Application of anming niuhuang in preparation of drugs for preventing or treating melanoma

Abstract

This invention relates to the field of pharmaceutical technology, specifically to the novel use of Angong Niuhuang in the preparation of drugs for the prevention or treatment of melanoma. The Angong Niuhuang is composed of eleven traditional Chinese medicines: bezoar, buffalo horn, musk or artificial musk, pearl, cinnabar, realgar, Coptis chinensis, Scutellaria baicalensis, Gardenia jasminoides, Curcuma longa, and borneol. Animal experiments have shown that Angong Niuhuang can significantly inhibit tumor growth, reduce the expression levels of tumor markers MMP-2, MMP-9, and VEGF, and improve routine blood tests and blood biochemical indicators in mice, without significant toxic side effects. It can inhibit the epithelial-mesenchymal transition (EMT) process by downregulating immune escape-related factors such as PD-L1, TGF-β, and CTLA-4, and regulating the expression of molecules such as E-cadherin and N-cadherin, thereby delaying tumor metastasis.

Description

Technical Field

[0001] The present invention relates to the field of medicine, in particular to technologies related to tumor therapy. Specifically, the present invention relates to the application of Angong Niuhuang in the treatment of melanoma, especially its inhibition of the diffusion and migration of melanoma cells via an external administration route Background Art

[0003] The main inducement of melanoma is closely related to ultraviolet (UV) radiation. Ultraviolet exposure is the most common environmental factor for melanoma, and in particular, ultraviolet B (UVB) radiation can directly damage the DNA of skin cells, induce mutations, and promote the carcinogenesis of melanocytes through long-term accumulation. In addition, genetic factors are also an important cause of melanoma. For example, people with a family history of melanoma or people with certain specific gene mutations (such as CDKN2A, BRAF, NRAS, etc.) have a significantly increased risk of developing melanoma. Age, gender, skin type (such as light skin), immune status, etc. are also factors affecting the onset of melanoma.

[0004] At present, the main treatment methods for melanoma include surgical treatment, radiotherapy, chemotherapy, immunotherapy, targeted therapy and the like. Surgical treatment is the main treatment method for early-stage melanoma, which achieves the purpose of cure by resecting the tumor and its surrounding healthy tissues. However, with the progression of the tumor, especially when the tumor metastasizes to other parts, surgery often cannot achieve a complete cure. For advanced melanoma, immune checkpoint inhibitors (such as PD-1 and PD-L1 inhibitors) and targeted therapies (such as BRAF and MEK inhibitors) have become important therapeutic advances in recent years, which can effectively prolong the survival period of patients. Chemotherapy has limited application effects in melanoma and has relatively large side effects, so it is usually used as an adjuvant therapy. Radiotherapy is mainly used for tumors that cannot be surgically resected or for relieving symptoms.

[0005] However, although modern medicine has made some progress in the treatment of melanoma, especially in immunotherapy, the therapeutic effect for advanced melanoma is still unsatisfactory, and most treatment methods have certain problems of side effects and drug resistance. Therefore, exploring more effective and safer treatment methods is still an important direction of current melanoma research.

[0006] Metastasis is a leading cause of death in melanoma, and the process involves the regulation of multiple cellular signaling pathways. Initially, the mechanism of melanoma metastasis was thought to be a simple entry of cancer cells from the primary site into the bloodstream or lymphatic system. However, recent studies have revealed that melanoma metastasis is a complex, multi-stage process involving multiple steps such as cancer cell escape, invasion, spread, and colonization in distant tissues. Several signaling pathways play important roles in melanoma metastasis, including the matrix metalloproteinase (MMP) pathway, the MAPK signaling pathway, the PI3K / Akt signaling pathway, the TGF-β pathway, and the Wnt / β-catenin signaling pathway.

[0007] Matrix metalloproteinases (MMPs) are a class of enzymes that degrade the extracellular matrix (ECM) and are involved in the invasion and metastasis of tumor cells. MMPs enable tumor cells to breach the basement membrane and enter blood vessels or lymphatic vessels by breaking down components of the ECM (such as collagen and elastin), thereby metastasizing to distant organs. Studies have shown that the overexpression of MMPs is closely related to tumor invasiveness and metastasis.

[0008] The MAPK pathway is one of the important signaling pathways regulating cell proliferation, differentiation, and migration, playing a crucial role, especially in the metastasis of tumor cells. The Ras / Raf / MEK / ERK signaling pathway is a classic MAPK pathway, playing a vital role in the proliferation, invasion, and metastasis of melanoma cells. Activation of this pathway enhances the metastatic ability of tumor cells by regulating cell motility, adhesion, and metabolism.

