good efficacy in improving glomerular filtration function. Comparison of the results of Example 1 and Comparative Examples 1-7 reveals that when some raw material components of the tonic Chinese medicine composition are not within the scope of this application, the fluorescence intensity of the zebrafish is significantly increased, indicating a decrease in glomerular filtration and renal function. These results indicate that the components in the tonic Chinese medicine composition provided by this application can produce a significant synergistic effect, thereby achieving the purpose of improving glomerular filtration function and having a protective effect on renal function.

[0202] c) Protective effect on renal function (incidence of renal edema)

[0203] Wild-type AB strain zebrafish (2 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Water-soluble samples (different sample concentrations are shown in Table 8) were administered, with a positive control of dexamethasone at a concentration of 9.81 µg / mL. A normal control group and a model control group were also set up, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were given aristolochic acid in water to establish a zebrafish renal function injury model. After treatment at 28℃ for 2 days, each experimental group was observed under a dissecting microscope, and the number of zebrafish with renal edema was counted. The incidence of renal edema (%) in each experimental group was calculated, and the nephroprotective efficacy (incidence of renal edema) was evaluated using statistical analysis of this indicator. Statistical analysis showed that p < 0.05 indicated a statistically significant difference.

[0204] The experimental results are shown in Table 8. Figure 7 and Figure 8 As shown. Among them, Figure 7 Typical images of renal edema in zebrafish after different sample treatments (red arrows indicate renal edema). Figure 8 The incidence of renal edema in zebrafish after different sample treatments is shown.

[0205] Table 8. Results of the experiment evaluating the renal protective efficacy of different samples (incidence of renal edema) (n=30)

[0206] Note: Compared with the model control group, ;

[0207] "-" indicates that it does not exist.

[0208] The results above show that the tonic Chinese medicine compositions prepared in Examples 1-3 of this application all have good renal protective effects. Comparison of the results of Example 1 and Comparative Examples 1-7 reveals that when some raw material components of the tonic Chinese medicine composition are not within the scope of this application, the incidence of renal edema in zebrafish is significantly increased, indicating a decline in renal function. These results indicate that the components in the tonic Chinese medicine composition provided in this application can produce a significant synergistic effect, thereby achieving the goal of reducing the incidence of renal edema.

[0209] The tonic Chinese medicine composition provided in this application has the effects of nourishing yin and strengthening yang, replenishing qi and blood. Furthermore, the efficacy experiment of zebrafish has proven that the Chinese medicine composition of this application has the effects of anti-fatigue, anti-aging and kidney function protection, such as reducing the incidence of renal edema and improving glomerular filtration function.

[0210] To further clarify the efficacy of the traditional Chinese medicine composition provided in this application in nourishing Yin and strengthening Yang, and in tonifying both Yin and Yang, a rat model of kidney Yin and Yang deficiency was constructed. The rats' body weight, spontaneous activity ability, and limb grip strength were measured to reflect the ameliorative effect of the traditional Chinese medicine composition on the syndrome of kidney Yin and Yang deficiency. Furthermore, based on the characteristics of the model, the effects of the traditional Chinese medicine composition on castration in rats were reflected through indicators such as sex hormone levels, uterine histopathology, and estrogen receptors. Combining traditional symptom manifestations with modern medical indicators, the efficacy of the traditional Chinese medicine composition in nourishing Yin and strengthening Yang is jointly elucidated.

[0211] Existing research indicates that hydrocortisone can cause symptoms of kidney yang deficiency. This application established mouse models of kidney yang deficiency and mouse models of kidney deficiency-induced abortion, using modern medical laboratory indicators to reflect the distribution of TCM syndromes. Urine tests can reflect the mice's "difficulty urinating"; spontaneous activity reflects the mice's "mental fatigue"; body temperature measurements reflect the mice's "cold limbs and aversion to cold". The combined effects of these indicators can reflect the ameliorative effect of the TCM composition of this application on various symptoms of kidney deficiency.

[0212] In addition, this application also uses intraperitoneal injection of acetylphenylhydrazine + cyclophosphamide to establish a mouse model of qi and blood deficiency, and detects hemoglobin to reflect the symptoms of blood deficiency in mice; grip strength test and weighted swimming test to reflect the symptoms of qi deficiency in mice. By combining the characterization indicators of qi and blood deficiency, the efficacy of the traditional Chinese medicine composition of this application in replenishing qi and blood is explained.

[0213] Through the construction of the above four models and the detection of relevant indicators, this application clarifies the pharmacodynamic effects of the traditional Chinese medicine composition in treating symptoms related to deficiency of both kidney yin and yang and deficiency of both qi and blood. It demonstrates the efficacy and characteristics of the traditional Chinese medicine composition in nourishing yin and strengthening yang, replenishing qi and blood, and provides a basis for the formulation development of the traditional Chinese medicine composition in this application.

[0214] For ease of description, in the following experiments, the high-dose group of Shibu Granules is also referred to as the "high-dose group of Shibu Granules", the medium-dose group of Shibu Granules is also referred to as the "medium-dose group of Shibu Granules", and the low-dose group of Shibu Granules is also referred to as the "low-dose group of Shibu Granules".

[0215] II. Efficacy test of Shibu Granules on rats with kidney yin and yang deficiency

[0216] To explore the core efficacy of Shibu Granules in nourishing Yin and strengthening Yang, and replenishing both Yin and Yang, this experiment used female rats to remove both ovaries and inject p-chlorophenylalanine (PCPA) into an ovariectomy model to simulate the complex pathological state of Yin and Yang deficiency in clinical patients.

[0217] In terms of experimental index detection, by regularly monitoring the changes in the body weight of rats and combining traditional disease symptoms such as spontaneous activity ability and forepaw grip strength, the improvement effect of Shibu Granules on the symptoms of kidney yin and yang deficiency was intuitively reflected; focusing on the pathological changes caused by castration, the serum sex hormone levels were measured, and pathological sections of uterine tissues were observed and the estrogen receptor expression levels were detected to explain the regulatory effect of the traditional Chinese medicine composition granule for tonifying deficiency (i.e., Shibu Granules) prepared in the embodiments of this application on the reproductive endocrine system of castrated rats.

[0218] Experiment 1:

[0219] 1. Experimental materials

[0220] (1)Animals

[0221] SPF-grade female SD rats, weighing (200±10) g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the experimental animal production license number is SCXK (Beijing) 2021-0006. They were housed in the SPF-grade animal laboratory of the Experimental Animal Center of Tianjin University of Traditional Chinese Medicine (the animal breeding license number is SYXK (Tianjin) 2020-0005), and they had free access to water and food. The animal experiment was approved by the Experimental Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (approval number: TCM-LAEC2025020Y0929).

