A traditional Chinese medicine composition for treating brain tumor and a preparation method and application thereof

Abstract

The application provides a traditional Chinese medicine composition for treating brain tumors and a preparation method and application thereof, and the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 3-5 parts of tianlong, 3-5 parts of centipede, 6-12 parts of sanleng, 6-12 parts of zedoary, 15-60 parts of belamcanda chinensis, 9-12 parts of acorus gramineus, 10-20 parts of dandelion, 9-12 parts of poria cocos and 9-12 parts of atractylodes. The composition is prepared through the strict combination of monarch, minister, assistant and messenger and the synergistic effect of specific medicinal ingredients, nine drugs are combined, warm and cool are in order, and elimination and tonification are combined and cancelled, so that the effects of removing blood stasis and resolving masses, dispelling wind and calming are realized; the unique combination mechanism effectively restrains the toxic and side effects of insect drugs while enhancing the treatment intensity of removing blood stasis and resolving masses, and ensures the safety of the medicine; in addition, based on the syndrome differentiation strategy of flexible adjustment of the amount of monarch drug, the composition can be widely used for various types of brain tumor patients, and significant progress is achieved in the aspects of efficacy, safety and clinical applicability.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine compound prescription technology, and specifically discloses a traditional Chinese medicine composition for treating brain tumors, its preparation method and application. Background Technology

[0002] Brain tumors are common tumors of the central nervous system, including primary brain tumors (such as gliomas and meningiomas) and metastatic brain tumors. Their incidence is increasing year by year, and treatment is challenging with a poor prognosis. Currently, conventional treatments for brain tumors mainly include surgical resection, radiotherapy, and chemotherapy. While surgical resection can directly remove tumor tissue, the complex structure of the brain makes surgery high-risk, easily leading to neurological damage, and it is difficult to completely remove invasive tumors (such as high-grade gliomas). Radiotherapy and chemotherapy are often accompanied by serious side effects, such as bone marrow suppression, decreased immune function, nausea, and vomiting, affecting the patient's quality of life and easily leading to drug resistance and high recurrence rates.

[0003] In the field of traditional Chinese medicine (TCM), several compositions have been proposed for adjuvant treatment of brain tumors, such as formulas based on the principles of promoting blood circulation, removing blood stasis, clearing heat and detoxifying, or strengthening the body's resistance. These TCM compositions typically consist of multiple herbs, such as Astragalus membranaceus, Angelica sinensis, and Hedyotis diffusa, aiming to regulate the body's immunity, inhibit tumor cell proliferation, or reduce the side effects of radiotherapy and chemotherapy through multiple targets. However, existing TCM treatments for brain tumors still have many limitations: First, most formulas lack optimization targeting the specific pathological mechanisms of brain tumors (such as blood-brain barrier penetration and tumor microenvironment regulation), leading to unstable efficacy or limited to adjuvant effects; second, the compatibility of existing TCM compositions is mostly based on traditional experience, lacking modern pharmacological evidence, and their active ingredients, mechanisms of action, and dose-effect relationships are unclear, making standardized production and clinical application difficult; in addition, some TCM compositions may interact with other treatment methods (such as chemotherapy drugs), increasing treatment risks.

[0004] Therefore, there is an urgent need in this field to develop a novel traditional Chinese medicine composition that can effectively target the pathological characteristics of brain tumors, improve treatment efficacy, reduce side effects, and possess a clear pharmacological basis and reproducibility. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a safe, efficient, and easily standardized traditional Chinese medicine treatment plan.

[0006] The purpose of this invention is to provide a traditional Chinese medicine composition for treating brain tumors, its preparation method, and its application.

[0007] On one hand, the present invention provides a traditional Chinese medicine composition for treating brain tumors, the composition comprising the following raw materials in parts by weight: 3 - 5 parts of Tianlong, 3 - 5 parts of Scolopendra, 6 - 12 parts of Sparganii Rhizoma, 6 - 12 parts of Curcumae Rhizoma, 15 - 60 parts of Hedyotis Diffusa, 9 - 12 parts of Acorus Tatarinowii Rhizome, 10 - 20 parts of Taraxacum Mongolicum Hand.-Mazz., 9 - 12 parts of Poria, 9 - 12 parts of Atractylodes Macrocephala Koidz.