[0009] The PI3K / Akt signaling pathway regulates cell survival, proliferation, migration, and invasion. In melanoma, activation of the PI3K / Akt pathway is often associated with increased tumor cell metastasis, drug resistance, and alterations in the tumor microenvironment. Upregulation of the PI3K / Akt pathway can promote tumor cell migration and invasion, and enhance tumor cell tolerance to chemotherapy drugs.

[0010] Currently, the pathways related to melanoma metastasis have become a hot topic in oncology research, especially in areas such as matrix metalloproteinases (MMPs), the MAPK signaling pathway, and the PI3K / Akt pathway, where significant progress has been made.

[0011] Studies have shown that melanoma melanin-producing macrophages (MMPs) not only play a crucial role in the metastasis of melanoma but are also closely related to the malignancy and metastatic potential of the tumor. By inhibiting the activity of MMPs, researchers have found that this can significantly reduce the invasiveness and metastasis of melanoma and delay tumor progression. Inhibitors of MMPs, such as peptide and small molecule inhibitors, are undergoing clinical trials and have shown potential therapeutic efficacy.

[0012] The role of the MAPK signaling pathway in tumor cell proliferation, differentiation, and metastasis is increasingly recognized. Studies have shown that activation of the MAPK pathway promotes tumor cell metastasis by encouraging melanoma cells to infiltrate blood vessels and lymphatic vessels. Inhibitors targeting the MAPK pathway (such as MEK inhibitors) have demonstrated efficacy in clinical trials for patients with advanced melanoma, but drug resistance remains a significant challenge that needs to be addressed.

[0013] Studies on the PI3K / Akt pathway have shown that inhibiting this pathway can significantly reduce the proliferation and metastasis of melanoma cells. Inhibitors of the PI3K / Akt pathway are already being tested in clinical trials, and some drugs, such as BKM120 and GDC-0980, can significantly inhibit melanoma metastasis and improve patient survival rates.

[0014] Despite the significant progress these studies have made in suppressing melanoma metastasis, the efficacy of single-target therapies remains limited due to challenges such as drug resistance, drug side effects, and other immune escape mechanisms. Therefore, combination therapy strategies, especially those incorporating traditional Chinese medicine compound therapies, have become a current research focus.

[0015] In order to inhibit melanoma metastasis, interventions based on the MAPK, MMPs, and PI3K / Akt signaling pathways are gradually becoming a new direction in the treatment of melanoma. Modern medicine mainly uses inhibitors targeting these pathways to intervene in tumor metastasis. For example, MEK inhibitors (such as trametinib) and BRAF inhibitors (such as dabrafenib) have achieved certain clinical efficacy, especially for melanoma patients with BRAF mutations.

[0016] However, single-target therapies often face challenges such as drug resistance and side effects. Therefore, current research is beginning to combine traditional Chinese medicine with modern targeted therapies, aiming to further inhibit melanoma metastasis through multi-target effects. The effects of traditional Chinese medicine in regulating the immune system, anti-oxidation, and anti-inflammation can provide a new treatment option for melanoma patients, especially in improving efficacy and reducing side effects. Combining traditional Chinese medicine with modern targeted therapy to inhibit melanoma metastasis has become an important direction for future melanoma treatment.

[0017] The combination of traditional Chinese medicine (TCM) and modern medical treatments can improve treatment outcomes, reduce side effects, and enhance patients' quality of life. Regarding tumor metastasis and drug resistance, TCM can exert its effects through multiple targets and pathways, regulating the cancer cell microenvironment and inhibiting cancer cell migration and metastasis, thus providing new treatment options for melanoma patients.

[0018] As a classic traditional Chinese medicine compound, Angong Niuhuang Pill is widely used in the field of traditional Chinese medicine. Its main components include bezoar, musk, pearl, borneol, etc., and it has multiple pharmacological effects such as clearing heat and detoxifying, calming convulsions and tranquilizing, anti-inflammation, and anti-oxidation. Its pharmacological studies show that Angong Niuhuang Pill can produce positive therapeutic effects on a variety of diseases by regulating the immune system, inhibiting inflammatory responses, and reducing oxidative stress. However, the application of Angong Niuhuang Pill in melanoma treatment has not been reported yet. Therefore, exploring the application of Angong Niuhuang in melanoma treatment has important clinical significance and scientific research value. Contents of the Invention

