Combination extract of tabernaemontana divaricata, chamaecyparis and paotoa caffra, preparation method and application

Abstract

This invention relates to the field of pharmaceutical preparation technology, providing a combined extract of *Euonymus alatus*, *Chamaeguta foetida*, and Brazilian ginseng, its preparation method, and applications. The combined extract of *Euonymus alatus*, *Chamaeguta foetida*, and Brazilian ginseng possesses anti-fatigue effects. It is a pure plant extract, and the composition and ratio of the *Euonymus alatus*, *Chamaeguta foetida*, and Brazilian ginseng extracts are carefully considered. The pharmacological effects of the combined extract, with each ingredient working synergistically, are superior to those of using *Euonymus alatus* alone, thus demonstrating the anti-fatigue, ulcerative colitis treatment, and anti-kidney yang deficiency effects of the combined extract.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, and particularly relates to the combined extract of Evodia rutaecarpa, Chamaegu and Brazilian ginseng, its preparation method and application. Background Technology

[0002] Ulcerative colitis (UC) is a common inflammatory disease of the gastrointestinal tract with complex etiologies and pathogenesis. Lesions are primarily located in the sigmoid colon and rectum, and in severe cases can extend to the descending colon or the entire colon. The disease has a long course and is prone to recurrence, seriously affecting patients' physical and mental health and financial burden. In recent years, with the deepening research into traditional Chinese medicine, its potential in treating gastrointestinal diseases has gradually attracted attention. During the pathogenesis of ulcerative colitis, local tissue damage in the colon, external pathogenic factors, and the influence of autoimmune factors often lead to significant abnormalities in immune function, such as extensive infiltration of immune cells into the intestinal mucosa, fluctuating levels of pro-inflammatory factors (such as IL-6, IL-1β, TNF-α, etc.), and massive proliferation of T cells, B cells, monocytes, and NK cells. The antibodies, cytokines, and inflammatory mediators released by these immune responses trigger severe inflammatory reactions around the intestines, directly damaging the intestinal mucosal epithelium and leading to long-term inflammatory lesions.

[0003] From the perspective of Traditional Chinese Medicine (TCM), chronic ulcerative colitis easily depletes the body's Yang energy, especially leading to spleen and kidney Yang deficiency. Kidney Yang deficiency manifests as symptoms such as lower back and knee weakness, aversion to cold, cold limbs, fatigue, and physical weakness, severely impacting the patient's quality of life. Furthermore, due to prolonged inflammation, impaired nutrient absorption, and immune system disorders, patients often experience significant fatigue, weakness, and other systemic symptoms of weakness, further exacerbating the pathological state of kidney Yang deficiency. Therefore, in the treatment of ulcerative colitis, warming and tonifying kidney Yang and invigorating Qi and the spleen have become important directions for TCM intervention. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a combined extract of *Euonymus alatus*, *Chamaecyparis edulis*, and Brazilian ginseng, its preparation method, and applications. The aim is to treat individuals with kidney yang deficiency, fatigue, and those for whom current treatments for ulcerative colitis often fail to effectively reach the lesions. The combined extract of *Euonymus alatus*, *Chamaecyparis edulis*, and Brazilian ginseng is a pure plant extract. In treating fatigue, kidney yang deficiency, and ulcerative colitis, its components exhibit synergistic effects, are non-addictive, convenient to carry, have a pleasant taste, and demonstrate significant efficacy.

[0005] This invention provides a combined extract of *Gnaphalium affine*, *Chamaegu* and Brazilian ginseng, composed of the following raw materials in parts by weight: 7-21 parts of *Gnaphalium affine*, 6-18 parts of *Chamaegu* and 1-3 parts of Brazilian ginseng.

[0006] Furthermore, 10-14 parts by weight of *Gnaphalium affine*, 8-12 parts by weight of *Chamaegu*, and 1.5-2.5 parts by weight of Brazilian ginseng.

[0007] Furthermore, 14 parts by weight of *Gnaphalium affine*, 12 parts by weight of *Chamaegu*, and 3 parts by weight of Brazilian ginseng.

[0008] Furthermore, 7 parts by weight of *Gnaphalium affine*, 6 parts by weight of *Chamaegu*, and 1 part by weight of Brazilian ginseng.