[0008] According to a specific embodiment of the present invention, preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 3 - 5 parts of Tianlong, 3 - 5 parts of Scolopendra, 6 - 12 parts of Sparganii Rhizoma, 6 - 12 parts of Curcumae Rhizoma, 20 - 40 parts of Hedyotis Diffusa, 9 - 12 parts of Acorus Tatarinowii Rhizome, 10 - 20 parts of Taraxacum Mongolicum Hand.-Mazz., 9 - 12 parts of Poria, 9 - 12 parts of Atractylodes Macrocephala Koidz.

[0009] According to a specific embodiment of the present invention, preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 3 parts of Tianlong, 3 parts of Scolopendra, 6 - 12 parts of Sparganii Rhizoma, 6 - 12 parts of Curcumae Rhizoma, 15 - 60 parts of Hedyotis Diffusa, 9 - 12 parts of Acorus Tatarinowii Rhizome, 15 parts of Taraxacum Mongolicum Hand.-Mazz., 9 - 12 parts of Poria, 9 - 12 parts of Atractylodes Macrocephala Koidz.

[0010] According to the traditional Chinese medicine composition described in claim 1, wherein the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 3 parts of Tianlong, 3 parts of Scolopendra, 9 parts of Sparganii Rhizoma, 9 parts of Curcumae Rhizoma, 30 parts of Hedyotis Diffusa, 12 parts of Acorus Tatarinowii Rhizome, 15 parts of Taraxacum Mongolicum Hand.-Mazz., 12 parts of Poria, 12 parts of Atractylodes Macrocephala Koidz.

[0011] According to a specific embodiment of the present invention, preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 3 parts of Tianlong, 3 parts of Scolopendra, 9 parts of Sparganii Rhizoma processed with vinegar, 9 parts of Curcumae Rhizoma processed with vinegar, 30 parts of Hedyotis Diffusa, 12 parts of Acorus Tatarinowii Rhizome, 15 parts of Taraxacum Mongolicum Hand.-Mazz., 12 parts of Poria, 12 parts of Atractylodes Macrocephala Koidz.

[0012] In the present invention, the preparation method of Sparganii Rhizoma processed with vinegar includes: taking the cleaned Sparganii Rhizoma and immersing it in a boiling water pot until it is five or six - tenths cooked, adding vinegar and then cooking until it is eight - tenths cooked, stopping adding water and stopping continuous heating, leaving it in the pot to be thoroughly steamed, sucking up the remaining soup, taking it out, drying until there is no moisture on the outer skin, slicing, and drying in the sun; wherein the mass ratio of Sparganii Rhizoma to vinegar is 100:20 - 40, preferably 100:30.

[0013] In the present invention, the preparation method of Curcumae Rhizoma processed with vinegar includes: taking the cleaned Curcumae Rhizoma slices, adding a certain amount of vinegar and mixing evenly, moistening until the vinegar liquid is completely absorbed; putting the Curcumae Rhizoma slices after absorbing vinegar into a frying container, heating with low heat until the surface is slightly yellow and slightly charred, taking it out, cooling and then sieving to remove debris; wherein the mass ratio of Curcumae Rhizoma to rice vinegar is 100:15 - 25; preferably 100:20.

[0014] According to a specific embodiment of the present invention, preferably, in the traditional Chinese medicine composition, the ratio of the parts by weight of Tianlong to Scolopendra is 1:1.

[0015] According to a specific embodiment of the present invention, preferably, the weight ratio of Sparganium rhizome to Curcuma rhizome in the traditional Chinese medicine composition is 1:1.

[0016] According to a specific embodiment of the present invention, preferably, the weight ratio of Poria cocos to Atractylodes macrocephala in the traditional Chinese medicine composition is 1:1.

[0017] According to a specific embodiment of the present invention, the traditional Chinese medicine composition further includes, by weight, parts of raw materials: Licorice root 3-9 parts, dried tangerine peel 3-9 parts, and roasted hawthorn, malt, and barley sprouts 9-15 parts.