[0019] The present invention provides a new use of Angong Niuhuang in the preparation of a medicament for treating melanoma. The prescription used is composed of eleven raw medicinal materials including bezoar, concentrated buffalo horn powder, musk or artificial musk, pearl, cinnabar, realgar, coptis, scutellaria, cape jasmine, turmeric root-tuber, and borneol. In the prescription, bezoar and concentrated buffalo horn powder are used as monarch drugs, which have the effects of clearing heat, detoxifying, cooling blood and resolving toxins; musk and borneol are used as minister drugs, which can induce resuscitation and refresh the mind, and improve local circulation; supplemented with pearl and cinnabar to calm the mind and tranquilize, scutellaria and cape jasmine to clear heat and cool blood, coptis to resist inflammation and detoxify, turmeric root-tuber to regulate qi and resolve dampness, and realgar to kill parasites and detoxify. The combined use of all the medicines synergistically achieves the comprehensive effects of clearing heat and detoxifying, dissipating tumor toxins, and inhibiting metastasis.

[0020] The present invention establishes a mouse model of transplanted melanoma and adopts external intervention with Angong Niuhuang, and finds that the agent can significantly inhibit the increase of tumor volume and weight, showing good anti-tumor activity. The results of blood routine and biochemical index analysis show that Angong Niuhuang can effectively improve the immune function and general state of melanoma mice, and does not cause obvious toxic and side effects, reflecting good safety. Further detection by ELISA and RT-qPCR techniques shows that Angong Niuhuang can significantly down-regulate the expression of molecular markers related to tumor metastasis, including angiogenesis and matrix degradation factors such as MMP-2, MMP-9 and VEGF, inhibit the expression of epithelial-mesenchymal transition-related molecules such as N-cadherin, increase the expression of E-cadherin, and reduce the levels of immune escape-related molecules such as PD-L1 and TGF-β, suggesting that it may exert a comprehensive therapeutic effect by inhibiting tumor metastasis and improving the immune microenvironment. In summary, the present invention firstly clearly proposes the new use of Angong Niuhuang in the treatment of melanoma, which is especially suitable for tumor suppression and anti-metastasis treatment of melanoma, has good pharmacological activity, safety and clinical transformation prospect for external local use, and can provide effective traditional Chinese medicine intervention strategies and technical support for the comprehensive treatment of melanoma. Brief Description of the Drawings

[0021] Figure 1 . Effects of Angong Niuhuang on tumor volume and weight of mice subcutaneously transplanted with melanoma cells. Compared with the control group, **** P < 0.0001,*** p < 0.001 ** P < 0.01, * P < 0.05 ns No significant difference.

[0022] Figure 2 Effects of Angong Niuhuang on tumor markers in a mouse model of melanoma. Compared with the control group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 ns No significant difference.

[0023] Figure 3 Effects of Angong Niuhuang on white blood cells, red blood cells, platelets, LDH, AST, and ALT in a melanoma model mouse. Compared with the control group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 ns No significant difference.

[0024] Figure 4 Effects of Angong Niuhuang on the protein expression of metastasis-related markers MMP-2, MMP-9, E-cadherin, and N-cadherin in a melanoma model mouse. Compared with the control group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 s No significant difference.

[0025] Figure 5 Effects of Angong Niuhuang on the transcriptional expression of N-cadherin, MMP-9, E-cadherin, MMP-2, PD-L1, and TGF-β in melanoma model mice. Compared with the control group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 ns No significant difference. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1: The intervention effect of Angong Niuhuang on a mouse model of melanoma xenograft.

[0028] 1. Experimental Objective

[0029] This experiment aimed to evaluate the interventional effect of Angong Niuhuang on a melanoma transplantation model mouse, observing its influence on tumor size, metastasis, and related tumor markers under topical administration. The main objective was to preliminarily explore the inhibitory effect of Angong Niuhuang on melanoma through a simple tumor intervention efficacy assessment, and to evaluate its anti-tumor potential by detecting changes in tumor markers using ELISA.

[0030] 2. Experimental Materials

[0031] Experimental animals: Male Balb / c mice aged 8-10 weeks were purchased from the experimental animal center and randomly divided into five groups (6 mice per group): normal group, control group, low-dose Angong Niuhuang group (125 mg / mL), medium-dose Angong Niuhuang group (250 mg / mL), and high-dose Angong Niuhuang group (500 mg / mL).

[0032] Experimental cells: B16-F10 melanoma cells, purchased from a cell bank, cultured using RPMI-1640 containing 10% fetal bovine serum, and cultured at 37°C and 5% CO2.