[0222] (2)Reagents and drugs

[0223] p-Chlorophenylalanine (Shanghai Macklin Biochemical Co., Ltd., product number: D831376); chloral hydrate solution (Tianjin Solomon Biotechnology Co., Ltd., product number: N0097-500); estradiol (E2, product number: CSB-E05110r), follicle-stimulating hormone (FSH, product number: CSB-E06869r), luteinizing hormone (LH, product number: CSB-E12654r), ELISA detection kits, all purchased from Wuhan Huamei Bioengineering Co., Ltd.; estrogen receptor α (ERα, product number: bs-0725R), estrogen receptor β (ERβ, product number: bs-0116R), purchased from Beijing Biosynthesis Biotechnology Co., Ltd.

[0224] (3)Instruments

[0225] Open field apparatus (Beijing Zhishu Duobao Biotechnology Co., Ltd.); grip strength meter (Jinan Yiyan Technology Development Co., Ltd.); multifunctional microplate reader (TECAN, Switzerland); gel imaging system (GE Company, USA).

[0226] 2. Experimental methods

[0227] (1)Animal modeling, grouping and drug administration

[0228] Fifty female SD rats were randomly divided into a sham-operated group (n=9) and a castration-operated group (n=41) after one week of acclimatization. The castration-operated group underwent bilateral ovariectomy to establish a castration model. Three rats died during model establishment; all were due to failure to awaken after anesthesia and were unrelated to the surgical procedure. Starting from the 5th day post-surgery, vaginal smears were performed once daily, with the absence of estrus response indicating successful model establishment. Rats with successful model establishment were intraperitoneally injected with p-chlorophenylalanine (PCPA, 450 mg / kg) suspension for two consecutive days to establish an insomnia model. The sham-operated group did not undergo bilateral ovariectomy or PCPA injection. The castration-operated rats with insomnia were randomly divided into a model group (n=10), a high-dose Shibu granules group (14.4 g crude drug / kg, n=9), a medium-dose Shibu granules group (7.2 g crude drug / kg, n=10), and a low-dose Shibu granules group (3.6 g crude drug / kg, n=9). Shibu granules were dissolved in pure water and prepared fresh daily. Mice in each treatment group were administered Shibu granules by gavage at the corresponding dose daily, while the sham-operated group and model group were administered the same amount of pure water by gavage daily for 4 consecutive weeks. Shibu granules are the granules of the tonic traditional Chinese medicine composition prepared in Example 1.

[0229] (2) Sampling and testing indicators

[0230] a) General observation

[0231] Regularly observe and record the mental state, stool condition, and daytime and nighttime activity levels of the rats in each group.

[0232] b) Open field experiment

[0233] After the last administration, rats from each group were placed sequentially in the center of the experimental open space box. Analysis software was used to statistically analyze indicators such as the rats' resting time and total distance traveled within 3 minutes to assess the behavioral performance of spontaneous movement in each group. After each test, the open space box was sprayed with 75 vol% ethanol aqueous solution for disinfection and wiped clean to prevent the odor from affecting the next rat.

[0234] c) Grip test

[0235] Using a grip strength tester, the grip strength of the forepaws of rats in each group was tested after the last administration of the drug. The instrument value was adjusted to zero, the rat was placed on the gripping board, and its tail was gently pulled backward. After the animal had a firm grip, the force was evenly applied and pulled backward until the animal released its grip. The grip strength value was recorded. The above operation was repeated, and the average value was taken for 3 measurements.

[0236] d) Serum hormone level detection

[0237] After the last administration, blood was collected from the abdominal aorta of rats, and serum was separated by centrifugation. Serum estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels were detected using an ELISA kit.

[0238] e) Pathological observation

[0239] Complete uterine tissues were collected from rats in each group. The tissues were fixed with formalin, dehydrated, embedded in paraffin, sectioned, dewaxed and placed in water, stained with hematoxylin for 10-20 min, differentiated with hydrochloric acid and ethanol, and after returning to blue, stained with eosin for 3-5 min. After dehydration, clearing and mounting, the tissues were observed and photographed under a pathological microscope.

[0240] (3) Statistical methods

[0241] Data analysis was performed, with continuous data expressed as mean ± standard deviation (X̄±s). One-way ANOVA was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0242] 3. Experimental Results

[0243] (1) General observation

[0244] The rats in the sham-operated group had normal drinking and eating habits, good mental state, normal stools, were active at night, and slept more during the day; the rats in the insomnia group had poor mental state, were active both day and night, were easily agitated, and had loose stools; the rats' condition improved after intervention with different doses of Shibu Granules.

[0245] (2) Independent activities

[0246] Compared with the sham-operated group, the total distance traveled by rats in the model group in the open field was significantly shortened (P<0.01). Compared with the model group, the medium dose of Shibu Granules significantly reduced the resting time of rats in the open field (P<0.05) and prolonged the total distance traveled (P<0.05), as shown in Table 9.

[0247] Table 9. Effects of Shibu Granules on Spontaneous Activity in Rats with Kidney Yin-Yang Deficiency

[0248] Note: Compared with the sham surgery group. Compared with the model group, .

[0249] (3) Grip force measurement

[0250] In the gripping force test, compared with the sham-operated group, the gripping force and gripping force / body weight of rats in the model group were significantly reduced (P<0.001). Compared with the model group, the gripping force and gripping force / body weight of rats in the high- and medium-dose groups of Shibu Granules were significantly increased (P<0.001); the gripping force of the low-dose group was significantly increased (P<0.01), and the gripping force / body weight showed an increasing trend. See Table 10.

[0251] Table 10. Effects of Shibu Granules on grip strength in rats with kidney yin and yang deficiency.

[0252] Note: Compared with the sham surgery group. Compared with the model group, .

[0253] (4) Serum hormone levels

[0254] Compared with the sham-operated group, the serum estradiol level in the model group rats was significantly decreased (P<0.001); compared with the model group, the serum estradiol level in the medium-dose group of Shibu Granules was significantly increased (P<0.001), and the high- and low-dose groups showed an increasing trend. Compared with the sham-operated group, the serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels in the model group rats were significantly increased (P<0.01, P<0.001), while the high-, medium-, and low-dose groups of Shibu Granules all significantly decreased serum FSH levels (P<0.05, P<0.01, P<0.001) and LH levels (P<0.01, P<0.001). See Table 11.

[0255] Table 11 Effects of Shibu Granules on Serum Hormone Levels in Rats with Kidney Yin-Yang Deficiency

[0256] Note: Compared with the sham surgery group. Compared with the model group, .

[0257] (5) Expression of ERα and ERβ proteins

[0258] Compared with the sham-operated group, there was no significant difference in uterine ERα expression in the model group rats. Compared with the model group, the high, medium, and low dose groups of Shibu Granules showed a trend of increasing uterine ERα expression in rats. Compared with the sham-operated group, uterine ERβ expression in the model group rats was significantly decreased (P<0.05). Compared with the model group, the high dose group of Shibu Granules significantly increased uterine ERβ expression (P<0.01), while the medium and low dose groups showed a trend of increasing uterine ERβ expression. See Table 12.