[0009] Furthermore, the preparation method of the combined extract of *Gnaphalium affine*, *Gnaphalium affine*, and Brazilian ginseng includes the following steps:

[0010] The raw materials, by weight, are 7-21 parts of *Gnaphalium affine*, 6-18 parts of *Chamaegu*, and 1-3 parts of Brazilian ginseng, mixed and then crushed into coarse powder. Add 8-12 times the total weight of the raw materials to water, heat and reflux for extraction 1-3 times, 1-2 hours each time, and collect the extract from each heating and reflux extraction; Combine the extracts, filter, and concentrate the filtrate under reduced pressure at 60-80℃ to a clear extract with a relative density of 1.10-1.25; The extract is vacuum dried or freeze-dried, then pulverized and sieved to obtain the combined extract of *Gnaphalium affine*, *Gnaphalium affine*, and Brazilian ginseng.

[0011] Furthermore, the heating and reflux extraction was performed twice. The first extraction was performed with 10 times the amount of water for 2 hours, and the second extraction was performed with 8 times the amount of water for 1.5 hours.

[0012] Furthermore, the extracts of *Gnaphalium affine*, *Gnaphalium affine*, and Brazilian ginseng are also formulated with pharmaceutically or food-grade excipients.

[0013] Furthermore, extracts of *Gnaphalium affine*, *Gnaphalium affine*, and Brazilian ginseng can be formulated into pharmaceutical or food products such as tablets, capsules, powders, mixtures, pills, granules, solutions, syrups, decoctions, beverages, biscuits, candies, or pastries.

[0014] Furthermore, the combined extracts of *Gnaphalium affine*, *Gnaphalium affine*, and Brazilian ginseng are used in the preparation of products with anti-fatigue, ulcerative colitis treatment, and / or anti-kidney yang deficiency effects.

[0015] Furthermore, the products include pharmaceuticals, health supplements, or food.

[0016] The present invention has the following technical effects: the combined extract of *Euonymus alatus*, *Chamaeguta* and Brazilian ginseng has anti-fatigue effects. It is a pure plant extract, and fully considers the composition and ratio of *Euonymus alatus* extract, *Chamaeguta* extract and Brazilian ginseng extract. The pharmacological effects of each raw material working together according to their ratio are better than those of using *Euonymus alatus* alone, thereby exerting the anti-fatigue, ulcerative colitis treatment and anti-kidney yang deficiency effects of the combined extract of *Euonymus alatus*, *Chamaeguta* and Brazilian ginseng. Attached Figure Description

[0017] The exemplary embodiments of the present invention can be more fully understood by referring to the following figures, where *** indicates P < 0.001, ** indicates P < 0.01, and * indicates P < 0.05: Figure 1 This is the result of the effect of the combined extract of Example 2 of the present invention on the general signs of mice with ulcerative colitis, wherein: Figure 1 In this context, A represents the DAI score; Figure 1 In this context, B represents the change in body mass.

[0018] Figure 2 This is an observation diagram of the intestinal and spleen phenotypes of mice with ulcerative colitis, based on the combined extract of Example 2 of this invention. Figure 2 In this context, A represents the gut phenotype; Figure 2 B in the text represents the spleen phenotype.

[0019] Figure 3 This is a statistical graph showing the effect of the combined extract of Example 2 of the present invention on the colon length and organ index of mice with ulcerative colitis, wherein: Figure 3 In this context, A represents the length of the colon. Figure 3 B in the figure represents the spleen index; Figure 3 C in the figure represents the kidney index.

[0020] Figure 4 This is the result of the combined extract of Example 2 of the present invention on the pathological effects of colon tissue in mice with ulcerative colitis.

[0021] Figure 5 This is the result of the effect of the combined extract of Example 2 of the present invention on the inflammatory response of mice with ulcerative colitis.

[0022] Figure 6 This is the result of the effect of the combined extract of Example 3 of the present invention on the inflammatory response of rats with kidney yang deficiency.

[0023] Figure 7This is the result of the combined extract of Example 3 of the present invention on the hormone levels of ACTH, CORT, T, T4, E2 and other hormones in rats with kidney yang deficiency.