[0018] In this invention, "Jiao San Xian" is a collective term for charred malt, charred Shenqu (a type of fermented wheat), and charred hawthorn.

[0019] On the other hand, the present invention also provides a method for preparing the above-mentioned traditional Chinese medicine composition, wherein the preparation method includes: Each raw material of the composition is pulverized and sieved to obtain raw material powder. A predetermined weight of each raw material powder is weighed and mixed to obtain the traditional Chinese medicine composition.

[0020] In some specific embodiments of the present invention, the preparation method includes: (1) After cleaning and cutting each raw material of the Chinese herbal medicine composition, dry it to a moisture content of less than 5%; (2) Thoroughly crush the powder and use an automated sieving system to control the powder particle size to ensure that it becomes fine powder. It must pass through the No. 5 sieve specified in the Chinese Pharmacopoeia. Among them, Tianlong, centipede and calamus are crushed separately and then mixed. (3) Mix thoroughly and evenly.

[0021] (4) Sterilize by cobalt-60 irradiation; (5) Packaging, and strictly in accordance with the standards of the Chinese Pharmacopoeia, conduct comprehensive testing on the properties, particle size, moisture, filling volume difference, microbial limit, and active ingredient content of the finished product. Only after passing the test can it be used.

[0022] On the other hand, the present invention also provides a pharmaceutical composition for treating brain tumors, which is prepared from the above-mentioned traditional Chinese medicine composition.

[0023] According to a specific embodiment of the present invention, preferably, the brain tumor includes a pituitary adenoma, a brain metastasis, or a glioma.

[0024] According to a specific embodiment of the present invention, preferably, the glioma includes glioblastoma.

[0025] According to a specific embodiment of the present invention, preferably, the pharmaceutical composition further includes one or more pharmaceutically acceptable excipients.

[0026] According to a specific embodiment of the present invention, preferably, the dosage form of the pharmaceutical composition includes one or more of the following: pills, capsules, tablets, powders, solutions or suspensions.

[0027] On the other hand, the present invention also provides the above-mentioned traditional Chinese medicine composition, the above-mentioned preparation method, and the application of the above-mentioned pharmaceutical composition in the preparation of a drug for treating brain tumors.

[0028] According to a specific embodiment of the present invention, preferably, the brain tumor includes a pituitary adenoma, a brain metastasis, or a glioma.

[0029] According to a specific embodiment of the present invention, preferably, the glioma is...

[0030] The traditional Chinese medicine composition described in this invention is a brain tumor powder, which is a potent agent for resolving blood stasis and eliminating symptoms.

[0031] The brain tumor powder formula of this invention uses Tianlong (also known as gecko) as the chief ingredient. Tianlong is mainly used to dispel wind, calm fright, disperse nodules, and detoxify. Because of its ability to move around and its ability to regenerate its tail after being cut off, it is particularly good at scraping away wind from the meridians and blood vessels. In addition, it is a substance with flesh and blood, and its taste is salty and cold. It is both nourishing and does not retain pathogens, so it is the chief ingredient (it is best to use the whole Tianlong when making the medicine, keeping the tail).

[0032] Centipede, Sparganium, and Curcuma are used as assistant herbs. Centipede can unblock the meridians, relieve pain, calm the nerves, and stop spasms. This herb has a pungent taste and is most effective at dispelling wind from the meridians, reaching every part of the body. Moreover, the other three herbs are warm in nature, which can both enhance the wind-dispelling, detoxifying, and calming effects of Sparganium and restrain its salty and cold nature. Unblocking the meridians requires breaking up blood stasis. Tumors are a cluster of abnormal proliferating foci that require a large network of blood vessels to support their growth. This is how strong herbs can cure serious illnesses; their ability to break up blood stasis is most effective. The most potent leeches are blister beetles and venomous leeches. However, blister beetles and leeches are extremely virulent and can easily cause systemic bleeding, damaging the spleen and stomach. At the same time, in order to dispel wind and remove blood stasis, it is necessary to have qi-regulating herbs. Sparganium rhizome promotes blood circulation and qi, while Curcuma zedoaria promotes qi circulation and blood circulation. These two herbs are essential for breaking up blood stasis and unblocking the meridians. The four herbs complement each other but also restrain each other, allowing the blood to flow within the vessels. Clinically, for patients with robust constitutions and masses, the original dosage can be used to counteract the poison with poison. For patients with weak constitutions, the dosage can be adjusted accordingly.