[0259] Table 12 Effects of Shibu Granules on the Expression of ERα and ERβ Proteins in Uterine Tissue of Rats with Kidney Yin-Yang Deficiency

[0260] Note: Compared with the sham surgery group. Compared with the model group, .

[0261] (6) Histopathological staining

[0262] Results of HE staining of the uterus as follows Figure 9As shown, the uterus of rats in the sham-operated group had a normal uterine morphology, with the endometrial epithelial cells being a single layer of columnar epithelial cells arranged neatly. The lamina propria contained a large number of uterine glands, and the endometrium and myometrium were thicker. In the model group, the uterine volume of rats was significantly reduced, the uterine cavity narrowed, the endometrial epithelial cells were flattened and irregularly arranged, the number of uterine glands in the lamina propria was fewer, and the endometrium and myometrium showed significant atrophy. The high, medium, and low dose groups of Shibu Granules all showed a certain degree of increase in the number of uterine glands, and the degree of atrophy of the endometrium and myometrium improved to some extent.

[0263] Experiment 2:

[0264] 1. Test materials

[0265] Same as Experiment 1.

[0266] 2. Test Methods

[0267] (1) Animal modeling, grouping and administration

[0268] Seventy-two female SD rats were randomly divided into a sham-operated group (n=8) and a castration-operated group (n=64) after one week of acclimatization. Rats in the castration-operated group underwent bilateral ovariectomy. Vaginal smears were taken daily starting on the 5th postoperative day, and successful modeling was defined as the absence of estrus response. Rats with successful modeling were intraperitoneally injected with p-chlorophenylalanine (PCPA, 450 mg / kg) suspension for two consecutive days to establish an insomnia model. The sham-operated group did not undergo bilateral ovariectomy or PCPA injection. The castrated rats with insomnia were randomly divided into a model group, Examples 1, 4-7, Comparative Examples 8 and 9 (clinically equivalent dose 3.6 g crude drug / kg, n=8). Shibu granules were dissolved in pure water and prepared fresh daily. Mice in each treatment group were administered Shibu granules by gavage daily at the corresponding dose, while the sham-operated group and model group were administered the same amount of pure water by gavage daily for four consecutive weeks.

[0269] (2) Sampling and testing indicators

[0270] a) Open field experiment

[0271] After the last administration, rats from each group were placed sequentially in the center of the experimental open space box. Analysis software was used to statistically analyze indicators such as the rats' resting time and total distance traveled within 3 minutes to assess the behavioral performance of spontaneous movement in each group. After each test, the open space box was sprayed with 75 vol% ethanol aqueous solution for disinfection and wiped clean to prevent the odor from affecting the next rat.

[0272] b) Grip test

[0273] Using a grip strength tester, the grip strength of the forepaws of rats in each group was tested after the last administration of the drug. The instrument value was adjusted to zero, the rat was placed on the gripping board, and its tail was gently pulled backward. After the animal had a firm grip, the force was evenly applied and pulled backward until the animal released its grip. The grip strength value was recorded. The above operation was repeated, and the average value was taken for 3 measurements.

[0274] 3. Test Results

[0275] (1) Independent activities

[0276] Compared with the sham-operated group, the total distance traveled by rats in the model group in the open field was significantly shortened (P<0.05). Compared with the model group, all prescription groups in each example significantly reduced the resting time of rats in the open field (P<0.05), with the reduction in Example 1 being greater than that in Examples 4-5, and the reduction in Examples 4-5 being greater than that in Examples 6-7. Compared with the model group, prescription groups 1 and 4-5 significantly prolonged the total distance traveled (P<0.05), and Examples 6-7 also showed a trend of prolongation, with the prolongation in Example 1 being greater than that in Examples 4-5, and the prolongation in Examples 4-5 being greater than that in Examples 6-7. Compared with the model group, the resting time of rats in Comparative Examples 8-9 was not significantly reduced, and the total distance traveled was not significantly increased. Specific results are shown in Table 13.

[0277] Table 13 Effects of Shibu Granules on Spontaneous Activity in Rats with Kidney Yin-Yang Deficiency

[0278] Note: Compared with the sham surgery group. Compared with the model group, .

[0279] (2) Grip force measurement

[0280] In the gripping force test, compared with the sham-operated group, the gripping force and gripping force / body weight of rats in the model group were significantly reduced (P<0.001, P<0.0001). Compared with the model group, the gripping force of rats in each prescription group was significantly increased (P<0.01, P<0.001), with the increase in Example 1 being greater than that in Examples 4-5, and the increase in Examples 4-5 being greater than that in Examples 6-7. Compared with the model group, the gripping force / body weight of rats in Examples 1 and 4-5 was significantly increased (P<0.05, P<0.01), and Examples 6-7 also showed an increasing trend, but the increase was less than that in Examples 1 and 4-5. Compared with the model group, the gripping force and gripping force / body weight of rats in Comparative Examples 8-9 were not significantly increased. Specific results are shown in Table 14.

[0281] Table 14 Effects of Shibu Granules on Grasping Strength in Rats with Kidney Yin-Yang Deficiency

[0282] Note: Compared with the sham operation group, ; compared with the model group, .

[0283] The experimental results show that Shibu Granules exhibit a definite positive regulatory effect in key dimensions such as enhancing the spontaneous activity ability of rats, strengthening the forepaw grip strength, regulating serum hormone homeostasis, repairing the pathological damage of uterine tissue, and regulating the expression of estrogen receptors. This provides an experimental basis for the application of this application to improve the symptoms of kidney yang and yin deficiency, and also lays a foundation for the further research and improvement of this application.

[0284] III. Pharmacodynamic Test of Shibu Granules on Mice with Kidney Yang Deficiency Induced by Hydrocortisone

[0285] In this experiment, a mouse model of kidney yang deficiency was constructed by subcutaneous injection of hydrocortisone to simulate the complex pathological state of kidney yang deficiency in clinical practice. Shibu Granules were administered by gavage to intervene in the model mice. At the same time, modern medical detection methods were used to compare the differences in syndromes, forepaw grip strength, body temperature changes, urine volume, organ indices, and tissue pathology of mice in each group. Comprehensive consideration of the above indicators can reflect the improvement effect of the traditional Chinese medicine composition granule for tonifying (Shibu Granules) prepared in Example 1 of this application on various symptoms of kidney yang deficiency, providing a reliable experimental basis for explaining the pharmacodynamic mechanism and clinical application of Shibu Granules.