[0033] Angong Niuhuang: Prepare Angong Niuhuang powder according to the prescription ratio of Angong Niuhuang Pill in the Chinese Pharmacopoeia, and then dissolve it with 0.9% sodium carboxymethyl cellulose to obtain Angong Niuhuang suspension.

[0034] Tumor marker detection: ELISA kits are used to detect tumor markers in the blood, such as S100 and MIA.

[0035] 3 Experimental Methods

[0036] 3.1 Establishment of a melanoma model

[0037] B16-F10 cells were cultured to the logarithmic growth phase, and the cell suspension was adjusted to 1×10⁻⁶ with PBS. 6 Cells / mL. Each mouse was subcutaneously injected with 100 μL of cell suspension (1 × 10⁻⁶ cells / mL). 6 A mouse model of melanoma was established by placing 10 cells in the right abdomen.

[0038] After waiting approximately 7 days for the tumor to form (the tumor size is approximately 100 mm) 3 Intervention was initiated. Treatment was administered in groups: the normal group received no treatment, the control group received an equal volume of physiological saline, and the Angong Niuhuang group received low, medium, and high doses.

[0039] Apply Angong Niuhuang Suspension externally to the tumor site at a rate of 0.2 mL / tumor / day for 21 consecutive days.

[0040] 3.2 Tumor volume measurement

[0041] Measure the tumor's long and short diameters with calipers every 3 days and calculate the tumor volume: Tumor volume = (long diameter × short diameter) 2 ) / 2. At the end of the experiment, the mice were euthanized and dissected to observe the size of the tumor and record its weight.

[0042] 3.3 ELISA detection of tumor markers

[0043] Peripheral blood samples were collected from experimental mice, and serum tumor markers, such as S100 and MIA, were measured using an ELISA kit to assess the effect of Angong Niuhuang on tumor-related markers. The ELISA experiment was performed according to the manufacturer's instructions.

[0044] 3.4 Complete blood count and blood biochemistry analysis

[0045] After the experiment, blood samples were collected from mice and routine blood tests were performed using a fully automated blood analyzer. Blood biochemical analysis included indicators such as ALT, AST, ALP, LDH, and blood glucose to assess the effects of Angong Niuhuang on the physiological state of mice.

[0046] 3.5 Statistical Analysis

[0047] The experimental results were statistically analyzed using SPSS software. One-way ANOVA was used for comparisons between groups, and the significance level was set at P < 0.05.

[0048] 4. Experimental Results

[0049] 4.1 Tumor growth status

[0050] Twenty-one days after administration, tumor volume increased in all groups of mice, but there were significant differences between the different treatment groups. The tumor volume increased the fastest in the normal group and the control group, while the tumor volume growth rate was significantly slowed in the low, medium, and high dose groups of Angong Niuhuang.

[0051] The high-dose group of mice showed the smallest tumor growth, with a tumor volume of (405±35) mm. 3 The value was significantly smaller than that of the control group (892±80) mm. 3(P < 0.01). The tumor volume in the medium-dose group of mice was (654 ± 50) mm. 3 The tumor volume in the low-dose group was (765±60) mm. 3 Both were significantly lower than the control group.

[0052] 4.2 Tumor weight

[0053] The tumor weight in the high-dose group was (1.05±0.08) g, significantly lower than that in the control group (2.27±0.12) g (P<0.01). The tumor weights in the medium-dose and low-dose groups were (1.57±0.10) g and (1.91±0.14) g, respectively, also significantly lower than those in the control group (P<0.05). These data indicate that Angong Niuhuang can effectively inhibit tumor growth and weight gain.

[0054] 4.3 Tumor marker ELISA detection

[0055] In the detection of tumor markers, the serum levels of S100 and MIA in the high-dose group mice were significantly lower than those in the control group. Specifically, the S100 level in the high-dose group mice was (98.5±8.7) pg / mL and the MIA level was (145.3±12.4) pg / mL, while the levels in the control group were (182.3±14.5) pg / mL and (236.8±18.2) pg / mL, respectively, and the differences were statistically significant (P<0.01).

[0056] The levels of S100 and MIA in the medium-dose and low-dose groups were also lower than those in the control group, but the differences were relatively small, indicating that the high-dose Angong Niuhuang had a more significant inhibitory effect on tumor markers.

[0057] 4.4 Complete Blood Count and Blood Biochemistry Analysis

[0058] Blood routine analysis showed that there were no significant changes in white blood cell count and red blood cell count in the high-dose group of mice, indicating that Angong Niuhuang had little effect on the immune system of mice.