[0024] Figure 8 The results of the combined extract of Example 3 of this invention on the pathological effects of kidneys on rats with kidney yang deficiency are shown in the figures: testis and epididymis. Scale bar: 200 μm.

[0025] Figure 9 This is the effect of the combined extract of Example 3 of the present invention on the pathological condition of the testes of rats with kidney yang deficiency. Scale bar: 200 μm.

[0026] Figure 10 This is the effect of the combined extract of Example 3 of the present invention on the pathology of the epididymis of rats with kidney yang deficiency. Scale bar: 200 μm. Detailed Implementation

[0027] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0029] This invention provides a combined extract of *Gnaphalium affine*, *Chamaegu*, and Brazilian ginseng, composed of the following raw materials in parts by weight: 7-21 parts of *Gnaphalium affine*, 6-18 parts of *Chamaegu*, and 1-3 parts of Brazilian ginseng.

[0030] In some embodiments, 10-14 parts by weight of *Gnaphalium affine*, 8-12 parts by weight of *Chamaegu*, and 1.5-2.5 parts by weight of Brazilian ginseng are used.

[0031] In some embodiments, 14 parts by weight of *Gnaphalium affine*, 12 parts by weight of *Chamma jasminoides*, and 3 parts by weight of *Gynostemma pentaphyllum* are used.

[0032] In some embodiments, 7 parts by weight of *Gnaphalium affine*, 6 parts by weight of *Chamma jasminoides*, and 1 part by weight of *Gnaphalium affine* are used.

[0033] In some embodiments, the preparation method of the combined extract of *Gnaphalium affine*, *Gnaphalium affine*, and Brazilian ginseng includes the following steps: The raw materials, by weight, are 7-21 parts of *Gnaphalium affine*, 6-18 parts of *Chamaegu*, and 1-3 parts of Brazilian ginseng, mixed and then crushed into coarse powder. Add 8-12 times the total weight of the raw materials to water, heat and reflux for extraction 1-3 times, 1-2 hours each time, and collect the extract from each heating and reflux extraction; Combine the extracts, filter, and concentrate the filtrate under reduced pressure at 60-80℃ to a clear extract with a relative density of 1.10-1.25; The extract is vacuum dried or freeze-dried, then pulverized and sieved to obtain the combined extract of *Gnaphalium affine*, *Gnaphalium affine*, and Brazilian ginseng.

[0034] In some embodiments, the heating and reflux extraction is performed twice: the first extraction is performed with 10 times the amount of water for 2 hours, and the second extraction is performed with 8 times the amount of water for 1.5 hours.

[0035] In some embodiments, the combined extract of *Gnaphalium affine*, *Gnaphalium affine*, and Brazilian ginseng is further mixed with pharmaceutically or food-grade excipients.

[0036] In some embodiments, extracts of *Gnaphalium affine*, *Gnaphalium affine*, and Brazilian ginseng are formulated into pharmaceutical or food-grade tablets, capsules, powders, mixtures, pills, granules, solutions, syrups, decoctions, beverages, biscuits, candies, or pastries.

[0037] In some embodiments, the combined extracts of *Gnaphalium affine*, *Gnaphalium affine*, and Brazilian ginseng are used in the preparation of products with anti-fatigue, ulcerative colitis treatment, and / or anti-kidney yang deficiency effects.

[0038] In some embodiments, the product includes pharmaceuticals, health products, or food.

[0039] Example 1: Preparation of Combined Extract Group 1 Ingredients: 7g of *Gnaphalium affine*, 6g of *Chamaegu*, and 1g of Brazilian ginseng.

[0040] Preparation method: (1) Weigh each raw material, mix them, and then crush them into coarse powder (pass through a 20-mesh sieve). (2) Add purified water in a volume of 10 times the total weight of the raw materials, heat and reflux for 2 hours, filter, and collect the filtrate; (3) Add 8 times the amount of purified water to the filter residue, heat and reflux for 1.5 hours, and then filter. (4) Combine the two filtrates and concentrate them under reduced pressure at 60°C to a clear extract with a relative density of 1.15; (5) Dry the clear extract at 60°C for 48 hours, then pulverize it through an 80-mesh sieve to obtain the powder of combined extract 1.

