Preparation and application method of traditional Chinese medicine composition for tumor immunotherapy combination and synergism

Abstract

The present application relates to pharmaceutical preparation technology, and aims to provide a preparation and application method of a traditional Chinese medicine composition for tumor immunotherapy synergism. The method comprises the following steps: freezing and grinding five grain insects, soaking in ethanol, and then freezing, crushing and drying again; extracting and concentrating gleditsia sinensis lindley alone; extracting and concentrating radix astragali, fried radix paeoniae alba, herba taraxaci, radix pinelliae, cox sanguis, radix ophiopogonis, rabdosia sasaoana, radix tetrastigme, and radix smilacifolia; mixing the two concentrated liquids, adding the five grain insect powder, and mixing uniformly to obtain the traditional Chinese medicine composition. The gleditsia sinensis lindley is extracted and concentrated alone, and then is prepared into a medicine together with the concentrated liquid of other medicinal materials, so that the loss of the medicine components can be avoided, and the effectiveness can be ensured. The traditional Chinese medicine is used to strengthen the body resistance and improve the constitution to improve the overall immunity. The traditional Chinese medicine composition is used as a synergist of the above single target tumor immunotherapy, can strengthen the body resistance and improve the constitution, enhance the tumor inhibition effect, and reduce the toxicity.

Description

Technical Field

[0001] This invention relates to drug preparation technology, and in particular to a method for preparing and applying a traditional Chinese medicine composition for synergistic effect in tumor immunotherapy. Background Technology

[0002] Cancer is currently one of the most difficult diseases to cure and remains one of the most challenging problems in modern medicine. For a long time, surgery, chemotherapy, and radiotherapy have significantly improved the survival rate of some cancer patients due to their remarkable anti-tumor effects, making them one of the first-line treatments for cancer. However, the cytotoxic effects associated with radiotherapy and chemotherapy can also affect normal cells, often disrupting the homeostasis of the patient's immune system and leading to a series of serious complications. This is the fundamental reason why cancer remains one of the most difficult diseases to cure. Therefore, further exploring safe and effective treatment methods has become a key direction in anti-cancer research.

[0003] Studies have shown that T cells become exhausted during tumor development. T cells that should be activated overexpress certain surface molecules that inhibit their activation, such as programmed death protein-1 and its ligand (PD-1 / PD-L1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), and lymphocyte activation gene-3 (LAG-3). These molecules are collectively known as immune checkpoints. Research indicates that immune checkpoints play a crucial role in immune regulation. Immune checkpoints play an important role in maintaining homeostasis and are highly expressed on the surface of tumor cells and various immune cells. The binding of immune checkpoints to their ligands can inhibit certain signaling pathways, thereby maintaining immune tolerance, which plays an important role in preventing autoimmune diseases. However, tumor cells often use these mechanisms to evade immune surveillance; this phenomenon is commonly referred to as "immune escape." Against this backdrop, researchers discovered that immune checkpoint inhibitors (ICIs) can prevent immune checkpoints from binding to their ligands or interfere with their functional activity after binding, thereby achieving an anti-tumor effect. This gave rise to the concept of immunotherapy. Immunotherapy enhances the body's ability to recognize tumor cells and utilizes the immune system to eliminate them. It has attracted widespread attention and is gradually becoming one of the main methods of anti-tumor treatment.

[0004] Immunotherapy offers a promising alternative to cancer treatment by enhancing the patient's own immune system's ability to recognize and eliminate tumor cells while minimizing damage to normal cells and tissues. This advancement has brought new hope to cancer patients. In recent years, tumor immunotherapy has made significant progress, particularly in the application of immune checkpoint inhibitors (ICIs). ICIs inhibit the body's immune system by preventing excessive overactivity, thus allowing the immune system to become unchecked and prone to overreacting. However, clinical evidence suggests that PD-1 and CTLA-4 inhibitors have some significant limitations as monotherapy for cancer. These limitations include low objective tumor response rates, increased risk of secondary resistance, and potential adverse toxicity. Furthermore, studies combining multiple tumor immunotherapies to improve tumor suppression have not made significant progress due to increased side effects far outweighing any improvement in tumor suppression efficacy.

[0005] It is generally believed that traditional Chinese medicine (TCM) can only focus on reducing the toxic side effects of chemotherapy and radiotherapy. In the past, most research on combining TCM with anti-tumor therapy has focused on enhancing the efficacy and reducing the toxicity of TCM in combination with chemotherapy and radiotherapy. Although some progress has been made, its value is limited.

[0006] This invention aims to address the above problems by proposing a novel technical solution. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for preparing and applying a traditional Chinese medicine composition for synergistic effect in tumor immunotherapy.

[0008] To solve the technical problem, the solution of the present invention is:

[0009] A method for preparing a traditional Chinese medicine composition for synergistic use in tumor immunotherapy is provided, comprising:

[0010] (1) In accordance with the requirements of the 2020 edition of the Chinese Pharmacopoeia, take 15-60 parts of Astragalus membranaceus, 5-20 parts of Gleditsia sinensis thorns, 5-24 parts of stir-fried Paeonia lactiflora, 10-40 parts of Taraxacum mongolicum, 6-24 parts of Pinellia ternata, 15-60 parts of Coix lacryma-jobi, and 6-30 parts of Ophiopogon japonicus; in accordance with the requirements of the 2015 edition of the Zhejiang Province Traditional Chinese Medicine Processing Standards, take 5-50 parts of Cordyceps militaris, 15-60 parts of Achyranthes bidentata, 10-40 parts of Clematis armandii, and 15-60 parts of Actinidia chinensis root; the parts mentioned are by weight.

[0011] (2) After freezing and grinding the grain insects into powder, soak them in 75% ethanol for 24 hours; after recovering the ethanol under reduced pressure, freeze and pulverize the solid again, and dry it for later use.

[0012] (3) Take soapberry thorns, add water and heat to boil twice to extract; combine the two extracts and concentrate to obtain soapberry thorn water extract concentrate;

[0013] (4) Take Astragalus membranaceus, stir-fried white peony root, dandelion, ginger-processed Pinellia ternata, coix seed, Ophiopogon japonicus, fragrant tea leaves, three leaves green, and Actinidia chinensis root, combine them, add water, heat and boil to extract twice; combine the two extracts and concentrate to obtain a concentrated water extract of multiple medicinal materials;

[0014] (5) Mix the two water extract concentrates, then add the five grain insect powder and mix evenly to obtain the traditional Chinese medicine composition.

[0015] The present invention further provides a method for preparing a drug for synergistic use in tumor immunotherapy, which involves adding excipients to a traditional Chinese medicine composition prepared by the aforementioned method according to conventional pharmaceutical processing methods; and then preparing it into traditional Chinese medicine pills, capsules or tablets to obtain the drug.

[0016] The present invention also provides a method for using the drug prepared by the aforementioned method, which is to take the prepared Chinese medicine pills, capsules or tablets orally, with the daily dosage for adults being equivalent to 107g to 468g of the total raw drug.

[0017] To suit cancer patients diagnosed with Yin deficiency and excessive Yang according to Traditional Chinese Medicine, this invention provides an optimized adjustment scheme that removes Astragalus membranaceus, while the other manufacturing methods remain the same. Specifically:

[0018] A method for preparing a traditional Chinese medicine composition for synergistic use in tumor immunotherapy is provided, comprising:

[0019] (1) In accordance with the requirements of the 2020 edition of the Chinese Pharmacopoeia, take 5-20 parts of Gleditsia sinensis thorns, 5-24 parts of stir-fried Paeonia lactiflora, 10-40 parts of Taraxacum mongolicum, 6-24 parts of ginger-processed Pinellia ternata, 15-60 parts of Coix lacryma-jobi, and 6-30 parts of Ophiopogon japonicus; in accordance with the requirements of the 2015 edition of the Zhejiang Province Traditional Chinese Medicine Processing Standards, take 5-50 parts of five-grain worm, 15-60 parts of fragrant tea vegetable, 10-40 parts of Trifolium repens, and 15-60 parts of Actinidia chinensis root; the parts mentioned are by weight.

[0020] (2) After freezing and grinding the grain insects into powder, soak them in 75% ethanol for 24 hours; after recovering the ethanol under reduced pressure, freeze-crush the solid and dry it;

[0021] (3) Take soapberry thorns, add water and heat to boil twice to extract; combine the two extracts and concentrate to obtain soapberry thorn water extract concentrate;

[0022] (4) Take stir-fried white peony root, dandelion, ginger-processed pinellia, coix seed, ophiopogon japonicus, fragrant tea vegetable, three-leaf green, and vine pear root, combine them, add water, heat and boil to extract twice; combine the two extracts and concentrate to obtain a concentrated water extract of multiple medicinal materials;

[0023] (5) Mix the two water extract concentrates, then add the five grain insect powder and mix evenly to obtain the traditional Chinese medicine composition.

[0024] Further, excipients are added to the prepared traditional Chinese medicine composition according to conventional pharmaceutical processing methods; then, it is made into pills, capsules, or tablets to obtain the corresponding drug. For use by tumor patients diagnosed with yin deficiency and excessive fire according to traditional Chinese medicine, it is administered orally. The daily adult dosage is equivalent to 92g to 408g of the total raw herb.