[0033] It is supplemented with Acorus tatarinowii, dandelion, and Hedyotis diffusa. Among them, the nine-jointed Acorus tatarinowii is the best, and the twelve-jointed variety is even better. The volatile oil of Acorus tatarinowii has been confirmed in published literature at home and abroad to cross the blood-brain barrier, help improve the integrity of the blood-brain barrier, and even enter the brain in synergy with the active ingredients of other drugs (doi: 10.1016 / j.jep.2025.119484), achieving the effect of "guiding the medicine upward". It can also work together with dandelion and Hedyotis diffusa to promote diuresis and eliminate phlegm, so that qi goes upward and water goes downward, clearing heat and detoxifying, dispersing swelling and lumps.

[0034] Poria cocos and Atractylodes macrocephala are used to regulate and tonify the spleen and stomach and nourish the heart spirit. In clinical practice, the overall anti-tumor formula always takes the orderly rise and fall of the spleen and stomach as the main responsibility. Therefore, considering the whole formula, the combination of nine herbs is in an orderly state of warmth and coolness, with elimination and supplementation combined, making it an excellent formula for dissipating stasis and masses and expelling wind to relieve convulsion.

[0035] For long-term use, it is necessary to prevent liver and kidney function damage, pay attention to regulating and tonifying the spleen and stomach, and licorice, dried tangerine peel, and stir-fried medicated leaven can be added to tonify the spleen and boost qi, resolve phlegm, and further neutralize the toxicity of insect drugs.

[0036] In terms of dosage, this invention also complies with the regulations of the Chinese Pharmacopoeia. The dosage ratio of Agkistrodon acutus and Scolopendra subspinipes is preferably 1:1, the dosage ratio of Sparganium stoloniferum and Curcuma zedoaria is 1:1, and the dosage ratio of Poria cocos and Atractylodes macrocephala is also 1:1. Since the "ministerial herbs" cannot exceed the "sovereign herb", Agkistrodon acutus is the sovereign herb. In clinical practice, for mild cases such as WHO grade I, II gliomas or astrocytomas, a relatively smaller dosage can be used, while for cases such as WHO grade III, WHO grade IV or patients who have undergone multiple brain tumor surgeries, a larger dosage can be used; for Scolopendra subspinipes and the like, after all, they contain toxins. According to the dosage of the sovereign herb Agkistrodon acutus, the ministerial herbs cannot increase the physical burden of patients, and there may be a little toxicity remaining during the decoction process because the process is not unified. For patients with a deficiency of healthy qi and preponderance of pathogenic factors, the healthy qi cannot be further depleted.

[0037] This invention discloses a traditional Chinese medicine composition for treating brain tumors, its preparation method and application. The provided brain tumor powder, through the rigorous compatibility of "sovereign, ministerial, adjuvant, and guiding herbs" and the synergistic effect of specific medicinal flavors, the combination of nine herbs is in an orderly state of warmth and coolness, with elimination and supplementation combined, achieving the effects of dissipating stasis and masses and expelling wind to relieve convulsion; its unique compatibility mechanism enhances the treatment intensity of promoting blood circulation to remove stasis and nodules while effectively restricting the toxic and side effects of insect drugs, protecting the spleen and stomach without damaging healthy qi, ensuring the safety of medication; in addition, based on the syndrome differentiation and treatment strategy of flexibly adjusting the dosage of the sovereign herb, this composition can be applied to various types of brain tumor patients, showing significant progress in terms of efficacy, safety, and clinical adaptability. Description of the Drawings

[0038] Figure 1 Results of CCK-8 assay.

[0039] Figure 2 Results of A172 EDU assay.

[0040] Figure 3 Results of T98G EDU assay.

[0041] Figure 4 Results of cell cloning.

[0042] Figure 5 Results of cell migration.

[0043] Figure 6 Results of cell invasion.

[0044] Figure 7This is the result of the ELISA test.