[0041] Example 2: Preparation of Combined Extract Group 2 Ingredients: 14g of *Gnaphalium affine*, 12g of *Chamaegu*, and 3g of Brazilian ginseng.

[0042] Preparation method: (1) Weigh each raw material, mix them, and then crush them into coarse powder (pass through a 20-mesh sieve). (2) Add purified water in a volume of 10 times the total weight of the raw materials, heat and reflux for 2 hours, filter, and collect the filtrate; (3) Add 8 times the amount of purified water to the filter residue, heat and reflux for 1.5 hours, and then filter. (4) Combine the two filtrates and concentrate them under reduced pressure at 70°C to a clear extract with a relative density of 1.25; (5) Dry the clear extract at 60°C for 48 hours, then pulverize it through an 80-mesh sieve to obtain the powder of combined extract 2.

[0043] Example 3: Preparation of Compound Beverage Ingredients: 14g of *Gnaphalium affine*, 12g of *Chamaegu*, and 3g of Brazilian ginseng.

[0044] Preparation method: (1) Add 10 times the total weight of the raw materials of purified water, heat and reflux for 2 hours, filter, and collect the filtrate; add 12 times the amount of purified water to the filter residue, heat and reflux for 1.5 hours, and filter; combine the two filtrates, filter and concentrate into a clear paste for later use. (2) Add 10% green tea juice, 12% white sugar, 0.15% citric acid, 0.025% potassium sorbate and food additives to the clear paste, and prepare it into a commercially acceptable compound beverage according to the conventional beverage preparation method.

[0045] Example 4: Tablet Preparation Ingredients: 14g of *Gnaphalium affine*, 12g of *Chamaegu*, and 3g of Brazilian ginseng.

[0046] Preparation method: (1) Add 8 times the total weight of the raw materials of purified water, heat and reflux for 2 hours, filter and collect the filtrate; add 10 times the amount of purified water to the filter residue, heat and reflux for 1.5 hours, filter; combine the two filtrates, filter and concentrate into a clear paste for later use.

[0047] (2) Dry the ointment, add an appropriate amount of excipients, mix well, and compress into tablets.

[0048] Example 5: Capsule Preparation Ingredients: 14g of *Gnaphalium affine*, 12g of *Chamaegu*, and 3g of Brazilian ginseng.

[0049] Preparation method: (1) Add 10 times the total weight of the raw materials of purified water, heat and reflux for 2 hours, filter and collect the filtrate; add 8 times the amount of purified water to the filter residue, heat and reflux for 1.5 hours, filter; combine the two filtrates, filter and concentrate into a clear paste for later use.

[0050] (2) Dry the paste, pulverize it, and fill it into capsules.

[0051] Example 6: Granule Preparation Ingredients: 14g of *Gnaphalium affine*, 12g of *Chamaegu*, and 3g of Brazilian ginseng.

[0052] Preparation method: (1) Add 10 times the total weight of the raw materials of purified water, heat and reflux for 2 hours, filter and collect the filtrate; add 10 times the amount of purified water to the filter residue, heat and reflux for 1.5 hours, filter; combine the two filtrates, filter and concentrate into a clear paste for later use.

[0053] (2) Add an appropriate amount of sucrose to the clear paste, make it into granules, and dry it to obtain the product.

[0054] Comparative Example 1: Preparation of *Gnaphalium affine* extract Raw material: 7g of *Gnaphalium affine* (this amount is the same as that of *Gnaphalium affine* in Example 1, for easy dosage comparison).

[0055] Preparation method: Refer to Example 1 to obtain the extract of *Gnaphalium affine*.

[0056] Application Example 1: Anti-fatigue effect of extracts from a combination of *Gnaphalium affine*, *Champa japonica*, and Brazilian ginseng. (1) Initial screening in experiments Rats were divided into three groups. Each group of rats had a 1.5g weight attached to its tail and were placed in a Morris-type water maze for a swimming test. The timing started when the rat entered the water and ended when the rat was taken out of the water five seconds after its nose was submerged and it could not resurface. The interval was recorded as an indicator of the rat's swimming endurance. The swimming endurance of the four groups of rats was statistically calculated. Rats were screened based on the absolute value of the difference between their swimming time and the average time, which was greater than twice the standard deviation. Rats with large differences in endurance were removed, and the remaining rats entered the experiment.