[0025] In this invention, the traditional Chinese medicine pills, capsules, or tablets can be used in combination with tumor immunotherapy drugs to enhance the efficacy of tumor treatment; alternatively, the traditional Chinese medicine pills, capsules, or tablets can be used alone to treat tumors. The tumor immunotherapy drugs are any one of: programmed death protein-1, a ligand of programmed death protein-1, cytotoxic T-lymphocyte antigen 4, or lymphocyte activation gene-3.

[0026] The implementation principle of this invention:

[0027] During its long-term research on tumor immunotherapy, the applicant recognized that ICI monotherapy is equivalent to single-target tumor immunomodulatory therapy, while strengthening the body's constitution through traditional Chinese medicine (TCM) is equivalent to enhancing overall immunity, which can actually be considered a multi-target immunomodulatory therapy, thus significantly increasing the potential for tumor immunomodulatory enhancement. Based on this concept, this invention innovatively proposes using TCM to strengthen the body's constitution and enhance overall immunity, combining it as an synergist for the aforementioned single-target tumor immunotherapy. By strengthening the body's constitution and enhancing overall immunity, it enhances tumor-suppressing efficacy and reduces toxicity. This innovative approach demonstrates the important value of traditional Chinese medicine in comprehensive tumor treatment strategies and provides a new perspective for the future development of TCM in the field of anti-tumor research.

[0028] According to traditional Chinese medicine theory, tumors often occur due to deficiency of vital energy (decreased physical condition, insufficient immunity, diagnosed as qi and yin deficiency in TCM) and the accumulation of phlegm and blood stasis. Among these, deficiency of vital energy is the internal cause and the key factor; the principle of TCM treatment is to tonify qi and nourish yin, and strengthen the body to improve its constitution. This overall approach aims to improve the immune level and boost vital energy to expel pathogens.

[0029] According to modern medical theory, tumor development is related to impaired immune function in the body, specifically the inability to promptly eliminate and kill tumor cells. Theoretically, strengthening the immune system to effectively eliminate and kill tumor cells can alleviate or cure the tumor. Common tumor immunotherapy drugs (such as PD-1 / PD-L1, CTLA-4, and LAG-3) enhance tumor immune function by targeting specific specific targets (e.g., by antagonizing excessively high levels of immune prophylaxis).

[0030] The innovative approach of this invention is to enhance the overall immune level through a multi-target approach using a traditional Chinese medicine composition (boosting the body's overall vital energy to expel pathogens), thus providing synergy for single-target immune enhancement in tumor immunotherapy drugs. Based on years of clinical experience, the prescription has been repeatedly optimized and the usage improved, resulting in a significant improvement in the efficacy of immunotherapy drugs.

[0031] Based on traditional Chinese medicine theory and practice, Astragalus membranaceus (Huangqi) tonifies Qi. Combined with Gleditsia sinensis (Zaojiao) thorns, it is traditionally used in TCM surgery to treat patients with Qi deficiency and weakness in expelling toxins, thus achieving pus drainage. This invention creatively and successfully applies it to the treatment of tumors, and enhances tumor-suppressing effects when used in combination with immunomodulatory drugs. Gnaphalium affine (Wuguchong) is traditionally used to treat infantile malnutrition. This invention creatively and successfully applies it to improving the constitution of tumor patients (improving cachexia). Its synergistic effect with other Qi- and Yin-tonifying herbs (Astragalus membranaceus, Ophiopogon japonicus, and Paeonia lactiflora) further enhances its therapeutic effect. Astragalus membranaceus, combined with Ophiopogon japonicus and Paeonia lactiflora, tonifies both Qi and Yin; Pinellia ternata (Jingbanxia) combined with Coix lacryma-jobi (Yiyiren) eliminates phlegm and dampness; Taraxacum mongolicum (Pueraria lobata) combined with Clematis armandii (Sanyeqing) clears heat and detoxifies (anti-inflammatory); and Gleditsia sinensis thorns, combined with Actinidia chinensis root and Citrus aurantium (Xiangchacai), promote blood circulation and remove blood stasis. The entire formula, while strengthening the body's constitution, resolves phlegm and removes blood stasis, promotes tumor suppression and eliminates toxins, and significantly enhances the efficacy of immunomodulatory drugs.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] 1. It is generally believed that traditional Chinese medicine (TCM) can only focus on reducing the toxic side effects of chemotherapy and radiotherapy. Previous research combining TCM with anti-tumor therapies has largely concentrated on enhancing the efficacy and reducing the toxicity of TCM in conjunction with chemotherapy and radiotherapy. While some progress has been made, its value is limited. ICIs monotherapy is equivalent to single-target tumor immunotherapy (enhancement). This application innovatively proposes combining TCM's ability to strengthen the body's constitution and improve overall health with the single-target tumor immunotherapy enhancement of ICIs monotherapy, significantly increasing the potential for enhancing tumor immunity. This patented solution uses TCM to strengthen the body's constitution and improve overall health, which is equivalent to enhancing overall immunity. This is used as an enhancer in the above-mentioned single-target tumor immunotherapy, strengthening the body's constitution and improving its overall health while enhancing tumor-suppressing efficacy and reducing toxicity, demonstrating the important value of traditional Chinese medicine in comprehensive tumor treatment strategies. It also provides a new perspective for the future development of TCM in the field of anti-tumor research.

[0034] In this invention, a method with outstanding efficacy equivalent to "treating both the symptoms and the root cause" is proposed in the field of tumor immunotherapy: traditional Chinese medicine strengthens the body's resistance and improves the constitution to increase immunity through multiple targets as a method of "treating the root cause"; ICIs monotherapy is equivalent to improving tumor immunity through a single target as a method of "treating the symptoms", and the combination of these two methods constitutes a method of "treating both the symptoms and the root cause".

[0035] The method proposed in this invention differs from the conventional approach of combining immunotherapy with chemotherapy (targeted chemotherapy) and radiotherapy to enhance tumor suppression. Chemotherapy (targeted chemotherapy) and radiotherapy combined with immunotherapy can lead to serious conflicts, such as damaging immunity and reducing immune cell function, including severe toxic side effects from tumor radiotherapy and chemotherapy. The method proposed in this invention (combining immunotherapy with tonifying traditional Chinese medicine) avoids this serious conflict of damaging immunity and reducing immune cell function, and eliminates the severe toxic side effects of tumor radiotherapy and chemotherapy. The combined traditional Chinese and Western medicine approach to tumor treatment in this invention does not have cytotoxicity issues. These are the main innovations, representing a significant increase in efficacy and a reduction in toxic side effects.

[0036] 2. While immunomodulatory drugs have the advantage of increasing the body's immunity without damaging normal cells, the experimental results of this invention demonstrate that the combined use of conventional immunomodulatory drugs not only fails to significantly increase efficacy but may also increase toxicity. For example, experimental data show that mice died when two immunomodulatory drugs were used together. This, in turn, proves that the innovative approach of using traditional Chinese medicine compositions / drugs as a means of enhancing the efficacy of immunomodulatory drugs, which transcends technical imagination and represents a completely different technical approach, also demonstrating its irreplaceable importance.

[0037] 3. It is generally believed that Astragalus membranaceus (Huangqi) tonifies Qi, and when combined with Gleditsia sinensis (Zaojiao) thorns, it is traditionally used in Traditional Chinese Medicine (TCM) surgery for draining pus and eliminating toxins when the body's vital energy is deficient and unable to expel them. This invention creatively and successfully applies this combination to the treatment of tumors, and also to its synergistic effect with immunomodulatory drugs in tumor treatment. TCM theory holds that tumors are caused by the internal accumulation of "blood stasis and toxins," therefore, this invention creatively and successfully applies this "drug pair" to the treatment of tumors, leveraging its ability to invigorate vital energy and expel toxins, thus significantly enhancing tumor suppression and efficacy.

[0038] 4. According to traditional Chinese medicine theory, the five-grain insect is commonly used to treat infantile malnutrition. This invention creatively and successfully applies it to the improvement of physical constitution (cachexia) in cancer patients. Its efficacy is even more significant when synergistically improved with other qi- and yin-tonifying herbs (Astragalus membranaceus, Ophiopogon japonicus, and Paeonia lactiflora). Astragalus membranaceus, combined with Ophiopogon japonicus and Paeonia lactiflora, tonifies both qi and yin; Pinellia ternata, combined with Coix lacryma-jobi, eliminates phlegm and dampness; Taraxacum mongolicum, combined with Clematis armandii, clears heat and detoxifies (anti-inflammatory); and Gleditsia sinensis thorns, combined with Actinidia chinensis root and Citrus aurantium, promote blood circulation and remove blood stasis. The entire formula, while strengthening the body and improving overall constitution, also resolves phlegm, removes blood stasis, supports toxins, and inhibits tumor growth, significantly enhancing the efficacy of immunomodulatory drugs.

[0039] 5. According to traditional Chinese medicine theory and practical processing techniques, a variety of Chinese medicinal herbs, such as Astragalus membranaceus, Gleditsia sinensis thorns, stir-fried Paeonia lactiflora, Taraxacum mongolicum, Pinellia ternata (processed with ginger), Coix lacryma-jobi, Ophiopogon japonicus, Rhizoma Citri Reticulatae, Clematis armandii, and Actinidia chinensis root, are usually decocted together and then extracted with water. In the course of long-term research on extraction processes and components, as well as the relationship between extraction processes and efficacy, this invention unexpectedly discovered the water-boiling extraction characteristics of Gleditsia sinensis thorns, which has not been publicly recorded in existing research.