[0059] Blood biochemical analysis results showed that the high-dose Angong Niuhuang group had a relatively mild effect on indicators such as LDH, AST, and ALT. In contrast, the low-dose and medium-dose groups showed some improvement in certain biochemical indicators, but none reached the levels of the control group, indicating that Angong Niuhuang helps reduce damage to tissues such as the liver and muscles.

[0060] 4.5 Tumor Histological Analysis:

[0061] Examination of the dissected tumor tissues shows that the tumor tissues in the high-dose group present obvious necrotic areas, and the tissue structure disorder and cell proliferation are significantly slowed down compared with the tumor tissues in the control group. Histological scores show that the proliferative activity of tumor cells in the high-dose group is significantly reduced, demonstrating a tumor inhibitory effect.

[0062] The tumor tissues of mice in the low- and medium-dose groups also show partial necrosis and a trend of slowed proliferation, indicating that Angong Niuhuang has certain anti-tumor activity, with the most significant effect observed in the high-dose group.

[0063] 5 Conclusion

[0064] The experimental results show that Angong Niuhuang exerts a significant anti-tumor effect in the treatment of melanoma, and tumor growth is significantly inhibited especially in the high-dose group. Angong Niuhuang can not only effectively inhibit the increase of tumor volume and weight, but also reduce the levels of tumor markers (S100 and MIA) in blood. Tumor histological analysis also shows that Angong Niuhuang has a certain inhibitory effect on tumor tissues, and particularly in the high-dose group, it exhibits strong anti-tumor activity. These results support the potential of Angong Niuhuang in melanoma treatment, and further provide experimental evidence for its clinical application.

[0065] Example 2: Interventional effect of Angong Niuhuang in a subcutaneous xenograft tumor model of melanoma cells

[0066] 1. Experimental objective

[0067] This experiment aims to explore the pharmacological mechanism of Angong Niuhuang on melanoma metastasis, with focus on analyzing its effects by inhibiting metastasis-related surface markers, matrix metalloproteinases (MMPs) and immune escape molecules, so as to further reveal the inhibitory effect of Angong Niuhuang on melanoma metastasis.

[0068] 2 Experimental materials

[0069] Mouse model: The melanoma xenograft model mice in Example 1 are used. The experimental mice are 6-8 week old male BALB / c nude mice.

[0070] Tumor cells: B16F10 melanoma cell line.

[0071] The drug preparation is the same as that in Example 1, including the high-dose group (200 mg / mL), the medium-dose group (100 mg / mL), and the low-dose group (50 mg / mL).

[0072] Reagents: ELISA kits are used for detecting tumor markers and metastasis-related indicators; RT-qPCR kits are used for analyzing the mRNA expression of metastasis-related surface markers, MMPs and immune escape molecules.

[0073] 3 Experimental methods

[0074] 3.1 Establishment of mouse model

[0075] Referring to the mouse model method in Example 1, a melanoma transplantation model was established by subcutaneous injection of B16F10 melanoma cell suspension.

[0076] Mice were divided into five groups: normal group, control group, high-dose group (200 mg / kg), medium-dose group (100 mg / kg), and low-dose group (50 mg / kg).

[0077] Intervention treatment began 7 days after tumor establishment in the transplanted mice. Mice were administered Angong Niuhuang Suspension by gavage once daily for 21 consecutive days. The control group received physiological saline.

[0078] At the end of the experiment, the mice were euthanized and tumor tissue was collected for further analysis.

[0079] 3.2 Analysis of transfer markers and MMPs

[0080] The levels of transfer-related surface markers (such as E-cadherin, N-cadherin, etc.) and MMPs (such as MMP-2, MMP-9) in mouse serum were detected using an ELISA kit.

[0081] 3.2 RT-qPCR analysis

[0082] RNA was extracted from tumor tissues, and the mRNA expression of metastasis-related surface markers, matrix metalloproteinases (MMP-2, MMP-9), and immune escape molecules (such as PD-L1, TGF-β) was analyzed by RT-qPCR.

[0083] 4 Experimental Results

[0084] 4.1. Analysis of transfer markers and MMPs

[0085] The levels of transfer-related surface markers and matrix metalloproteinases (MMP-2 and MMP-9) in serum were detected using an EISA kit. The results showed that compared with the normal group and the Angong Niuhuang intervention group, the serum levels of MMP-2 and MMP-9 in the model group mice were significantly increased (P < 0.05). However, in the high-dose and medium-dose Angong Niuhuang groups, the levels of MMP-2 and MMP-9 were significantly decreased (P < 0.01), and the high-dose group showed a more significant inhibitory effect than the medium- and low-dose groups.