[0057] (2) Experimental grouping and drug administration Rats were randomly divided into four groups according to their body weight: blank group (physiological saline), Evodia rutaecarpa extract group (comparative example 1), combined extract group 1 (experimental example 1), and combined extract group 2 (experimental example 2). The corresponding drugs were administered by gavage according to their body weight.

[0058] (3) Fatigue exhaustion test Before drug administration, an adaptive weight-bearing swimming experiment was conducted for one day. Rats were loaded with a weight (made of wire) equal to 5% of their body weight at the base of their tails and swam in a Morris-type water maze at a depth of 30 cm and a temperature of 20 ± 2℃. Timing was started immediately after the rats were placed in the water, and their behavior at exhaustion was observed and recorded. The formal experiment began after the adaptive experiment. Before each experiment, the rats were fasted for 12 hours, weighed, and their weight adjusted. Thirty minutes after drug administration, the rats were placed in the water as described above. They were retrieved after sinking 5 seconds below the surface and failing to resurface, exhibiting the exhaustion behavior recorded earlier. This time was recorded as the exhaustion swimming time.

[0059] The results of the anti-fatigue exhaustion experiment are shown in Tables 1 and 2. On the first day, compared with the normal group, each drug administration group had no significant effect on the first exhaustion swimming time of rats. On the 10th day, the swimming time of the *Euonymus alatus* extract group was significantly longer than that of the blank group (P<0.05), indicating that *Euonymus alatus* itself has a certain anti-fatigue effect. The combined extract group 1 and combined extract group 2 significantly prolonged the exhaustion swimming time of rats compared with the blank group and the *Euonymus alatus* extract group (P<0.05), indicating that combined extract 1 and combined extract 2 can effectively combat fatigue and are more effective than single *Euonymus alatus* extract.

[0060] Table 1. Swimming exhaustion time (seconds, x̄±SD, n=8) of rats in each group after 10 days

[0061] Table 2. Comparison of swimming exhaustion time between groups on day 10 (seconds, x̄±SD, n=8)

[0062] Note: * indicates P<0.05, ** indicates P<0.01.

[0063] Application Example 2: Treatment of Ulcerative Colitis with Combined Extracts Mice were randomly divided into 6 groups according to body weight: control group, model group, mesalazine group, *Eriocaulon buergerianum* extract group, combined extract group 1, and combined extract group 2. After 7 days of acclimatization, mice in the control group were given free access to deionized water, while mice in the other groups were given free access to 3% sodium dextran sulfate solution for 7 consecutive days to induce an ulcerative colitis model. During the drug administration period, mice in the control group and model group were given an equal volume of physiological saline by gavage, while mice in the mesalazine group were given mesalazine (100 mg / kg) by gavage. -1 The extract groups (Comparative Example 1), combined extract group 1 (Experimental Example 1), and combined extract group 2 (Experimental Example 2) were administered the corresponding drugs by gavage according to body weight.

[0064] (1) The experimental results of treating ulcerative colitis were as follows: ① The mice in the blank group were in good physiological condition, showing that they were active, responsive, had smooth and glossy fur, regular eating and drinking behavior, normal fecal shape and healthy color, and stable weight gain. ② After the mice in the model group were induced to develop an ulcerative colitis model with sodium dextran sulfate, their general condition deteriorated significantly: the mice were lethargic, sluggish, less active and curled up; their fur became rough, dull, sparse and messy; their food intake and water intake decreased significantly; and they gradually developed diarrhea and visible bloody stools. ③ Compared with the model group, mice in the mesalazine group, the Eriocaulon buergerianum extract group, and the combined extract group 1 and combined extract group 2 showed a positive improvement trend after drug treatment: their mental state gradually recovered, their activity level and food intake increased, their fur gradually changed from sparse and dry to soft and shiny, and their fecal bleeding symptoms also gradually decreased; among them, compared with Eriocaulon buergerianum extract, mice in combined extract group 2 showed more obvious effects in terms of improvement in mental state, activity and food intake, fur condition, and fecal shape and color.