[0040] Extracting Gleditsia sinensis thorns with other drugs via water significantly reduces the variety and content of the extracted medicinal components, resulting in a substantial decrease in antitumor efficacy, whether used alone or in combination with immunomodulatory drugs. Furthermore, even when Gleditsia sinensis thorns are extracted separately with hot water, and other medicinal herbs are extracted separately with hot water, the contents of the two water extracts appear normal when analyzed separately by liquid chromatography-mass spectrometry (LC-MS). However, when the two extracts are mixed and analyzed by LC-MS, the contents are significantly lower compared to the separate analysis results. To address this issue, this invention creatively proposes a method of extracting Gleditsia sinensis thorns separately with hot water and then concentrating the extract, while simultaneously extracting and concentrating multiple other medicinal herbs (excluding Gleditsia sinensis thorns) together with hot water. The two concentrates are then used to prepare the drug. This innovative approach avoids significant loss of medicinal components, thus ensuring efficacy.

[0041] 6. According to traditional Chinese medicine theory, *Eriocheir sinensis* is traditionally prepared by stir-frying before consumption. However, the applicant's research has found batch-to-batch instability in the microbial control of *Eriocheir sinensis* during the production of traditional Chinese medicine preparations, with some batches exhibiting excessive microbial levels. This invention, based on extensive research into the microbial control process and components of *Eriocheir sinensis*, as well as its relationship to efficacy, creatively proposes a superior improved traditional processing technique for *Eriocheir sinensis* used in the production of traditional Chinese medicine preparations. This technique facilitates the achievement of adequate microbial control after *Eriocheir sinensis* is added to the medicine. The traditional processing method of *Eriocheir sinensis*, which involves stir-frying before consumption, carries the risk of incomplete microbial control within the *Eriocheir sinensis*, leading to substandard microbial control in the prepared medicine. Attached Figure Description

[0042] Figure 1 This is the ion chromatogram of the positive ion mode base peak of sample A in Example 1.

[0043] Figure 2 This is the negative ion mode base peak ion chromatogram of sample A in Example 1.

[0044] Figure 3 The image shows the ion chromatogram of the positive ion mode base peak of the sample in Comparative Example 1.

[0045] Figure 4 The image shows the base peak ion chromatogram of the negative ion mode sample in Comparative Example 1. Detailed Implementation

[0046] The present invention will now be described in detail with reference to specific embodiments.

[0047] Part One: Examples and Comparative Cases

[0048] The Chinese herbal medicine pieces used in Example 1 and Comparative Example 1 of this application are the same, but the water extraction and concentration methods are different. They are used to study the changes in the effective components of the drugs in the concentrated liquid obtained when the soapberry thorn is extracted and concentrated alone and when it is extracted and concentrated in combination with other Chinese herbal medicine pieces.

[0049] The Chinese herbal medicine slices in Example 5 do not contain Astragalus membranaceus.

[0050] Example 1

[0051] According to the requirements of the 2020 edition of the Chinese Pharmacopoeia, take 30 parts of Astragalus membranaceus, 10 parts of Gleditsia sinensis thorns, 12 parts of stir-fried Paeonia lactiflora, 21 parts of Taraxacum mongolicum, 12 parts of ginger-processed Pinellia ternata, 30 parts of Coix lacryma-jobi, and 15 parts of Ophiopogon japonicus. The parts mentioned are by weight. In accordance with the requirements of the 2015 edition of the Zhejiang Provincial Standard for Processing Traditional Chinese Medicine, take 30 parts of Eclipta prostrata, 18 parts of Clematis armandii, and 30 parts of Actinidia chinensis root. The parts mentioned are by weight.

[0052] Of the above 10 kinds of Chinese medicinal herbs, except for the Gleditsia sinensis thorns, the other 9 kinds of Chinese medicinal herbs were boiled together and extracted twice. The extracts were combined and concentrated to obtain the water extract concentrate (Sample A in Example 1) for liquid chromatography-mass spectrometry (LC-MS) analysis.

[0053] The medicinal slices of Gleditsia sinensis were extracted twice by heating and boiling water. The two extracts were combined and then concentrated to obtain a concentrated water extract (Sample B in Example 1).

[0054] Comparative Example 1

[0055] According to the requirements of the 2020 edition of the Chinese Pharmacopoeia, take 30 parts of Astragalus membranaceus, 10 parts of Gleditsia sinensis thorns, 12 parts of stir-fried Paeonia lactiflora, 21 parts of Taraxacum mongolicum, 12 parts of ginger-processed Pinellia ternata, 30 parts of Coix lacryma-jobi, and 15 parts of Ophiopogon japonicus. The parts mentioned are by weight. In accordance with the requirements of the 2015 edition of the Zhejiang Provincial Standard for Processing Traditional Chinese Medicine, take 30 parts of Eclipta prostrata, 18 parts of Clematis armandii, and 30 parts of Actinidia chinensis root. The parts mentioned are by weight.

[0056] The above 10 kinds of Chinese medicinal herbs were heated together and boiled in water to extract twice. The extracts were combined and concentrated to obtain a concentrated water extract (sample of Comparative Example 1) for liquid chromatography-mass spectrometry (LC-MS) analysis.

[0057] Example 2

[0058] (1) In accordance with the requirements of the 2020 edition of the Chinese Pharmacopoeia, take 30 parts of Astragalus membranaceus, 10 parts of Gleditsia sinensis thorns, 12 parts of stir-fried Paeonia lactiflora, 21 parts of Taraxacum mongolicum, 12 parts of ginger-processed Pinellia ternata, 30 parts of Coix lacryma-jobi, and 15 parts of Ophiopogon japonicus. The number of parts is by weight. In accordance with the requirements of the 2015 edition of the Zhejiang Province Traditional Chinese Medicine Processing Standards, take 30 parts of Fragrant Tea Vegetable, 18 parts of Clematis armandii, 30 parts of Actinidia chinensis root, and 18 parts of Five Grains Worm. The number of parts is by weight.

[0059] (2) After freezing and grinding the grain insects into powder, soak them in 75% ethanol for 24 hours; after recovering the ethanol under reduced pressure, freeze and pulverize the solid again, and dry it for later use.

[0060] (3) The soapberry thorns were extracted twice by heating and boiling water. The two extracts were combined and concentrated to obtain the soapberry thorn water extract concentrate.

[0061] (4) The remaining 9 kinds of Chinese medicinal materials were heated and boiled twice to extract water. The extracts were combined and concentrated to obtain the concentrated water extract of the 9 kinds of Chinese medicinal materials.

[0062] (5) Mix the two water extract concentrates, then add the five grain insect powder and mix evenly to obtain the traditional Chinese medicine composition.

[0063] Then, excipients are added according to conventional pharmaceutical processing methods to produce traditional Chinese medicine pills, capsules, or tablets, thus obtaining a drug for enhancing the efficacy of tumor immunotherapy (Sample of Example 2).

[0064] The medication is taken orally. The daily adult dose is equivalent to 113g to 452g of the total raw medicinal material.

[0065] Example 3

[0066] (1) In accordance with the requirements of the 2020 edition of the Chinese Pharmacopoeia, take 60 parts of Astragalus membranaceus, 20 parts of Gleditsia sinensis thorns, 24 parts of stir-fried Paeonia lactiflora, 40 parts of Taraxacum mongolicum, 24 parts of ginger-processed Pinellia ternata, 60 parts of Coix lacryma-jobi, and 30 parts of Ophiopogon japonicus. The number of parts is by weight. In accordance with the requirements of the 2015 edition of the Zhejiang Province Traditional Chinese Medicine Processing Standards, take 60 parts of Fragrant Tea Vegetable, 40 parts of Clematis armandii, 60 parts of Actinidia chinensis root, and 50 parts of Five Grains Worm. The number of parts is by weight.

[0067] (2) After freezing and grinding the grain insects into powder, soak them in 75% ethanol for 24 hours; after recovering the ethanol under reduced pressure, freeze and pulverize the solid again, and dry it for later use.

[0068] (3) The soapberry thorns were extracted twice by heating and boiling water. The two extracts were combined and concentrated to obtain the soapberry thorn water extract concentrate.

[0069] (4) The remaining 9 kinds of Chinese medicinal materials were heated and boiled twice to extract water. The extracts were combined and concentrated to obtain the concentrated water extract of the 9 kinds of Chinese medicinal materials.

[0070] (5) Mix the two water extract concentrates, then add the five grain insect powder and mix evenly to obtain the traditional Chinese medicine composition.

[0071] Then, excipients are added according to conventional pharmaceutical processing methods to produce traditional Chinese medicine pills, capsules, or tablets, thus obtaining a drug for enhancing the efficacy of tumor immunotherapy (Sample of Example 3).

[0072] The medication is taken orally. The daily dosage for adults is equivalent to 234g to 468g of the total raw medicinal material.

[0073] Example 4

[0074] (1) In accordance with the requirements of the 2020 edition of the Chinese Pharmacopoeia, take 15 parts of Astragalus membranaceus, 5 parts of Gleditsia sinensis thorns, 5 parts of stir-fried Paeonia lactiflora, 10 parts of Taraxacum mongolicum, 6 parts of ginger-processed Pinellia ternata, 15 parts of Coix lacryma-jobi, and 6 parts of Ophiopogon japonicus. The number of parts is by weight. In accordance with the requirements of the 2015 edition of the Zhejiang Province Traditional Chinese Medicine Processing Standards, take 15 parts of Fragrant Tea Vegetable, 10 parts of Clematis armandii, 15 parts of Actinidia chinensis root, and 5 parts of Five Grains Worm. The number of parts is by weight.