[0286] 1. Test Materials

[0287] (1) Animals

[0288] SPF-grade male C57BL / 6J mice, weighing (20±2) g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The experimental animal production license number is SCXK (Beijing) 2021-0006. They were housed in the SPF-grade animal laboratory of the Experimental Animal Center of Tianjin University of Traditional Chinese Medicine (the animal breeding license number is SYXK (Tianjin) 2020-0005), with free access to water and food. The animal experiment was approved by the Experimental Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (approval number: TCM-LAEC2025020Y0929).

[0289] (2) Reagents and Drugs

[0290] Hydrocortisone (Beijing Solarbio Science & Technology Co., Ltd., product number: G8450).

[0291] (3) Instruments

[0292] Grip strength meter (Jinan Yiyan Technology Development Co., Ltd.); EMKA conscious animal pulmonary function detection system (EMKA Technologies, France); Multifunctional microplate reader (TECAN, Switzerland).

[0293] 2. Test Methods

[0294] (1) Animal modeling, grouping and administration

[0295] Male mice were acclimatized for 7 days and then randomly divided into a control group (n=10), a model group (n=10), a Jin Kui Shen Qi Wan (5.2 g crude drug / kg, n=9) group, a high-dose Shi Bu Granules group (20.8 g crude drug / kg, n=10), a medium-dose Shi Bu Granules group (10.4 g crude drug / kg, n=10), and a low-dose Shi Bu Granules group (5.2 g crude drug / kg, n=10). The drug-treated groups were administered the corresponding drugs by gavage daily, while the control and model groups were administered pure water by gavage for 3 consecutive weeks. Simultaneously with gavage, mice in the model and drug-treated groups were subcutaneously injected with 12.5 mg / kg hydrocortisone to establish a kidney-yang deficiency model, while the control group was subcutaneously injected with an equal volume of physiological saline.

[0296] (2) Sampling and testing indicators

[0297] a) General condition observation and syndrome scoring

[0298] Observe and record the signs and symptoms of the mice daily, such as their mental state, activity, and fur. After the last administration, score the mice in each group according to their symptoms. Observe whether the mice in each group exhibit a series of symptoms similar to those of kidney yang deficiency, including: mental state (lethargy, sluggishness, emaciation), spontaneous activity (curling up, huddling together and moving little), urination and defecation (filthy perianal area, loose stools), and fur (sparse, messy, and dull fur). Score each mouse based on the above 9 conditions, with 1 point for each condition and 0 points for none. The higher the score, the more obvious the symptoms.

[0299] b) Grip test

[0300] Using a grip strength tester, the grip strength of the forepaws of mice in each group was tested after the last administration. The instrument value was adjusted to zero, the mouse was placed on the gripping board, and its tail was gently pulled backward. After the animal had a firm grip, the force was evenly applied and pulled backward until the animal released its grip. The grip strength value was recorded. The above operation was repeated, and the average value was taken for 3 measurements.

[0301] c) Body temperature detection

[0302] After the last administration, the basal body temperature of the mice was measured and recorded using an infrared thermometer.

[0303] d) Urine volume measurement

[0304] After the last administration, each mouse was given 1 mL / 10g body weight of pure water by gavage, and the lower abdomen was gently pressed to expel any remaining urine, thus establishing a saline loading model. After 30 minutes, the mice were placed in metabolic cages, and urine output was continuously observed and recorded for 5 hours.

[0305] e) Organ Index

[0306] After blood was collected from mice in each group, intact kidney and adrenal gland tissues were directly harvested, dried with filter paper, and weighed on an electronic balance to calculate the organ index. The formula for calculating the organ index is: Organ Index (mg / g) -1 = Organ mass (mg) / Corresponding mouse body mass (g).

[0307] f) Histopathological examination

[0308] Mice were euthanized, and adrenal gland tissue was immediately removed and fixed with 10% neutral formalin. The tissue was then graded for dehydration, cleared, and embedded in paraffin to form paraffin blocks. Paraffin sections were dewaxed to water, graded for dewaxing with xylene and alcohol, stained with hematoxylin, separated by 1% hydrochloric acid and ethanol, rinsed with tap water for a blue inversion, stained with eosin, graded for dehydration, cleared, and mounted with neutral resin for observation.

[0309] (3) Statistical methods

[0310] Data analysis was performed, with continuous data expressed as mean ± standard deviation (X̄±s). One-way ANOVA was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0311] 3. Experimental Results

[0312] (1) General condition observation and syndrome scoring

[0313] Compared with the control group, the model group mice exhibited symptoms of kidney yang deficiency, such as huddling together with reduced movement, sparse body hair, curled-up back, slow movement, and dull fur. The kidney yang deficiency syndrome score was significantly higher (P<0.001). Compared with the model group, the mice in the medium and low dose groups of Shibu Granules showed significant improvement, with a significant decrease in the kidney yang deficiency syndrome score (P<0.05). The Jin Kui Shen Qi Wan group and the high dose group of Shibu Granules also showed a trend of improvement. See Table 15.

[0314] Table 15 Effects of Shibu Granules on the Syndrome Scores of Mice with Kidney Yang Deficiency

[0315] Note: Compared with the control group, Compared with the model group, .

[0316] (2) Grip force measurement

[0317] In the grip strength test, compared with the control group, the grip strength and grip strength / body weight of mice in the model group were significantly reduced (P<0.001). Compared with the model group, the grip strength (P<0.001) and grip strength / body weight of mice in the Jin Kui Shen Qi Wan group and the high, medium, and low dose groups of Shi Bu Granules were significantly increased (P<0.05, P<0.01, P<0.001). See Table 16.

[0318] Table 16 Effects of Shibu Granules on the grip strength of mice with kidney-yang deficiency

[0319] Note: Compared with the control group, Compared with the model group, .

[0320] (3) Basal body temperature measurement

[0321] Compared with the control group, the basal body temperature of mice in the model group was significantly lower (P<0.001). Compared with the model group, the basal body temperature of mice in the high, medium and low dose groups of Shibu Granules was significantly higher (P<0.001), as shown in Table 17.

[0322] Table 17 Effects of Shibu Granules on Body Temperature in Mice with Kidney Yang Deficiency

[0323] Note: Compared with the control group, Compared with the model group, .

[0324] (4) Urine volume measurement

[0325] Compared with the control group, the 5-hour urine output of mice in the model group was significantly reduced (P<0.001); compared with the model group, the 5-hour urine output of mice in the high- and medium-dose groups of Shibu Granules was significantly increased (P<0.01, P<0.001), and the 5-hour urine output of mice in the low-dose groups of Jinkui Shenqi Pills and Shibu Granules showed an increasing trend. See Table 18.

[0326] Table 18 Effects of Shibu Granules on Urine Output in Mice with Kidney Yang Deficiency

[0327] Note: Compared with the control group, Compared with the model group, , .

[0328] (5) Organ index

[0329] Compared with the control group, the kidney index of mice in the model group was significantly increased (P<0.001). Compared with the model group, the kidney index of mice in the high, medium and low dose groups of Shibu Granules was significantly decreased (P<0.05, P<0.01, P<0.001). See Table 19.