[0065] The results of the experiment on the general signs of ulcerative colitis in mice are as follows: Figure 1 As shown in the results, compared with the blank group, the DAI score of mice in the model group was significantly increased; compared with the model group, the DAI scores of mice in the *Euonymus alatus* extract group and combined extract group 1 and combined extract group 2 were decreased, and the combined extract group 1 and combined extract group 2 were lower than those in the *Euonymus alatus* extract group, with combined extract group 2 showing the best performance. This indicates that *Euonymus alatus* extract, combined extract 1, and combined extract 2 can alleviate the symptoms of ulcerative colitis in mice, and combined extract 1 and combined extract 2 have better therapeutic effects than *Euonymus alatus* extract.

[0066] (2) The results of the experiment on the effects of colon length and organ index in mice with ulcerative colitis are as follows: Figure 2 and Figure 3 As shown, the results indicated that compared with the blank group, the colon length of mice in the model group was significantly shortened; compared with the model group, the colon length of mice in the mesalazine group, the *Euonymus alatus* extract group, and the combined extract group 1 and combined extract group 2 was increased. Figure 2 and Figure 3 (A) Ulcerative colitis patients often experience extraintestinal complications, which can affect multiple organ systems, including splenomegaly. After modeling, the spleen and kidneys of mice in the model group became enlarged, and treatment with *Euonymus alatus* extract, combined extract 1, and combined extract 2 significantly improved the degree of spleen and kidney enlargement (P<0.05). Figure 2 B and Figure 3 (B and C in the text).

[0067] (3) The results of the experiment on the histopathology of colon tissue in mice with ulcerative colitis are as follows: Figure 4As shown, the results indicated that the colonic mucosa of mice in the blank control group was intact, with no inflammatory cell infiltration. In the model group, the colonic tissue of mice showed extensive ulceration, loss of mucosal epithelium, disappearance of intestinal glands, and replacement by connective tissue; the cytoplasm of myofibroblasts in the muscle layer was loose or vacuolated; the colonic mucosal epithelial structure was significantly damaged, with loose epithelial cell connections, deformed and reduced gland numbers, and destroyed and irregularly arranged crypt structures. The mucosal tissue structure loss was reduced in the *Euonymus alatus* extract group, combined extract group 1, and combined extract group 2, while the degree of inflammatory cell infiltration was alleviated. Furthermore, combined extract 1 and combined extract 2 showed a greater degree of improvement in the pathological changes of colonic tissue in mice with ulcerative colitis than using *Euonymus alatus* extract alone.

[0068] (4) The results of the experiment on the inflammatory response in mice with ulcerative colitis are as follows: Figure 5 As shown, the results indicated that compared with the blank group, the serum IL-6 and TNF-α levels in the model group mice were significantly increased, while the IL-10 level was significantly decreased. Compared with the model group, the serum TNF-α and IL-6 levels in the mesalazine group, the *Euonymus alatus* extract group, and the combined extract group 1 and combined extract group 2 were significantly decreased (P<0.05), and the serum IL-6 levels in each treatment group were significantly decreased compared with the model group (P<0.05). Compared with the model group, the serum IL-10 levels in the mesalazine group, the *Euonymus alatus* extract group, and the combined extract group 1 and combined extract group 2 were significantly increased (P<0.05).

[0069] Application Example 3: Anti-Kidney Yang Deficiency Effect of Combined Extracts SPF-grade male SD rats, 6 weeks old, weighing (200±20) g, were acclimatized for 3 days under a 20-23℃ temperature, 12h light cycle, and free access to water and food. On the 4th day, after 12 hours of fasting with free access to water, they were weighed and randomly divided into 6 groups of 8 rats each: blank group, model group, Gui Fu Di Huang Wan group, Eriocaulon buergerianum extract group, combined extract group 1, and combined extract group 2. Every morning, the Gui Fu Di Huang Wan group was administered a solution of Gui Fu Di Huang Wan by gavage (… The *Euonymus alatus* extract group (Comparative Example 1), combined extract group 1 (Experimental Example 1), and combined extract group 2 (Experimental Example 2) were administered the corresponding drugs via gavage according to body weight. Each drug group and model group received adenine solution via gavage (dosage...) every afternoon. The control group was administered physiological saline by gavage for a total of 21 days. Behavioral changes in rats were observed during the treatment period, and body weight was measured every 3 days, with dosage adjusted accordingly.