[0075] (2) After freezing and grinding the grain insects into powder, soak them in 75% ethanol for 24 hours; after recovering the ethanol under reduced pressure, freeze and pulverize the solid again, and dry it for later use.

[0076] (3) The soapberry thorns were extracted twice by heating and boiling water. The two extracts were combined and concentrated to obtain the soapberry thorn water extract concentrate.

[0077] (4) The remaining 9 kinds of Chinese medicinal materials were heated and boiled twice to extract water. The extracts were combined and concentrated to obtain the concentrated water extract of the 9 kinds of Chinese medicinal materials.

[0078] (5) Mix the two water extract concentrates, then add the five grain insect powder and mix evenly to obtain the traditional Chinese medicine composition.

[0079] Then, excipients are added according to conventional pharmaceutical processing methods to produce traditional Chinese medicine pills, capsules, or tablets, thus obtaining a drug for enhancing the efficacy of tumor immunotherapy (Example 4 sample).

[0080] The medication is taken orally. The daily adult dose is equivalent to 107g to 214g of the total raw medicinal material.

[0081] For cancer patients with normal physical condition, the traditional Chinese medicine pills, capsules, or tablets prepared in Examples 2-4 above can be used orally. They can be used alone for cancer treatment, but are more recommended to be used in combination with cancer immunotherapy drugs (such as PD-1 / PD-L1, CTLA-4, LAG-3, etc.) for synergistic effect. The daily dosage for adults is equivalent to 107g to 468g of total raw medicinal materials.

[0082] Example 5

[0083] (1) In accordance with the requirements of the 2020 edition of the Chinese Pharmacopoeia, take 10 parts of Gleditsia sinensis thorns, 12 parts of stir-fried white peony root, 21 parts of dandelion, 12 parts of ginger-processed Pinellia ternata, 30 parts of Coix seed, and 15 parts of Ophiopogon japonicus. The number of parts is by weight. In accordance with the requirements of the 2015 edition of the Zhejiang Province Traditional Chinese Medicine Processing Standards, take 30 parts of fragrant tea vegetable, 18 parts of Clematis armandii, 30 parts of Actinidia chinensis root, and 18 parts of five-grain insect. The number of parts is by weight.

[0084] (2) After freezing and grinding the grain insects into powder, soak them in 75% ethanol for 24 hours; after recovering the ethanol under reduced pressure, freeze and pulverize the solid again, and dry it for later use.

[0085] (3) The soapberry thorns were extracted twice by heating and boiling water. The two extracts were combined and concentrated to obtain the soapberry thorn water extract concentrate.

[0086] (4) The remaining 8 kinds of Chinese medicinal herbs were boiled in water twice and extracted twice. The extracts were combined and concentrated to obtain the concentrated water extract of the 8 kinds of Chinese medicinal herbs.

[0087] (5) Mix the two water extract concentrates, then add the five grain insect powder and mix evenly to obtain the traditional Chinese medicine composition.

[0088] Then, excipients are added according to conventional pharmaceutical processing methods to produce traditional Chinese medicine pills, capsules, or tablets, thus obtaining a drug for enhancing the efficacy of tumor immunotherapy (sample of Example 5).

[0089] The medication is taken orally. The daily adult dose is equivalent to 196g to 392g of the total raw medicinal material.

[0090] Example 6

[0091] (1) In accordance with the requirements of the 2020 edition of the Chinese Pharmacopoeia, take 20 parts of Gleditsia sinensis thorns, 24 parts of stir-fried white peony root, 40 parts of dandelion, 24 parts of ginger-processed Pinellia ternata, 60 parts of Coix seed, and 30 parts of Ophiopogon japonicus, where the number of parts is by weight; and in accordance with the requirements of the 2015 edition of the Zhejiang Province Traditional Chinese Medicine Processing Standards, take 60 parts of fragrant tea vegetable, 40 parts of three-leaf green, 60 parts of vine-actyly root, and 50 parts of five-grain insect, where the number of parts is by weight.

[0092] (2) After freezing and grinding the grain insects into powder, soak them in 75% ethanol for 24 hours; after recovering the ethanol under reduced pressure, freeze and pulverize the solid again, and dry it for later use.

[0093] (3) The soapberry thorns were extracted twice by heating and boiling water. The two extracts were combined and concentrated to obtain the soapberry thorn water extract concentrate.

[0094] (4) The remaining 8 kinds of Chinese medicinal materials were heated and boiled twice to extract the water. The two extracts were combined and concentrated to obtain a concentrated water extract of 9 kinds of Chinese medicinal materials.

[0095] (5) Mix the two water extract concentrates, then add the five grain insect powder and mix evenly to obtain the traditional Chinese medicine composition.

[0096] Then, excipients are added according to conventional pharmaceutical processing methods to produce traditional Chinese medicine pills, capsules, or tablets, thus obtaining a drug for enhancing the efficacy of tumor immunotherapy (sample of Example 6).

[0097] The medication is taken orally. The daily dosage for adults is equivalent to 204g to 408g of the total raw medicinal material.

[0098] Example 7

[0099] (1) In accordance with the requirements of the 2020 edition of the Chinese Pharmacopoeia, take 5 parts of Gleditsia sinensis thorns, 5 parts of stir-fried white peony root, 10 parts of dandelion, 6 parts of ginger-processed Pinellia ternata, 15 parts of Coix seed, and 6 parts of Ophiopogon japonicus, the number of parts being by weight; and in accordance with the requirements of the 2015 edition of the Zhejiang Province Traditional Chinese Medicine Processing Standards, take 15 parts of fragrant tea vegetable, 10 parts of three-leaf green, 15 parts of vine-actinia root, and 5 parts of five-grain insect, the number of parts being by weight.

[0100] (2) After freezing and grinding the grain insects into powder, soak them in 75% ethanol for 24 hours; after recovering the ethanol under reduced pressure, freeze and pulverize the solid again, and dry it for later use.

[0101] (3) The soapberry thorns were extracted twice by heating and boiling water. The two extracts were combined and concentrated to obtain the soapberry thorn water extract concentrate.

[0102] (4) The remaining 8 kinds of Chinese medicinal materials were heated and boiled twice to extract the water. The two extracts were combined and concentrated to obtain a concentrated water extract of 9 kinds of Chinese medicinal materials.

[0103] (5) Mix the two water extract concentrates, then add the five grain insect powder and mix evenly to obtain the traditional Chinese medicine composition.

[0104] Then, excipients are added according to conventional pharmaceutical processing methods to produce traditional Chinese medicine pills, capsules, or tablets, thus obtaining a drug for enhancing the efficacy of tumor immunotherapy (Example 4 sample).

[0105] The medication is taken orally. The daily adult dose is equivalent to 92g to 184g of the total raw drug.

[0106] For cancer patients diagnosed with Yin deficiency and excessive Yang, this invention proposes using a drug without Astragalus (as described in Examples 5-7). This is primarily based on the traditional Chinese medicine theory that Astragalus tonifies Qi but is prone to causing "heatiness." Combined with clinical experience, this approach optimizes the treatment for cancer patients with Yin deficiency and excessive Yang by removing Astragalus. The drug is administered orally and can be used alone for cancer treatment. It is also recommended for synergistic use in combination with cancer immunotherapy drugs (such as PD-1 / PD-L1, CTLA-4, LAG-3, etc.). The daily adult dosage is equivalent to 92g to 408g of total raw medicinal material.

[0107] Part Two: Experimental Records and Results Analysis

[0108] Experiment 1

[0109] Qualitative Detection of Components in Samples of Example 1 and Comparative Example 1

[0110] 1. Experimental Materials and Methods

[0111] 1.1 Instruments and Reagents

[0112] Samples: Sample A and Sample B from Example 1, and Sample 1 from Comparative Example 1.

[0113] Instruments: Waters SYNAPT G2-Si (Waters, Milford, MA, USA) QTOF high-resolution mass spectrometer, equipped with ESI electrospray ionization source; connected to Waters ACQUITY UPLC (I-Class) ultra-high performance liquid chromatograph, equipped with Acquity I-Class Binary Solvent Manager (BSM) binary pump, Acquity IClass SampleManager FL autosampler, CH-A column oven; analytical balance, ultrasonic instrument, centrifuge, etc.

[0114] Reagents: Formic acid (>98%, chromatographic grade, Shanghai Aladdin Reagent), acetonitrile (HPLC-MS grade, Tedia, USA).

[0115] 1.2 Chromatographic conditions

[0116] Chromatographic column: Waters CORTECS T3 UPLC column (2.1 × 100 mm, 1.6 μm)

[0117] Mobile phases: Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile.

[0118] Gradient program: 0-2 min: 5% B; 2-32 min: 5%-100% B; 32-33 min: 100% B; 33.5 min: 5% B; 33.5-35 min: 5% B. Flow rate: 0.3 mL / min, injection volume: 2 μL, column temperature: 35℃, sample chamber temperature: 10℃.