[0330] Table 19 Effects of Shibu Granules on Organ Indices in Mice with Kidney Yang Deficiency

[0331] Note: Compared with the control group, Compared with the model group, .

[0332] (7) Histopathological staining

[0333] like Figure 10 As shown, in the control group mice, adrenal cells exhibited regular morphology, uniform nucleus size, even chromatin distribution, and clear structures in each zone of the adrenal cortex (zona glomerulosa, zona fasciculata, and zona reticularis), with orderly cell arrangement and relatively stable thickness in each zone. In the model group mice, adrenal cortical cells showed irregular morphology, deformed nuclei, reduced cell number in each zone, widened intercellular spaces, and disordered cell arrangement. The Jin Kui Shen Qi Wan group and the high, medium, and low dose groups of Shi Bu Granules all improved the pathological condition of mouse adrenal tissue to a certain extent, specifically manifested as more regular cell morphology, uniform nucleus size, clearer structures in each zone of the adrenal cortex, orderly cell arrangement, and relatively stable thickness in each zone.

[0334] This experiment established a mouse model of kidney-yang deficiency by subcutaneous injection of hydrocortisone to simulate the complex pathological state of clinical kidney-yang deficiency. Subsequently, the model mice were treated with Shibu granules via gavage. Simultaneously, multidimensional modern medical detection methods were used to systematically compare the differences among the groups of mice in terms of symptom manifestations, forepaw grip strength, body temperature changes, urine output, organ indices, and histopathology, in order to explore the effect of Shibu granules on improving the symptoms of kidney-yang deficiency.

[0335] Experimental comparisons revealed that Shibu Granules significantly improved the symptoms of kidney-yang deficiency in mice, enhanced forelimb grip strength, increased basal body temperature, increased urine output within 5 hours, and improved the pathological state of adrenal tissue. The results suggest that Shibu Granules can, to some extent, improve relevant indicators in mice with kidney-yang deficiency.

[0336] IV. Efficacy test of Shibu Granules in treating a mouse model of renal deficiency and miscarriage induced by hydrocortisone combined with mifepristone.

[0337] This experiment investigates the effects of the tonifying traditional Chinese medicine composition granules (i.e., Shibu Granules) prepared according to the embodiments of this application on a mouse model of kidney deficiency and miscarriage. Female mice were administered hydrocortisone by gavage to simulate the pathological state of kidney yang deficiency. After successful conception, miscarriage was induced by gavage with mifepristone, thus replicating a mouse model closely resembling the pathological characteristics of clinical patients with kidney deficiency and miscarriage. The effects of Shibu Granules on the mouse model of kidney deficiency and miscarriage were examined by detecting indicators such as embryo loss rate and tissue morphological changes.

[0338] Experiment 1:

[0339] 1. Experimental materials

[0340] (1) Animals

[0341] SPF-grade female ICR mice, weighing (20±2) g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The production license number for experimental animals was SCXK (Beijing) 2021-0006. They were housed in the SPF-grade animal laboratory of the Experimental Animal Center of Tianjin University of Traditional Chinese Medicine (the animal breeding license number was SYXK (Tianjin) 2020-0005), with free access to water and food. The animal experiment was approved by the Experimental Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (approval number: TCM-LAEC2025020Y0929).

[0342] (2)Reagents and drugs

[0343] Hydrocortisone (Beijing Solarbio Science & Technology Co., Ltd., product number: G8450); Mifepristone (Beijing Solarbio Science & Technology Co., Ltd., product number: M7250). The Shibu Granules used were the granules of the traditional Chinese medicine composition for tonifying prepared in Example 1.

[0344] (3)Instruments

[0345] Multifunctional microplate reader (TECAN, Switzerland).

[0346] 2. Experimental methods

[0347] (1)Animal modeling, grouping and drug administration

[0348] After 7 days of adaptive feeding of female ICR mice, they were caged with male mice at a ratio of 1:1 for mating. The day when a white vaginal plug was found or a large number of sperm were seen in the vaginal smear was recorded as the first day of pregnancy in female mice. The pregnant mice were randomly divided into a control group, a model group, a high-dose Shibu Granules group (20.8 g / kg, calculated by raw drug amount), a medium-dose Shibu Granules group (10.4 g / kg, calculated by raw drug amount), and a low-dose Shibu Granules group (5.2 g / kg, calculated by raw drug amount, clinically equivalent). From the first day of pregnancy, the drug administration groups were given the corresponding dose of drugs by gavage every morning, and the control group and the model group were given pure water by gavage, for 9 consecutive days. The model was replicated every afternoon. Except for the control group given pure water by gavage, the other groups were given 25 mg / kg of hydrocortisone by gavage for 9 consecutive days, and 4 mg / kg of mifepristone by gavage on the 10th day to replicate the kidney deficiency abortion model.

[0349] (2)Sampling and detection indexes

[0350] a)Embryo loss rate

[0351] Mice were euthanized, and the uterus was dissected to observe embryo loss. Specific criteria for judgment: normal embryos were large and bead-like, with no visible bleeding in the uterus, and appeared pale red; aborted embryos showed obvious bleeding in the uterus, were dark brown, or exhibited a bamboo-like uterine structure, and showed complete or partial embryo disappearance. The uterus was longitudinally dissected, and the total number of fetuses, the number of surviving embryos, the number of aborted embryos, and the embryo loss rate were calculated. Embryo loss rate = (Number of aborted embryos / (Number of aborted embryos + Number of surviving embryos)) × 100%.

[0352] b) Histopathological observation

[0353] Kidney and uterine tissues were collected, fixed in formalin, dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and sectioned to obtain 4 μm paraffin sections. After HE staining, the lesions in the kidney and uterine tissues were observed.

[0354] (3) Statistical methods

[0355] Data analysis was performed, with continuous data expressed as mean ± standard deviation (X̄±s). One-way ANOVA was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0356] 3. Experimental Results

[0357] (1) Embryo loss rate

[0358] The embryo loss rate results are shown in Table 20. Compared with the control group, the embryo loss rate of the model group mice was significantly increased (P<0.001); compared with the model group, the embryo loss rate of the high, medium and low dose groups of Shibu Granules was significantly decreased (P<0.001).

[0359] Table 20. Effects of Shibu Granules on Embryo Loss Rate in Mice with Kidney Deficiency and Abortion

[0360] Note: Compared with the control group, Compared with the model group, .

[0361] (2) Histopathological staining

[0362] Kidney HE staining results as follows Figure 11 As shown, in the control group mice, the renal tubules and glomeruli had normal and clear structures, and there was no inflammatory cell infiltration in the tubulointerstitium. In the model group mice, the renal tubules had irregular lumens and a large number of inflammatory cells infiltrated. The high, medium, and low doses of Shibu Granules all improved the kidney lesions, reduced the number of irregular renal tubules, and alleviated inflammatory cell infiltration.