[0070] (1) The results of the experiment on the inflammatory response of rats with kidney yang deficiency are as follows: Figure 6As shown, the results indicated that compared with the blank group, the serum IL-6 and TNF-α levels of rats in the model group were increased, while the IL-10 level was decreased; compared with the model group, the serum IL-6 and TNF-α levels of mice in the Gui Fu Di Huang Wan group, the Evodia rutaecarpa extract group, and the combined extract group (1 and 2) were decreased; compared with the model group, the serum IL-10 level of mice in the Gui Fu Di Huang Wan group, the Evodia rutaecarpa extract group, and the combined extract group (1 and 2) was increased, which is consistent with the treatment of ulcerative colitis.

[0071] (2) The effects of the drug on improving kidney-yang deficiency syndrome were evaluated by detecting the serum levels of adrenocorticotropic hormone (ACTH), corticosterone (CORT), estradiol (E2), testosterone (T), and thyroxine (T4) in each group of rats. The results are as follows: Figure 7 As shown, compared with the blank group, the levels of ACTH, CORT, T, and T4 in the model group rats were significantly reduced, while the level of E2 was increased, consistent with the pathological characteristics of endocrine disorders in kidney-yang deficiency syndrome. The intervention with *Euonymus alatus* extract (Comparative Example 1) had a relatively mild effect on improving the levels of various hormones. In contrast, both combined extract group 1 (Experimental Example 1) and combined extract group 2 (Experimental Example 2) could increase the levels of ACTH, CORT, E2, T, and T4 to varying degrees. Among them, combined extract group 2 showed better regulatory effects on most indicators, and its overall effect was better than that of *Euonymus alatus* single extract. This indicates that the combined use of *Euonymus alatus*, *Chamaecyparis edulis*, and Brazilian ginseng has a synergistic effect on improving kidney-yang deficiency syndrome, and can effectively regulate the functions of the hypothalamus-pituitary-adrenal axis, gonadal axis, and thyroid axis, with combined extract group 2 showing a more significant effect.

[0072] (3) The effects of *Euonymus alatus* extract on the pathology of the kidney, testis, and epididymis in rats with kidney-yang deficiency are as follows: Figure 8 , Figure 9 , Figure 10 As shown, the results are as follows: ① Kidney: In the blank group, the kidney tissue structure of rats was clear, the boundary between the cortex and medulla was relatively clear, the renal tubular structure was relatively intact, no obvious degeneration or necrosis was observed, and there was no congestion or inflammatory cell infiltration in the interstitium; in the model group, the pathological sections showed obvious dilation of the renal tubules, flattened renal tubular epithelial cells, enlarged renal tubular lumen, partial renal tubular necrosis, and a large amount of fibrous tissue proliferation in the interstitium, interspersed with inflammatory cell infiltration; in the pathological sections of the combined extract group 1 and combined extract group 2, there was only a small amount of renal tubular necrosis and slight fibrous tissue proliferation in the interstitium; in the *Euonymus alatus* extract group, the renal tubules were more significantly dilated, some renal tubules were necrotic, a small amount of fibrous tissue proliferation in the interstitium, accompanied by inflammatory cell infiltration. Figure 8 ).

[0073] ② Testis: In the blank group, the seminiferous tubule basement membrane of rats was intact, the spermatogenic cells at all levels were regularly arranged, blood vessels were abundant, and interstitial cells were well developed. A large number of sperm were visible near the lumen. In the model group, most seminiferous tubules were significantly thinner in diameter, with wider spacing, thinner seminiferous epithelium, and disordered arrangement of spermatogenic cells, with reduced layers and numbers, indicating that adenine caused testicular tissue damage. In the *Euonymus alatus* extract group and the combined extract group of *Euonymus alatus*, *Chamaecyparis* and Brazilian ginseng, the seminiferous tubule basement membrane was thickened, the seminiferous epithelium was thinned, the spermatogenic cells were regular, a few epithelial cells were separated from the base, sloughed spermatogenic cells were visible in the lumen, some supporting cells were separated from spermatogenic cells, and mature sperm were visible in the lumen. This indicates that *Euonymus alatus* extract and combined extract group 1 and combined extract group 2 can improve the pathological state of the rat testis. Figure 9 ).