[0119] 1.3 Mass Spectrometry Conditions

[0120] Electrospray ionization (ESI) source, scanning in positive and negative ion modes separately, MS E Continue mode, scan time 0.2s, scan range 50–1200. In MS... E Collision energies were used, with low collision energies at 6 V and high collision energies at 15-45 V. Sodium formate was used for mass spectrometry calibration, and leucine enkephalin (positive ion mode m / z 556.2771, negative ion mode m / z 554.2615) was used for real-time mass calibration.

[0121] 1.4 Sample Pretreatment

[0122] Extraction method: After centrifuging the compound sample (12000 rpm, 10 min), the supernatant was filtered through a 0.45 μm filter membrane.

[0123] 2. Experimental Results

[0124] 2.1 Base Peak Chromatogram (BPC) of Sample A in Example 1

[0125] 2.1.1 Ion mode of positive sample A in Example 1: see Figure 1 .

[0126] 2.1.2 Negative ion mode of sample A in Example 1: See Figure 2 .

[0127] 2.1.3 A total of 30 compounds were detected in sample A of Example 1. The mass spectrometry information of each component is shown in Table 1.

[0128] Table 1. Mass spectrometry information of 30 compounds in sample A of Example 1

[0129]

[0130] 2.2 Base Peak Chromatogram (BPC) of Comparative Example 1 Sample

[0131] 2.2.1 Positive ion mode of Comparative Example 1 sample: see Figure 3 .

[0132] 2.2.2 Negative ion mode of Comparative Example 1 sample: see Figure 4 .

[0133] 2.2.3 A total of 17 compounds were detected in the Comparative Example 1 sample. The mass spectrometry information of each component is shown in Table 2.

[0134] Table 2. Mass spectrometry information of 17 compounds in Comparative Example 1 sample

[0135]

[0136] Conclusion: The baseline ion chromatogram of Sample A (without Gleditsia sinensis thorns) in Example 1 showed a greater variety of components and relatively higher content; in contrast, the baseline ion chromatogram of Sample A in Comparative Example 1 showed a smaller variety of components and relatively lower content. This experimental result confirms that Gleditsia sinensis thorns cannot be extracted with water along with the other nine traditional Chinese medicines.

[0137] Experiment 2

[0138] Experiments on tumor inhibition of MFC gastric cancer in 615 mice using samples from Example 2, Example 3, and Example 4.

[0139] 1. Experimental Materials and Methods

[0140] 1.1 Laboratory Animals

[0141] Animal species: 615 mice; Number of animals: 48; Sex: Male; Weight range: 23-28g; Source: Produced by Hangzhou Medical College, Production License No.: SCXX (Zhejiang) 2024-0002; Certificate No.: 20251016Abba01000180048; Animal housing conditions: Temperature 22-26℃, humidity 40%-70%, light cycle 12h light / 12h darkness, free access to food and water, feed is standard mouse pellet feed, drinking water is sterile distilled water.

[0142] 1.2 Experimental Drugs

[0143] Test drugs: Low-dose group: Sample of Example 4; Medium-dose group: Sample of Example 2; High-dose group: Sample of Example 3;

[0144] Chemotherapy control drug: 5-fluorouracil (5-fu): provided by MCE1, batch number: HY-90006; specification: 200mg, prepared as a 25mg / kg solution, stored at 4℃.

[0145] 1.3 Tumor inoculation with implanted blocks

[0146] Approximately 30 mg of MFC tumor tissue was administered subcutaneously to each mouse. The tumor was allowed to grow to approximately 100 mm. 3 When the child is large, medication and treatment begin.

[0147] 1.4 Experimental grouping and drug administration

[0148] Forty-eight 615 mice were randomly divided into groups of eight:

[0149] Control group: No MFC gastric cancer vaccine;

[0150] Model group: Inoculated with MFC, no treatment for gastric cancer. Control group and model group: Administered the same volume of normal saline by gavage daily;

[0151] High-dose group: Sample 29.38 g / kg from Example 3, administered by gavage;

[0152] Medium dose group: 14.69 g / kg of sample from Example 2, administered by gavage;

[0153] Low-dose group: Sample 7.345 g / kg from Example 4, administered by gavage;

[0154] 5-FU group: 5-FU 25mg / kg body weight, every other day, intraperitoneal injection.

[0155] When the tumor grows to about 100 mm 3 When the tumor is large, treatment begins. Tumor volume is measured as usual. The experiment is terminated after 17 days of treatment. The tumor is then dissected, weighed, and the tumor inhibition rate is calculated.

[0156] 1.5 Statistics

[0157] Data analysis was performed using SPSS 25.0 software. Normality tests (Shapiro-Wilk) were employed. If the data were normally distributed, an independent samples t-test was used between two groups, and a Levene test was used for homogeneity of variance among multiple groups. If variances were homogeneous, one-way ANOVA was used with LSD and Tamhane's T2 correction; if variances were unequal, Dunnett's T3 test was used. If the data were not normally distributed, a Wilcoxon rank-sum test was used between two groups, and a Kruskal-Wallis rank-sum test was used for multiple groups. For changes in weight levels at different time points across multiple groups, repeated measures ANOVA was used if the data were normally distributed; otherwise, a generalized estimation equation was used. All data are expressed as mean ± standard deviation. (±s), P<0.05 or P<0.01 were considered statistically significant.

[0158] 2. Experimental Results

[0159] 2.1 Status of each observation indicator

[0160] During the experiment, the 615 mice had a good diet, shiny fur, and normal daily activities. The changes in body weight before and after treatment in each experimental group are shown in the table below. Regarding body weight, the control group and model group showed an increase in body weight after treatment (P<0.01, P<0.05), the 5-fu group showed a decrease in body weight (P<0.05), and the remaining groups showed no significant changes in body weight. See Table 3.

[0161]

[0162] 2.1 Gastric cancer MFC tumor growth, tumor weight, and tumor suppression rate

[0163] In terms of tumor weight, the tumor weight of each experimental group was lower than that of the model group. Compared with the model group, the high-dose group of Example 3 (P<0.01), the medium-dose group of Example 2 (P<0.05), and the 5-fu group (P<0.001) showed statistical significance. The tumor inhibition rates of each group, from highest to lowest, were: 5-fu group, high-dose group of Example 3, medium-dose group of Example 2, and low-dose group of Example 4. See Table 4 for details.

[0164]

[0165] Conclusion: In Example 3, the high-dose group of samples was equivalent to a human dose of approximately 234g of total raw medicinal material. The tumor weight of MFC gastric cancer was significantly smaller than that of the model group (P<0.01), and the tumor inhibition rate of traditional Chinese medicine alone was 32.9%. In Example 2, the medium-dose group of samples was equivalent to a human dose of approximately 113g of total raw medicinal material. The tumor weight of MFC gastric cancer was significantly smaller than that of the model group (P<0.05), and the tumor inhibition rate of traditional Chinese medicine alone was 24.8%.

[0166] Experiment 3

[0167] Example 2: Enhanced tumor suppression effect of combined immunotherapy drugs CTLA-4 and PD-1 inhibitors on MFC gastric cancer in 615 mice.

[0168] 1. Experimental Materials and Methods

[0169] 1.1 Laboratory Animals

[0170] Animal species: 615 mice; Number of animals: 60; Sex: Male; Weight range: 23-30g; Source: Produced by Hangzhou Qizhen Experimental Animal Technology Co., Ltd., Production License No.: SCXK (Zhejiang) 2022-0005; Certificate No.: 20250812Abzz01009990009; Animal housing conditions: Temperature 22-26℃, humidity 40%-70%, light cycle 12h light / 12h darkness, free access to food and water, feed is standard mouse pellet feed, drinking water is sterile distilled water.

[0171] 1.2 Experimental Drugs

[0172] Test drug: High-dose group: Sample from Example 2, total crude drug content 29.38 g / kg;

[0173] Combination immunotherapy:

[0174] CTLA-4 inhibitor: supplied by BioXcell, batch number: BE0131-25MG; specification: 25mg, prepared as 1ug / ul solution, stored at 4°C;

[0175] PD-1 inhibitor: provided by BioXcell, batch number: BP0146-100MG; specification: 100mg, prepared as 1ug / ul solution, stored at 4℃.

[0176] 1.3 Tumor inoculation with implanted blocks

[0177] Approximately 30 mg of MFC tumor tissue was administered subcutaneously to each mouse. The tumor was allowed to grow to approximately 100 mm. 3 When the child is large, medication and treatment begin.

[0178] 1.4 Experimental grouping and drug administration

[0179] Fifty-six 615 mice were randomly divided into groups of eight:

[0180] Control group: No MFC gastric cancer vaccine;

[0181] Model group: Inoculated with MFC gastric cancer and administered physiological saline by gavage;

[0182] Example 2 High-dose group of samples: Total crude drug content 29.38 g / kg, administered by gavage;

[0183] CTLA-4 inhibitor group: CTLA-4 inhibitors were injected intraperitoneally every two days at a dose of 5 mg / kg;

[0184] PD-1+CTLA-4 inhibitor group: CTLA-4 and PD-1 inhibitors were injected intraperitoneally every two days, at a dose of 5 mg / kg for both.

[0185] CTLA-4 inhibitor + high-dose group of sample from Example 2: The dosage of the two drugs was the same as that of the two groups above;

[0186] PD-1 + CTLA-4 inhibitor + high-dose group of sample from Example 2: The dosage of the three drugs is the same as that of the two groups above.