[0363] Results of HE staining of the uterus as follows Figure 12As shown, the endometrium of the mice in the control group developed well, the blood vessel morphology was intact, and the decidual cells were closely arranged. In the model group, the cell arrangement was irregular and angiogenesis decreased. In the high-, medium-, and low-dose groups of Shibu Granules, the endometrium of the mice developed to varying degrees, the cell arrangement was neat, and angiogenesis increased.

[0364] Experiment 2:

[0365] 1. Experimental materials

[0366] (1)Animals

[0367] SPF-grade female ICR mice, weighing (20±2) g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The production license number of experimental animals was SCXK (Beijing) 2021-0006. They were housed in the SPF-grade animal laboratory of the Experimental Animal Center of Tianjin University of Traditional Chinese Medicine (the breeding license number was SYXK (Tianjin) 2020-0005), with free access to water and food. The animal experiment was approved by the Experimental Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (approval number: TCM-LAEC2025020Y0929).

[0368] (2)Reagents and drugs

[0369] Hydrocortisone (Beijing Solarbio Science & Technology Co., Ltd., product number: G8450); Mifepristone (Beijing Solarbio Science & Technology Co., Ltd., product number: M7250).

[0370] (3)Instruments

[0371] Multifunctional microplate reader (TECAN, Switzerland).

[0372] 2. Experimental methods

[0373] (1)Animal modeling, grouping and drug administration

[0374] After 7 days of adaptive feeding of female ICR mice, they were caged with male mice at a ratio of 1:1 for mating. The day when a white vaginal plug was found or a large number of sperm were seen in the vaginal smear was recorded as the first day of pregnancy of the female mouse. The pregnant mice were randomly divided into a control group, a model group, Example 1, Examples 4-7, Comparative Example 8 and Comparative Example 9 (5.2 g / kg, clinical equivalent dose). From the first day of pregnancy, the drug administration groups were intragastrically administered the corresponding dose of the drug every morning, and the control group and the model group were intragastrically administered pure water for 9 consecutive days. The model was replicated every afternoon. Except for the control group intragastrically administered pure water, the other groups were intragastrically administered 25 mg / kg of hydrocortisone for 9 consecutive days, and 4 mg / kg of mifepristone was intragastrically administered on the 10th day to replicate the kidney deficiency abortion model.

[0375] (2)Sampling and detection indexes

[0376] Embryo loss rate:

[0377] Mice were euthanized, and the uterus was dissected to observe embryo loss. Specific criteria for judgment: normal embryos were large and bead-like, with no visible bleeding in the uterus, and appeared pale red; aborted embryos showed obvious bleeding in the uterus, were dark brown, or exhibited a bamboo-like uterine structure, and showed complete or partial disappearance of the embryo. The uterus was longitudinally dissected, and the total number of fetuses, the number of surviving embryos, the number of aborted embryos, and the embryo loss rate were calculated. Embryo loss rate = (Number of aborted embryos / (Number of aborted embryos + Number of surviving embryos)) × 100%.

[0378] (3) Statistical methods

[0379] Data analysis was performed, with continuous data expressed as mean ± standard deviation (X̄±s). One-way ANOVA was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0380] 3. Experimental Results

[0381] (1) Embryo loss rate

[0382] The embryo loss rate results are shown in Table 21. Compared with the control group, the embryo loss rate in the model group mice was significantly increased (P<0.0001). Compared with the model group, the embryo loss rates in Examples 1 and 4-7 were significantly reduced (P<0.01, P<0.001, P<0.0001), with the reduction in Example 1 being greater than that in Examples 4-5, and the reduction in Examples 4-5 being greater than that in Examples 6-7. Compared with the model group, the embryo loss rates in Comparative Examples 8-9 were not significantly reduced.

[0383] Table 21 Effects of Shibu Granules on Embryo Loss Rate in Mice with Kidney Deficiency and Abortion

[0384] Note: Compared with the control group, Compared with the model group, .

[0385] Kidney deficiency-induced abortion is a common clinical condition in traditional Chinese medicine. This experiment established a kidney deficiency-induced abortion model induced by hydrocortisone combined with mifepristone, which is consistent with the traditional Chinese medicine theory of "the kidney governs reproduction". The effects of Shibu Granules on the kidney deficiency-induced abortion model mice were systematically investigated.

[0386] Experimental results showed that, compared with the model group, high, medium, and low doses of Shibu Granules all exhibited significant effects in preventing miscarriage. Regarding the core indicators, all dose groups significantly reduced the embryo loss rate in mice, directly demonstrating a positive effect on maintaining pregnancy. Simultaneously, Shibu Granules could alleviate pathological changes in uterine tissue. These results suggest that Shibu Granules have a certain therapeutic effect on mice with kidney deficiency and miscarriage.

[0387] V. Efficacy test of Shibu Granules in treating a mouse model of qi and blood deficiency

[0388] In this experiment, a mouse model of qi and blood deficiency was established by intraperitoneal injection of cyclophosphamide combined with acetylphenylhydrazine. The effects of the traditional Chinese medicine composition granule for tonifying (i.e., Shibu Granule) prepared in Example 1 of this application on indicators such as the body weight and grasping force of mice with qi and blood deficiency were observed through experiments, and the pharmacodynamic effects of Shibu Granule on mice with qi and blood deficiency were investigated.

[0389] 1. Test materials

[0390] (1)Animals

[0391] SPF-grade female ICR mice, weighing (20±2) g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The experimental animal production license number is SCXK (Beijing) 2021-0006. They were housed in the SPF-grade animal laboratory of the Experimental Animal Center of Tianjin University of Traditional Chinese Medicine (the animal housing license number is SYXK (Tianjin) 2020-0005), with free access to water and food. The animal experiment was approved by the Experimental Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (approval number: TCM-LAEC2025020Y0929).

[0392] (2)Reagents and drugs

[0393] Cyclophosphamide for injection (Shanxi Pude Pharmaceutical Co., Ltd., batch number: 04240705), acetylphenylhydrazine (Shanghai Macklin Biochemical Co., Ltd., product number: A800026).

[0394] (3)Instruments

[0395] Grasping force meter (Jinan Yiyan Technology Development Co., Ltd.).

[0396] 2. Test methods

[0397] (1)Animal model establishment, grouping and administration

[0398] The mice were randomly divided into a blank group, a model group, and high, medium, and low dose groups of Shibu Granule. The administration groups were intragastrically administered with the high dose group of Shibu Granule (20.8 g / kg, calculated by crude drug amount), the medium dose group of Shibu Granule (10.4 g / kg, calculated by crude drug amount), and the low dose group of Shibu Granule (5.2 g / kg, calculated by crude drug amount) every day. The control group and the model group were intragastrically administered with pure water. The mice in the model group and the administration groups were intraperitoneally injected with acetylphenylhydrazine solution (20 mg / kg, 40 mg / kg) on the 1st and 4th days, and intraperitoneally injected with cyclophosphamide (25 mg / kg) on the 1st - 4th days to prepare an animal model of qi and blood deficiency. The control group was intraperitoneally injected with an equal volume of normal saline for 7 consecutive days.