[0074] ③ Epididymis: In the blank group, the epididymal ducts of rats were tightly and regularly arranged, with a large number of sperm evenly distributed within the lumen. In the model group, the epididymal duct walls were thickened, loosely arranged, with enlarged interstitium, and the lumen showed hyperplasia and atrophy. Sperm within the lumen aggregated into clusters, indicating that adenine significantly affected the physiological function of the epididymis. The thickening of the epididymal duct walls was significantly improved in the *Euonymus alatus* extract group and the combined extract groups 1 and 2, with tighter arrangement and increased, evenly distributed sperm within the lumen. This indicates that the combined extract can improve the pathological state of the rat epididymis. Figure 10 ).

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An extract composed of *Gnaphalium affine*, *Chamaegu*, and Brazilian ginseng, characterized in that... It is composed of the following raw materials in parts by weight: 7-21 parts of Evodia rutaecarpa, 6-18 parts of Chamaegu, and 1-3 parts of Brazilian ginseng.

2. The extract of *Gnaphalium affine*, *Gnaphalium affine*, and Brazilian ginseng as described in claim 1, characterized in that... 10-14 parts by weight of *Gnaphalium affine*, 8-12 parts by weight of *Chamaegu*, and 1.5-2.5 parts by weight of Brazilian ginseng.

3. The extract of *Gnaphalium affine*, *Champa japonica*, and Brazilian ginseng as described in claim 1, characterized in that... 14 parts by weight of *Gnaphalium affine*, 12 parts by weight of *Chamma jasminoides*, and 3 parts by weight of Brazilian ginseng.

4. The extract of *Gnaphalium affine*, *Champa japonica*, and Brazilian ginseng as described in claim 1, characterized in that... 7 parts by weight of *Gnaphalium affine*, 6 parts by weight of *Chamma jasminoides*, and 1 part by weight of Brazilian ginseng.

5. The extract of *Gnaphalium affine*, *Champa japonica*, and Brazilian ginseng as described in claim 1, characterized in that... The preparation method of the extract of the combination of *Gnaphalium affine*, *Gnaphalium affine*, and Brazilian ginseng includes the following steps: The raw materials, by weight, are 7-21 parts of *Gnaphalium affine*, 6-18 parts of *Chamaegu*, and 1-3 parts of Brazilian ginseng, mixed and then crushed into coarse powder. Add 8-12 times the total weight of the raw materials to water, heat and reflux for extraction 1-3 times, 1-2 hours each time, and collect the extract from each heating and reflux extraction; combine the extracts, filter, and concentrate the filtrate under reduced pressure at 60-80℃ to a clear extract with a relative density of 1.10-1.25; The extract is vacuum dried or freeze-dried, then pulverized and sieved to obtain the combined extract of *Gnaphalium affine*, *Gnaphalium affine*, and Brazilian ginseng.

6. The extract of *Gnaphalium affine*, *Champa japonica*, and Brazilian ginseng as described in claim 5, characterized in that... The extraction was performed twice: the first extraction was performed with 10 times the amount of water for 2 hours, and the second extraction was performed with 8 times the amount of water for 1.5 hours.

7. The extract of *Gnaphalium affine*, *Champa japonica*, and Brazilian ginseng as described in claim 6, characterized in that... The extracts of *Gnaphalium affine*, *Gnaphalium affine*, and Brazilian ginseng are also formulated with pharmaceutically or food-grade excipients.

8. The extract of *Gnaphalium affine*, *Champa japonica*, and Brazilian ginseng as described in claim 7, characterized in that... Extracts from a combination of *Gnaphalium affine*, *Gnaphalium affine*, and Brazilian ginseng can be made into tablets, capsules, powders, mixtures, pills, granules, solutions, syrups, decoctions, beverages, biscuits, candies, or pastries for pharmaceutical or food applications.

9. The use of the combined extract of *Gnaphalium affine*, *Gnaphalium affine* and Brazilian ginseng as described in any one of claims 1-8 in the preparation of products having anti-fatigue, anti-ulcerative colitis and / or anti-kidney yang deficiency effects.

10. The application as described in claim 9, characterized in that, Products include pharmaceuticals, health supplements, or food.

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