[0187] When the tumor grows to about 100 mm 3 When the tumor is large, treatment begins. Tumor volume is measured as usual. The experiment is terminated after 17 days of treatment. The tumor is then dissected, weighed, and the tumor inhibition rate is calculated.

[0188] 1.5 Statistics

[0189] Data analysis was performed using SPSS 25.0 software. Normality tests (Shapiro-Wilk) were employed. If the data were normally distributed, an independent samples t-test was used between two groups, and a Levene test was used for homogeneity of variance among multiple groups. If variances were homogeneous, one-way ANOVA was used with LSD and Tamhane's T2 correction; if variances were unequal, Dunnett's T3 test was used. If the data were not normally distributed, a Wilcoxon rank-sum test was used between two groups, and a Kruskal-Wallis rank-sum test was used for multiple groups. For changes in weight levels at different time points across multiple groups, repeated measures ANOVA was used if the data were normally distributed; otherwise, a generalized estimation equation was used. All data are expressed as mean ± standard deviation. (±s), P<0.05 or P<0.01 were considered statistically significant.

[0190] 2. Experimental Results

[0191] 2.1 Status of each observation indicator

[0192] During the experiment, two mice died in each of the PD-1 inhibitor group + CTLA-4 inhibitor group and the PD-1 inhibitor + CTLA-4 inhibitor group + high-dose sample from Example 2. The remaining mice had a good diet, normal coat color, and normal activity. The changes in body weight before and after treatment in each experimental group are shown in the table below. The model group showed an increase in body weight after treatment (P<0.05), while the other groups showed no significant change in body weight. See Table 5.

[0193]

[0194] 2.2 Gastric cancer MFC tumor growth, tumor weight, and tumor inhibition rate

[0195] In terms of tumor weight, the tumor weight of each experimental group was lower than that of the model group. Compared with the model group, the high-dose group of Example 2 sample (P<0.05), the CTLA-4 inhibitor group + PD-1 inhibitor group (P<0.05), the CTLA-4 inhibitor + high-dose group of Example 2 sample (P<0.01), and the CTLA-4 inhibitor + PD-1 inhibitor group + high-dose group of Example 2 sample (P<0.01) were statistically significant. The tumor inhibition rate of each group from highest to lowest was as follows: CTLA-4 inhibitor + high-dose group of Example 2 sample, CTLA-4 inhibitor + PD-1 inhibitor group + high-dose group of Example 2 sample, CTLA-4 inhibitor group + PD-1 inhibitor group, high-dose group of Example 2 sample, and CTLA-4 inhibitor group. See Table 6 for details.

[0196] The tumor inhibition rate of the CTLA-4 inhibitor group + PD-1 inhibitor group was basically not improved compared with that of the CTLA-4 inhibitor group with single immunotherapy; the tumor inhibition efficacy of the CTLA-4 inhibitor group with single immunotherapy combined with traditional Chinese medicine was significantly improved, but the combination of two immunotherapy drugs with traditional Chinese medicine did not improve it further.

[0197]

[0198] 2.3 Flow cytometry detection of gastric cancer MFC tumor tissue

[0199] Subcutaneous gastric cancer (MFC) tumor tissues from four groups of mice were prepared into single-cell suspensions and analyzed by flow cytometry. The results are as follows:

[0200] D8 / CD3 T killer cells: In Example 2, the high-dose group and the CTLA-4 group showed significantly higher levels than the model group (P < 0.05). The high-dose + CTLA-4 group in Example 2 showed a very significant increase compared to the model group (P < 0.01). The high-dose + CTLA-4 group in Example 2 also showed a significant increase compared to the two groups used alone (P < 0.05). See Table 7.

[0201]

[0202] Conclusion:

[0203] In the high-dose group of the sample in Example 2, the equivalent human dose was approximately 226 g of total crude drug. The weight of the MFC gastric cancer tumors was significantly smaller than that of the model group (P < 0.01). The tumor inhibition rate of the traditional Chinese medicine alone was 33.32%; the tumor inhibition rate of the CTLA-4 immune drug alone was 32.3%, which was close to that of the traditional Chinese medicine alone; the combined use of the CTLA-4 inhibitor and the PD-1 inhibitor did not significantly improve the tumor inhibition efficacy; the tumor inhibition efficacy of the combination of a single immune drug and traditional Chinese medicine (high-dose sample in Example 2) in the CTLA-4 inhibitor group had been significantly improved, but the combination of adding one more immune drug (two immune drugs) and traditional Chinese medicine did not further improve.

[0204] The combined use of two immune drugs did not significantly increase the efficacy, but may have increased the toxicity, so 2 / 8 mice in each of the two groups involving the combined use of two immune drugs died. Traditional Chinese medicine is of irreplaceable importance as a means to enhance the efficacy of immune drugs.

[0205] T killer cells of CD8 / CD3 in the local tumor tissue of MFC gastric cancer in mice: The high-dose group of the sample in Example 2 and the CTLA-4 group both significantly increased the proportion of T killer cells. The data also showed that the combination of these two further significantly enhanced this effect. This should also be one of the main mechanisms for obtaining synergistic effects in combination.

[0206] Experiment 4

[0207] Tumor inhibition and synergistic effect test of the combination of the sample in Example 2, the medium-dose sample in Example 5, and the immune drugs CTLA-4 and PD-1 inhibitors on S180 malignant sarcoma in BALB / c mice

[0208] 1. Experimental materials and methods

[0209] 1.1 Experimental animals

[0210] Animal species: BALB / c mice; Number of animals: 56; Animal gender: male; Animal weight range: 18 - 20 g; Animal source: Produced by Hangzhou Qizhen Laboratory Animal Technology Co., Ltd.; Production license number: SCXK(Zhe)2022 - 0005; Certificate number: 202500708Abzz01009990041; Animal feeding conditions: Temperature 22 - 26°C, humidity 40% - 70%, light cycle 12 h light / 12 h dark, free access to food and water, feed is standard mouse pellet feed, and drinking water is sterilized distilled water.

[0211] 1.2 Experimental drugs

[0212] Test drugs: Medium-dose group of the sample in Example 2: Dosage is 14.69 g / kg body weight of total crude drug;

[0213] Example 5: Dosage group of sample: Total amount of raw drug 12.74 g / kg body weight.

[0214] Combination immunotherapy:

[0215] CTLA-4 inhibitor: supplied by BioXcell, batch number: BE0131-25MG; specification: 25mg, prepared as 1ug / ul solution, stored at 4°C;

[0216] PD-1 inhibitor: provided by BioXcell, batch number: BP0146-100MG; specification: 100mg, prepared as 1ug / ul solution, stored at 4℃.

[0217] 1.3 Tumor inoculation with implanted blocks

[0218] Approximately 30 mg of tumor tissue from mouse S180 malignant sarcoma was administered subcutaneously to each mouse. The tumor was allowed to grow to approximately 100 mm. 3 When the child is large, medication and treatment begin.

[0219] 1.4 Experimental grouping and drug administration

[0220] Fifty-six BALB / c mice were randomly divided into groups of seven:

[0221] Control group: No S180 sarcoma injection;

[0222] Model group: Inoculated with S180 sarcoma and administered physiological saline by gavage;

[0223] PD-1 inhibitor group: Inoculated with S180 sarcoma, PD-1 inhibitor was injected intraperitoneally every two days at a dose of 5 mg / kg body weight;

[0224] CTLA-4 inhibitor group: S180 sarcoma was inoculated and CTLA-4 inhibitor was injected intraperitoneally every two days at a dose of 5 mg / kg body weight;

[0225] Example 2 sample medium dose group: Inoculated with S180 sarcoma, the total crude drug content of the Example 2 sample medium dose was 14.69 g / kg body weight by gavage;

[0226] In Example 5, the medium-dose group was inoculated with S180 sarcoma. The total crude drug content in the medium-dose group of Example 5 was 12.74 g / kg body weight.

[0227] Example 5: Medium dose group + PD-1 inhibitor group: S180 sarcoma was inoculated, and the above two groups were administered drugs together;

[0228] Example 2: Sample mid-dose group + CTLA-4 group: S180 sarcoma was inoculated, and the above two groups were administered drugs together.

[0229] When the tumor grows to about 100 mm 3 When the tumor is large, treatment begins. Tumor volume is measured as usual. After 17 days of treatment, the experiment is terminated, the tumor is dissected and weighed, and the tumor inhibition rate is calculated.

[0230] 1.5 Statistics

[0231] Data analysis was performed using SPSS 25.0 software. Normality tests (Shapiro-Wilk) were employed. If the data were normally distributed, an independent samples t-test was used between two groups, and a Levene test was used for homogeneity of variance among multiple groups. If variances were homogeneous, one-way ANOVA was used with LSD and Tamhane's T2 correction; if variances were unequal, Dunnett's T3 test was used. If the data were not normally distributed, a Wilcoxon rank-sum test was used between two groups, and a Kruskal-Wallis rank-sum test was used for multiple groups. For changes in weight levels at different time points across multiple groups, repeated measures ANOVA was used if the data were normally distributed; otherwise, a generalized estimation equation was used. All data are expressed as mean ± standard deviation. (±s), P<0.05 or P<0.01 were considered statistically significant.

[0232] 2. Experimental Results

[0233] 2.1 Status of each observation indicator

[0234] During the experiment, the BALB / c mice had a good diet, normal coat color, and normal daily activities. The changes in body weight before and after treatment in each experimental group are shown in Table 8.