[0399] (2)Sampling and detection indicators

[0400] a)Body weight change

[0401] Mice were weighed daily, and the weight changes of each group were dynamically observed. Percentage change in body weight (%) = (daily body weight - initial body weight) / daily body weight × 100%.

[0402] b) Grip test

[0403] Using a grip strength tester, the grip strength of the forepaws of mice in each group was tested after the last administration. The instrument value was adjusted to zero, the mouse was placed on the gripping board, and its tail was gently pulled backward. After the mouse had a firm grip, the force was evenly applied and pulled backward until the mouse released its grip. The grip strength value was recorded. The above operation was repeated, and the results were taken as the average value of 3 measurements.

[0404] c) Weighted swimming experiment

[0405] After the last administration, each group of mice had a stainless steel wire of 6% of their body weight tied around their tails and were placed in a swimming tank to record their swimming time. The swimming time was defined as the time when the mouse's head could not float to the surface within 10 seconds. The duration from entry into the water to the end of swimming was recorded.

[0406] (3) Statistical methods

[0407] Data analysis was performed, with continuous data expressed as mean ± standard deviation (X̄±s). One-way ANOVA was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0408] 3. Experimental Results

[0409] (1) Weight changes

[0410] Compared with the control group, the body weight of mice in the model group was significantly lower on days 6 and 7 (P<0.05, P<0.01). Compared with the model group, the body weight of mice in the high- and medium-dose groups of Shibu Granules increased from day 5, especially in the medium-dose group on day 7, which showed the most significant increase (P<0.05). See Table 22 and... Figure 13 . Figure 13 In comparison with the control group on the same day, Compared with the model group on the same day, .

[0411] Table 22 Effects of Shibu Granules on Body Weight Changes in Mice with Qi and Blood Deficiency

[0412] Note: Compared with the control group on the same day, Compared with the model group on the same day, .

[0413] (2) Grip test

[0414] The results of the mouse gripping strength are shown in Table 23. Compared with the control group, the gripping strength and gripping strength / body weight of the model group mice were significantly reduced (P<0.001). Compared with the model group, the gripping strength and gripping strength / body weight of the high-dose group of Shibu Granules were significantly increased (P<0.01, P<0.001), and the gripping strength of the medium-dose group was significantly increased (P<0.01).

[0415] Table 23 Effects of Shibu Granules on grip strength in mice with qi and blood deficiency

[0416] Note: Compared with the control group, Compared with the model group, .

[0417] (3) Swimming time with weight

[0418] Compared with the control group, the weight-bearing swimming time of mice in the model group was significantly reduced (P<0.001); compared with the model group, the weight-bearing swimming time of mice in the high, medium and low dose groups of Shibu Granules was significantly prolonged (P<0.05, P<0.001). See Table 24.

[0419] Table 24 Effects of Shibu Granules on Weight-Bearing Swimming Time in Qi-Deficient Mice

[0420] Note: Compared with the control group, Compared with the model group, , .

[0421] This experiment established a mouse model of qi and blood deficiency by intraperitoneal injection of cyclophosphamide combined with acetylphenylhydrazine, and then used gavage to administer Shibu granules to investigate their pharmacological effects on qi and blood deficiency.

[0422] After gavage administration of Shibu granules, mice with qi deficiency showed increased body weight, enhanced grip strength, and prolonged swimming time under load, suggesting that Shibu granules can improve symptoms related to qi and blood deficiency in mice and exert an anti-fatigue effect. In conclusion, Shibu granules have a certain therapeutic effect on mice with qi and blood deficiency.

[0423] VI. Clinical efficacy trial of the Ten-Tonifying Decoction for treating kidney yin and yang deficiency

[0424] One hundred patients with kidney deficiency who met the inclusion criteria were selected as the study subjects. They were given oral administration of a decoction of tonifying Chinese herbal medicine for one month. The study used a self-controlled before-and-after method to observe the improvement of different TCM syndrome scores and the safety of the medication before and after treatment.

[0425] 1. Materials and Methods

[0426] 1.1 General Information

[0427] One hundred patients with symptoms of kidney deficiency were selected, including 28 males and 72 females, aged 30 to 80 years, with a mean age of (51.95±11.80) years.

[0428] 1.2 Diagnostic Criteria

[0429] The specific diagnostic criteria for kidney deficiency syndrome are as follows: cold hands and feet, cold pain in the navel and abdomen, soreness and weakness in the lower back and knees, palpitations and shortness of breath, tinnitus, fatigue, loss of appetite, and difficulty urinating.

[0430] 1.3 Inclusion Criteria

[0431] (1) Meets the criteria of kidney yang deficiency and kidney yin deficiency in traditional Chinese medicine; (2) is between 30 and 80 years old; (3) has not used other tonifying Chinese patent medicines (Chinese patent medicines and decoctions) in the past week; (4) has signed the patient's informed consent form.

[0432] 1.4 Exclusion Criteria

[0433] (1) Severe hemodynamic instability; (2) Severe liver and kidney dysfunction and liver and kidney metabolic disorders; (3) Malignant arrhythmia; (4) Recently underwent upper gastrointestinal surgery such as esophageal and gastric surgery; (5) Have contraindications to enteral nutrition; (6) Participated in other clinical studies within 2 months before the start of the study; (7) The patient or their authorized representative is unwilling to sign a written informed consent form or is unwilling to comply with the study protocol; (8) Pregnant women; (9) Those deemed unsuitable to participate in this study by the researchers.

[0434] 1.5 Treatment Methods

[0435] All enrolled patients were treated with the tonic Chinese herbal decoction (Shi Bu Tang) prepared in Example 8. The ingredients were: 11.70g Rehmannia glutinosa (processed), 7.02g Dioscorea opposita (fried), 4.68g Poria cocos, 5.85g Cornus officinalis (processed with wine), 1.40g Schisandra chinensis (processed with wine), 4.68g Eucommia ulmoides (processed with salt), and 4.68g Achyranthes bidentata (processed with wine). One dose was taken orally daily for one month.

[0436] 1.6 Observation Indicators

[0437] Using a self-controlled before-and-after research method, we observed changes in patients' clinical symptoms (cold hands and feet, cold pain in the navel and abdomen, soreness and weakness in the lower back and knees, palpitations, shortness of breath, tinnitus, etc.), tongue appearance, and pulse before and after treatment. Based on the scoring criteria for different symptoms (see Table 25), patients with a disease assessment score of 3-9 were defined as mild patients; patients with a disease assessment score of 10-15 were defined as moderate patients; and patients with a disease assessment score of 15 or above were defined as severe patients.