[0086] The expression of transfer-related biomarkers E-cadherin and N-cadherin also showed a similar trend. In the model group, the level of E-cadherin decreased significantly and the level of N-cadherin increased significantly, while in the Angong Niuhuang intervention group, the expression of E-cadherin increased and the expression of N-cadherin decreased, especially in the high-dose group.

[0087] 4.2. RT-qPCR analysis

[0088] RT-qPCR results showed that the gene expression of metastasis-related surface markers was significantly altered in tumor tissues of mice treated with Angong Niuhuang. The mRNA expression levels of N-cadherin and MMP-9 in the tumor tissues of the model group mice were significantly higher than those in the normal group (P<0.05), while the mRNA expression levels of E-cadherin and MMP-2 were significantly lower than those in the normal group (P<0.05). In the Angong Niuhuang intervention group, the high-dose group showed significantly upregulated mRNA levels of E-cadherin and MMP-2, and significantly downregulated mRNA levels of N-cadherin and MMP-9 in the tumor tissues of mice (P<0.01), while the medium- and low-dose groups showed relatively mild inhibitory effects.

[0089] Furthermore, the expression of immune escape molecules such as PD-L1 and TGF-β was also altered. The mRNA expression levels of PD-L1 and TGF-β in the model group were significantly higher than those in the normal group, while in the high-dose Angong Niuhuang group, the mRNA levels of PD-L1 and TGF-β were significantly decreased (P < 0.05). These results suggest that Angong Niuhuang inhibits melanoma metastasis by regulating related signaling pathways and may enhance the immune response by inhibiting the expression of immune escape molecules.

[0090] 5. Conclusion

[0091] Analysis using ELISA and RT-qPCR showed that Angong Niuhuang can effectively inhibit melanoma metastasis. Specifically, Angong Niuhuang significantly reduced the expression of tumor metastasis-related surface markers and matrix metalloproteinases (MMP-2 and MMP-9), upregulated E-cadherin expression, and downregulated N-cadherin expression. Angong Niuhuang also inhibited the expression of immune escape molecules PD-L1 and TGF-β, suggesting that it regulates the melanoma metastasis process through multiple mechanisms. Therefore, Angong Niuhuang has an inhibitory effect on melanoma metastasis, providing a theoretical basis for further clinical research.

Claims

1. The use of Angong Niuhuang in the preparation of drugs for treating melanoma, characterized in that, The Angong Niuhuang is made from eleven Chinese medicinal materials: bezoar, buffalo horn, musk or artificial musk, pearl, cinnabar, realgar, coptis, scutellaria, gardenia, turmeric, and borneol.

2. The use according to claim 1, characterized in that, The melanoma is a malignant tumor originating from the skin and is highly metastatic.

3. The use according to claim 1, characterized in that, Angong Niuhuang is applied topically to the tumor area on the body surface of a melanoma transplantation model mouse, simulating a clinical topical treatment method.

4. The use according to claim 1, characterized in that, Angong Niuhuang inhibits the invasiveness and angiogenesis of melanoma by downregulating the expression of metastasis-related proteins MMP-2 and MMP-9 and angiogenesis factor VEGF.

5. The use according to claim 1, characterized in that, Angong Niuhuang can downregulate the expression of immune escape-related factors PD-L1, TGF-β, and CTLA-4, and regulate the balance of E-cadherin and N-cadherin, thereby inhibiting the epithelial-mesenchymal transition (EMT) process.

6. The use according to claim 1, characterized in that, Angong Niuhuang significantly improved blood routine and blood biochemical indicators in melanoma transplantation model mice, including white blood cell, red blood cell, and platelet counts, as well as liver and kidney function indicators, without obvious toxic reactions.

7. The use according to claim 1, characterized in that, The Angong Niuhuang medicine can be prepared as a paste, patch, or ointment for external use on the skin surface.

8. The use according to claim 1, characterized in that, The Angong Niuhuang drug can be prepared as a solid dosage form, a semi-solid dosage form, and a liquid dosage form for oral administration.

9. The use according to any one of claims 1 to 8, characterized in that, The concentration range for external use of the prescribed Angong Niuhuang is 0.01g to 0.5g / mL; the dosage range for oral administration is 0.5-5g / 60kg / day.

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