[0235]

[0236] 2.2 Tumor growth, tumor weight, and tumor inhibition rate of S180 malignant sarcoma in mice

[0237] In terms of tumor weight, the tumor weight of each experimental group was lower than that of the model group. The tumor weights of the PD-1 inhibitor group, CTLA-4 inhibitor group, medium-dose group of sample 5 in Example 5, medium-dose group of sample 2 in Example 2, medium-dose + PD-1 group of sample 5 in Example 5, and medium-dose + CTLA-4 group of sample 2 in Example 2 were all significantly smaller than those of the model group, and the differences were statistically significant. The tumor weights of the medium-dose + PD-1 group of sample 5 and the medium-dose + CTLA-4 group of sample 2 in Example 2 were significantly smaller than those of the immunotherapy monotherapy group, and the differences were statistically significant; they were also significantly smaller than those of the two traditional Chinese medicine monotherapy groups, and the differences were statistically significant. See Table 9 for details.

[0238]

[0239] Conclusion: The PD-1 inhibitor, CTLA-4 inhibitor, medium dose of the sample in Example 5, and medium dose of the sample in Example 2 all have significant anti-tumor effects on S180 malignant sarcoma in BALB / c mice, and the curative effects are similar; the medium dose of the sample in Example 2 is combined with the CTLA-4 inhibitor, and the medium dose of the sample in Example 5 is combined with the PD-1 inhibitor. The combination of the two traditional Chinese medicines and the two immune drugs both shows a relatively significant anti-tumor synergistic effect.

[0240] Experiment 5

[0241] Anti-tumor Synergistic Test of Samples in Example 5, Example 6, Example 7, and Example 2 Combined with Immune Drugs CTLA-4 and PD-1 Inhibitors on S180 Malignant Sarcoma in BALB / c Mice

[0242] 1. Experimental Materials and Methods

[0243] 1.1 Experimental Animals

[0244] Animal species: BALB / c mice; Number of animals: 110; Animal gender: male; Animal weight range: 18 - 20 g; Animal source: Produced by Hangzhou Qizhen Laboratory Animal Technology Co., Ltd., Production License Number: SCXK(Zhe)2022 - 0005; Certificate Number: 20250826Abzz01009990003; Animal feeding conditions: Temperature 22 - 26 °C, humidity 40% - 70%, light cycle 12 h light / 12 h darkness, free access to food and water, feed is standard mouse pellet feed, and drinking water is sterilized distilled water.

[0245] 1.2 Experimental Drugs

[0246] Test drugs: Sample in Example 2; Samples in Example 5, Example 6, and Example 7.

[0247] Combined immune drugs:

[0248] CTLA-4 inhibitor: Provided by BioXcell, batch number: BE0131 - 25MG; Specification: 25 mg, prepared into a 1 μg / μl solution and stored at 4 °C;

[0249] PD-1 inhibitor: Provided by BioXcell, batch number: BP0146 - 100MG; Specification: 100 mg, prepared into a 1 μg / μl solution and stored at 4 °C.

[0250] 1.3 Inoculating Tumor with Tumor Tissue Blocks

[0251] About 30 mg of tumor tissue blocks of mouse S180 malignant sarcoma were taken for each mouse and inoculated subcutaneously. When the tumor grew to about 100 MM 3 in size, drug treatment was started.

[0252] 1.4 Experimental grouping and drug administration

[0253] Seventy-two BALB / c mice were randomly divided into groups of eight:

[0254] Control group: No S180 sarcoma injection;

[0255] Model group: Inoculated with S180 sarcoma and administered physiological saline by gavage;

[0256] PD-1 inhibitor group: Inoculated with S180 sarcoma, PD-1 inhibitor was injected intraperitoneally every two days at a dose of 5 mg / kg body weight;

[0257] PD-1 inhibitor + TLA-4 inhibitor group: S180 sarcoma was injected, and CTLA-4 inhibitor 5 mg / kg body weight + PD-1 inhibitor 5 mg / kg body weight was injected intraperitoneally every two days;

[0258] PD-1 inhibitor + TLA-4 inhibitor + high-dose group of sample from Example 2: Inoculated with S180 sarcoma, CTLA-4 inhibitor 5mg / kg body weight + PD-1 inhibitor 5mg / kg body weight were injected intraperitoneally every two days + the total crude drug content of high-dose sample from Example 2 was 29.38g / kg body weight by gavage;

[0259] Example 6 High-dose group: Inoculated with S180 sarcoma, and administered via gavage the total crude drug content of the high-dose sample of Example 6 was 25.48 g / kg body weight;

[0260] Example 5 sample medium dose group: Inoculated with S180 sarcoma, the total crude drug content of the sample medium dose in Example 5 was 12.74 g / kg body weight by gavage;

[0261] Example 7 sample low-dose group: Inoculated with S180 sarcoma, and administered via gavage the total crude drug content of the low-dose sample of Example 7 was 6.37 g / kg body weight;

[0262] PD-1 inhibitor + high-dose group of sample 6 in Example 6: Inoculated with S180 sarcoma, PD-1 inhibitor 5mg / kg body weight was injected intraperitoneally every two days + total crude drug amount of sample 6 in Example 6 was 25.48g / kg body weight by gavage.

[0263] When the tumor grows to about 100 mm 3 When the tumor is large, treatment begins. Tumor volume is measured as usual. After 15 days of treatment, the experiment is terminated, the tumor is dissected and weighed, and the tumor inhibition rate is calculated.

[0264] 1.5 Statistics

[0265] Data analysis was performed using SPSS 25.0 analysis software. Normality test (Shapiro-Wilk) was adopted. If the data was normally distributed, independent samples t-test was used between two groups, and Levene test was used to test the homogeneity of variance among multiple groups. If the variance was homogeneous, one-way ANOVA was used with LSD and Tamhane’s T2 correction. If the variance was inhomogeneous, Dunnetts T3 test was used. If the data was not normally distributed, Wilcoxon rank sum test was used between two groups, and Kruskal-Wallis rank sum test was used among multiple groups. For the change in body weight levels at multiple time points in multiple groups, if the data was normally distributed, repeated measures ANOVA was used. If the data was not normally distributed, generalized estimating equations analysis was used. The data was expressed as mean ± standard deviation ( ±s), and P<0.05 or P<0.01 was considered statistically significant.

[0266] 2. Experimental results

[0267] 2.1 Conditions of each observation index

[0268] During the experiment, the BALB / c mice had good diet, normal hair color, and normal daily activities. The changes in body weight before and after treatment in each experimental group are shown in Table 10.

[0269]

[0270] 2.2 Tumor growth, tumor weight, and tumor inhibition rate of S180 malignant sarcoma in mice

[0271] In terms of tumor weight, the tumor weights of each experimental group were lower than those of the model group. Compared with the model group, there were statistical significances in the medium-dose group of Example 5 sample, the high-dose group of Example 6 sample, the PD-1 inhibitor group, the PD-1 inhibitor + high-dose group of Example 6 sample, the PD-1 + CTLA-4 inhibitor group, and the PD-1 + CTLA-4 inhibitor group + Example 2 sample group. The tumor weight of the high-dose group of Example 6 sample was significantly smaller than that of the low-dose group of Example 7 sample. The tumor weight of the PD-1 inhibitor + high-dose group of Example 6 sample was significantly smaller than that of the high-dose group of Example 6 sample. The tumor inhibition rates of each group from large to small were PD-1 inhibitor + high-dose group of Example 6 sample < PD-1 + CTLA-4 inhibitor group + Example 2 sample group < PD-1 + CTLA-4 inhibitor group < PD-1 inhibitor group < high-dose group of Example 6 sample < medium-dose group of Example 5 sample < low-dose group of Example 7 sample. The specific situation is shown in Table 11.

[0272]

[0273] Conclusion:

[0274] The medium dose of the sample in Example 5 and the high dose of the sample in Example 6 both had significant tumor inhibitory effects on murine S180 malignant sarcoma; after the high dose of the sample in Example 6 was combined with the PD-1 inhibitor, the tumor inhibition rate for murine S180 malignant sarcoma reached 71.95%, which was significantly higher than the high dose of the sample in Example 6 and also 22.21% higher than the 49.74% tumor inhibition rate of the PD-1 inhibitor.

[0275] The combination of the PD-1 inhibitor and the CTLA-4 inhibitor did not show significant synergistic effects on murine S180 malignant sarcoma; the tumor inhibition efficacy of adding the high dose of the sample in Example 2 based on the combination of the PD-1 inhibitor and the CTLA-4 inhibitor was 62.58%, and the synergistic amplitude did not reach that of the combination of the single immune drug PD-1 inhibitor and the high dose of the sample in Example 6 (the tumor inhibition rate for murine S180 malignant sarcoma reached 71.95% after combination). This further shows that the combination of multiple immune drugs and the combination of multiple immune drugs with the high dose of the sample in Example 2 of traditional Chinese medicine did not further increase the synergistic amplitude.