[0438] Table 25 Scoring criteria for different symptoms

[0439] 1.7 Efficacy Criteria

[0440] Based on the change in the patient's condition assessment score before and after medication, three outcomes were established: "significantly effective," "effective," and "ineffective." Patients with a change of 5 points or more in their assessment score were defined as "significantly effective"; those with a change of 3-4 points were defined as "effective"; and those with a change of less than 3 points were defined as "ineffective." The overall effective rate was calculated as (significantly effective + effective) / n × 100%.

[0441] 1.8 Statistical Analysis

[0442] Data analysis showed that the measurement data were expressed as “x̄±s”, and the t-test was used. P<0.05 was considered statistically significant.

[0443] 2. Results

[0444] 2.1 Clinical efficacy results

[0445] Based on observation and data analysis, the clinical efficacy results after treatment are shown in Table 26. As can be seen from Table 26, after taking the Shi Bu Tang decoction, 5 cases showed significant effects within 30 days, 82 cases showed effectiveness, and the total effective rate reached 87%.

[0446] Table 26 Clinical efficacy results after treatment (n, %)

[0447] 2.2 Improvement of clinical symptoms before and after treatment

[0448] The 100 patients included all had varying degrees of symptoms such as cold hands and feet, cold pain in the navel and abdomen, soreness and weakness of the lower back and knees, palpitations and shortness of breath, and tinnitus. The efficacy results showed that the patients had a good therapeutic effect on the symptoms of cold hands and feet, cold pain in the navel and abdomen, soreness and weakness of the lower back and knees, palpitations and shortness of breath after taking the Ten-Tonifying Decoction (P<0.05).

[0449] 2.3 Safety Observation

[0450] No adverse drug reactions were observed in any patient during or after treatment.

[0451] The above results indicate that the Shi Bu Tang formula can effectively improve the clinical symptoms of patients with kidney yin and yang deficiency, and its clinical application is safe and reliable.

[0452] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A tonic Chinese herbal composition, wherein, The raw material components of the traditional Chinese medicine composition, by weight, include: 57-106 parts of Rehmannia glutinosa (processed), 34-64 parts of Dioscorea opposita, 23-43 parts of Poria cocos, 29-53 parts of Cornus officinalis (processed with wine), 7-13 parts of Schisandra chinensis (processed with wine), 23-43 parts of Eucommia ulmoides (processed with salt), and 23-43 parts of Achyranthes bidentata (processed with wine), wherein the Dioscorea opposita is stir-fried or stir-fried with wheat bran.

2. The traditional Chinese medicine composition according to claim 1, wherein, The raw material components of the traditional Chinese medicine composition, by weight, include: 66-98 parts of Rehmannia glutinosa (processed), 39-59 parts of Dioscorea opposita (fried), 26-39 parts of Poria cocos, 33-49 parts of Cornus officinalis (processed with wine), 8-12 parts of Schisandra chinensis (processed with wine), 26-39 parts of Eucommia ulmoides (processed with salt), and 26-39 parts of Achyranthes bidentata (processed with wine).

3. The traditional Chinese medicine composition according to claim 1, wherein, The traditional Chinese medicine composition also includes pharmaceutically acceptable carriers or excipients; Preferably, the pharmaceutically acceptable carrier or excipient is selected from at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, flow aids, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavorings, sweeteners, ion exchangers, release agents, coating agents, flavoring agents, and antioxidants; Preferably, the traditional Chinese medicine composition is formulated into a pharmaceutically acceptable preparation; preferably, the traditional Chinese medicine composition is formulated into any one of the following dosage forms: granules, decoction, pills, tablets, or capsules.

4. A method for preparing a tonic Chinese herbal composition, wherein, Including the following steps: All the raw material components according to any one of claims 1-3 are boiled with water to extract the extract, and then the filtrate is collected by filtration to obtain a decoction of tonifying Chinese herbal medicine composition. Preferably, the weight of the water is 4-10 times the total weight of all raw material components; The decoction and extraction process is repeated 2-3 times, with each decoction and extraction lasting 0.5-3 hours.

5. The preparation method according to claim 4, wherein, It also includes the following steps: (1) Concentrate the filtrate into a clear paste; then add the clear paste to the excipients and granulate to obtain granules of the tonic Chinese medicine composition; or, (1') Concentrate the filtrate into a clear paste, dry the clear paste, pulverize it, then add excipients and wetting agents, mix well, granulate, and then dry again to obtain granules of the tonic Chinese medicine composition; or, (1'') The filtrate is concentrated into a clear paste, the clear paste is dried and pulverized, and then excipients are added for granulation to obtain granules of the tonic Chinese medicine composition.

6. The preparation method according to claim 5, wherein, In step (1), step (1'), or step (1''), the concentration is vacuum concentration, and the temperature of vacuum concentration is 65-85℃; the relative density of the clear extract is 1.05-1.20; the mass ratio of the excipients to the clear extract is 1:(1-2.5); the excipients include dextrin and lactose in a mass ratio of (1.5-2.5):

1. In step (1), the granulation process parameters include: air inlet temperature of 80-100℃, atomization pressure of 0.3-0.4 bar, and liquid inlet speed of 7-20 rpm; In step (1'), the drying method is selected from at least one of vacuum drying, belt drying and spray drying; the wetting agent is selected from water or an aqueous solution of ethanol with a volume fraction of 70%-95%; the temperature of the re-drying is 40℃-60℃; In step (1''), the drying method is selected from at least one of vacuum drying, belt drying and spray drying.

7. The preparation method according to claim 4, wherein, It also includes the following steps: The filtrate was concentrated into a clear extract; the clear extract was dried and then pulverized to obtain an extract powder. The extract powder and excipients are mixed evenly, then water is added for granulation, magnesium stearate is added and mixed evenly, and then tableted to obtain a tonic Chinese medicine composition tablet.

8. The preparation method according to claim 7, wherein, The concentration is a vacuum concentration, and the vacuum concentration temperature is 65-85℃; The relative density of the ointment is 1.05-1.20; The drying process is vacuum drying, and the vacuum drying temperature is 65-85℃. The mass ratio of the excipients to the extract powder is (1.5-2.5):1; The excipients include dextrin and lactose in a mass ratio of (1-3):1; The mass of the magnesium stearate is 0.4%-0.6% of the mass of the granulated particles.

9. Use of the traditional Chinese medicine composition according to any one of claims 1-3 in the preparation of anti-fatigue, anti-aging or renal function protection drugs.

10. The use of the traditional Chinese medicine composition according to any one of claims 1-3 in the preparation of a medicine for treating diseases caused by kidney yang deficiency, diseases caused by kidney yin and yang deficiency, miscarriage caused by kidney deficiency, or diseases caused by qi and blood deficiency.

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