[0276] Experiment 6

[0277] Tumor inhibition and synergistic test of the sample in Example 6 combined with the immune drug PD-1 inhibitor on BALB / c murine S180 malignant sarcoma

[0278] 1. Experimental materials and methods

[0279] 1.1 Experimental animals

[0280] Animal species: BALB / c mice; Number of animals: 50; Animal gender: male; Animal weight range: 18 - 20 g; Animal source: Produced by Hangzhou Medical College, production license number: SCXK(Zhe)2024 - 0002; Certificate number: 20251103Aazz01000180045; Animal feeding conditions: Temperature 22 - 26°C, humidity 40% - 70%, light cycle 12 h light / 12 h darkness, free access to food and water, feed is standard mouse pellet feed, drinking water is sterilized distilled water.

[0281] 1.2 Experimental drugs

[0282] Test drug: The sample in Example 6.

[0283] Combined immune drugs:

[0284] PD-1 inhibitor: Provided by BioXcell, batch number: BP0146 - 100MG; Specification: 100 mg, configured into 1 μg / μl solution, stored at 4°C.

[0285] 1.3 Inoculating tumors with tumor blocks

[0286] Approximately 30 mg of tumor tissue from mouse S180 malignant sarcoma was administered subcutaneously to each mouse. The tumor was allowed to grow to approximately 100 mm. 3 When the child is large, medication and treatment begin.

[0287] 1.4 Experimental grouping and drug administration

[0288] Fifty BALB / c mice were randomly divided into groups of 10 each:

[0289] Control group: No S180 sarcoma injection;

[0290] Model group: Inoculated with S180 sarcoma and administered physiological saline by gavage;

[0291] PD-1 inhibitor group: Inoculated with S180 sarcoma, PD-1 inhibitor was injected intraperitoneally every two days at a dose of 5 mg / kg body weight;

[0292] Example 6 High-dose group: Inoculated with S180 sarcoma, and administered via gavage the total crude drug content of the high-dose sample of Example 6 was 25.48 g / kg body weight;

[0293] PD-1 inhibitor + high-dose group of sample 6 in Example 6: Inoculated with S180 sarcoma, PD-1 inhibitor 5mg / kg body weight was injected intraperitoneally every two days + total crude drug amount of sample 6 in Example 6 was 25.48g / kg body weight by gavage.

[0294] When the tumor grows to about 100 mm 3 When the tumor is large, treatment begins. Tumor volume is measured as usual. After 15 days of treatment, the experiment is terminated, the tumor is dissected and weighed, and the tumor inhibition rate is calculated.

[0295] 1.5 Statistics

[0296] Data analysis was performed using SPSS 25.0 software. Normality tests (Shapiro-Wilk) were employed. If the data were normally distributed, an independent samples t-test was used between two groups, and a Levene test was used for homogeneity of variance among multiple groups. If variances were homogeneous, one-way ANOVA was used with LSD and Tamhane's T2 correction; if variances were unequal, Dunnett's T3 test was used. If the data were not normally distributed, a Wilcoxon rank-sum test was used between two groups, and a Kruskal-Wallis rank-sum test was used for multiple groups. For changes in weight levels at different time points across multiple groups, repeated measures ANOVA was used if the data were normally distributed; otherwise, a generalized estimation equation was used. All data are expressed as mean ± standard deviation. (±s), P<0.05 or P<0.01 were considered statistically significant.

[0297] 2. Experimental Results

[0298] 2.1 Status of each observation indicator

[0299] During the experiment, the BALB / c mice had a good diet, normal fur color, and normal daily activities. The changes in body weight before and after treatment in each experimental group are shown in Table 12.

[0300]

[0301] 2.2 Tumor growth, tumor weight, and tumor inhibition rate of S180 malignant sarcoma in mice

[0302] In terms of tumor weight, the tumor weights of the high-dose sample group (Example 6), the PD-1 inhibitor group, and the PD-1 inhibitor + high-dose sample group (Example 6) were all smaller than those of the model group (P < 0.01 or P < 0.05). Among these, the tumor weight of the PD-1 inhibitor + high-dose sample group (Example 6) was significantly smaller than that of the high-dose sample group (Example 6) and the PD-1 inhibitor group (Example 6) (P < 0.05). The tumor inhibition rates of each group, from highest to lowest, were: PD-1 inhibitor + high-dose sample group (Example 6) > PD-1 inhibitor group > high-dose sample group (Example 6). See Table 13 for details.

[0303]

[0304] in conclusion:

[0305] In Example 6, the high-dose group, the PD-1 inhibitor group, and the group using both drugs together all showed significant tumor-suppressing effects. The tumor-suppressing efficacy of the high-dose group and the group using the PD-1 inhibitor together was significantly higher than that of the two drugs used alone, and the tumor-suppressing effect was significantly enhanced after the two drugs were used together.

[0306] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for preparing a traditional Chinese medicine composition for synergistic effect in combination with tumor immunotherapy, characterized in that, include: (1) In accordance with the requirements of the 2020 edition of the Chinese Pharmacopoeia, take 15-60 parts of Astragalus membranaceus, 5-20 parts of Gleditsia sinensis thorns, 5-24 parts of stir-fried Paeonia lactiflora, 10-40 parts of Taraxacum mongolicum, 6-24 parts of Pinellia ternata, 15-60 parts of Coix lacryma-jobi, and 6-30 parts of Ophiopogon japonicus; in accordance with the requirements of the 2015 edition of the Zhejiang Province Traditional Chinese Medicine Processing Standards, take 5-50 parts of Cordyceps militaris, 15-60 parts of Achyranthes bidentata, 10-40 parts of Clematis armandii, and 15-60 parts of Actinidia chinensis root; the parts mentioned are by weight. (2) After freezing and grinding the grain insects into powder, soak them in 75% ethanol for 24 hours; after recovering the ethanol under reduced pressure, freeze and pulverize the solid again, and dry it for later use. (3) Take soapberry thorns, add water and heat to boil twice to extract; combine the two extracts and concentrate to obtain soapberry thorn water extract concentrate; (4) Take Astragalus membranaceus, stir-fried white peony root, dandelion, ginger-processed Pinellia ternata, coix seed, Ophiopogon japonicus, fragrant tea leaves, three leaves green, and Actinidia chinensis root, combine them, add water, heat and boil to extract twice; combine the two extracts and concentrate to obtain a concentrated water extract of multiple medicinal materials; (5) Mix the two water extract concentrates, then add the five grain insect powder and mix evenly to obtain the traditional Chinese medicine composition.

2. A method for preparing a drug for synergistic effect in combination with tumor immunotherapy, characterized in that, The process involves adding excipients to the traditional Chinese medicine composition prepared by the method described in claim 1 according to conventional pharmaceutical processing methods; then preparing it into pills, capsules, or tablets to obtain the drug.

3. The method of using the drug prepared by the method of claim 2, characterized in that, It involves administering the prepared Chinese medicine pills, capsules, or tablets orally. The daily dosage for adults is equivalent to 107g to 468g of the total raw medicinal material.

4. A method for preparing a traditional Chinese medicine composition for synergistic effect in combination with tumor immunotherapy, characterized in that, include: (1) In accordance with the requirements of the 2020 edition of the Chinese Pharmacopoeia, take 5-20 parts of Gleditsia sinensis thorns, 5-24 parts of stir-fried Paeonia lactiflora, 10-40 parts of Taraxacum mongolicum, 6-24 parts of ginger-processed Pinellia ternata, 15-60 parts of Coix lacryma-jobi, and 6-30 parts of Ophiopogon japonicus; in accordance with the requirements of the 2015 edition of the Zhejiang Province Traditional Chinese Medicine Processing Standards, take 5-50 parts of five-grain worm, 15-60 parts of fragrant tea vegetable, 10-40 parts of Trifolium repens, and 15-60 parts of Actinidia chinensis root; the parts mentioned are by weight. (2) After freezing and grinding the grain insects into powder, soak them in 75% ethanol for 24 hours; after recovering the ethanol under reduced pressure, freeze-crush the solid and dry it; (3) Take soapberry thorns, add water and heat to boil twice to extract; combine the two extracts and concentrate to obtain soapberry thorn water extract concentrate; (4) Take stir-fried white peony root, dandelion, ginger-processed pinellia, coix seed, ophiopogon japonicus, fragrant tea vegetable, three-leaf green, and vine pear root, combine them, add water, heat and boil to extract twice; combine the two extracts and concentrate to obtain a concentrated water extract of multiple medicinal materials; (5) Mix the two water extract concentrates, then add the five grain insect powder and mix evenly to obtain the traditional Chinese medicine composition.

5. A method for preparing a drug for synergistic effect in combination with tumor immunotherapy, characterized in that, The process involves adding excipients to the traditional Chinese medicine composition prepared by the method described in claim 4 according to conventional pharmaceutical processing methods; then preparing it into pills, capsules, or tablets to obtain the drug.

6. The method of using the drug prepared by the method of claim 5, characterized in that, It involves administering the prepared Chinese medicine pills, capsules, or tablets orally. The daily dosage for adults is equivalent to 92g to 408g of the total raw medicinal material.

7. The method of use according to any one of claims 3 or 6, characterized in that, The traditional Chinese medicine pills, capsules, or tablets can be used in combination with tumor immunotherapy drugs to enhance the efficacy of tumor treatment; or the traditional Chinese medicine pills, capsules, or tablets can be used alone to treat tumors.

8. The method according to claim 7, characterized in that, The tumor immunotherapy drug is any one of programmed death protein-1, a ligand of programmed death protein-1, cytotoxic T lymphocyte antigen 4, or lymphocyte activation gene-3.

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