[0045] Figure 8 This is the result of the A172 Q-PCR test.

[0046] Figure 9 This is the result of T98G q-PCR detection.

[0047] Figure 10 The image shows the results of enhanced MRI of the head in Case 1.

[0048] Figure 11 The results of enhanced MRI of the head in Case 2. Detailed Implementation

[0049] The present invention will be further described in detail below through specific embodiments, but this does not limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained commercially.

[0050] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0051] The raw materials used to prepare the traditional Chinese medicine composition of the present invention are formulated according to the mass ratio. During production, the ratio can be increased or decreased accordingly. For example, for large-scale production, the unit can be kilograms or tons, while for small-scale production, the unit can be grams. The weight can be increased or decreased, but the mass ratio of the raw materials between each component follows the prescribed ratio.

[0052] The experimental instruments and reagents used in the following examples are shown below: 1. Instruments

[0053] 2. Main reagents

[0054] Example 1

[0055] This embodiment provides a method for preparing a traditional Chinese medicine composition, which includes the following raw materials in parts by weight: 3 parts of Tianlong, 3 parts of centipede, 9 parts of Sanleng, 9 parts of Ezhu, 30 parts of Baihuasheshecao, 12 parts of Shichangpu, 15 parts of Pugongying, 12 parts of Fuling, and 12 parts of Baizhu.

[0056] Detailed production steps: (1) After cleaning and cutting each raw material of the composition, dry it to a moisture content of less than 5%; (2) Thoroughly crush the powder and use an automated sieving system to control the powder particle size to ensure that it becomes fine powder and passes through the No. 5 sieve specified in the Chinese Pharmacopoeia; among them, Tianlong, centipede and calamus are crushed separately and then mixed. (3) Weigh the above-mentioned raw materials by weight and mix them thoroughly and evenly to obtain the powder; (4) Sterilize by cobalt-60 irradiation; (5) Packaging, and strictly in accordance with the standards of the Chinese Pharmacopoeia, conduct comprehensive testing on the properties, particle size, moisture, filling weight difference, microbial limit, and active ingredient content of the finished product. If the test results are qualified, the mixed powder of the Chinese medicine composition is prepared.

[0057] Example 2

[0058] This embodiment provides a method for preparing the mixed powder of traditional Chinese medicine composition obtained in Example 1 into a lyophilized powder for brain tumor powder. The specific preparation process is as follows: Raw material processing: The mixed powder prepared as in Example 1 was used; Pre-freezing stage: Load the raw materials into a freeze dryer and rapidly cool them to below -30°C to completely freeze the moisture into ice crystals; Sublimation drying (single drying): Vacuum is drawn, and ice crystals are directly sublimated into water vapor by heating, removing most of the moisture; Analysis and drying (secondary drying): The temperature is raised to 40℃ to further remove residual moisture and bound water, reducing the moisture content to below 3%; secondary drying time is 3 hours; Post-processing; pulverization: The freeze-dried lumps are processed into fine powder (2000 mesh) using a pulverizer. Packaging: Double-sealed container, moisture-proof and oxidation-proof.

[0059] Example 3: In vitro observation of the inhibitory effect of the traditional Chinese medicine composition of the present invention on the growth of glioma.

[0060] I. Experimental Procedure

[0061] 1. Preparation of serum containing the traditional Chinese medicine composition (compound NLP) of this application

[0062] Compound NLP: 3g of Tianlong (a type of medicinal herb), 3g of Wugong (a type of centipede), 9g of Sanleng (a type of herb), 9g of Ezhu (a type of herb), 30g of Hedyotis diffusa (a type of herb), 12g of Shichangpu (a type of herb), 15g of Pugongying (a type of herb), 12g of Fuling (a type of herb), and 12g of Baizhu (a type of herb). The lyophilized powder of the brain tumor powder was prepared according to the preparation methods of Examples 1 and 2.

[0063] SD rats (200±20g) were randomly divided into an NLP group and a control group. The daily dose for rats was calculated using the equivalent dose conversion formula based on body surface area: coefficient = (human weight / rat weight) 1 / 3. Human weight was estimated at 70kg and rat weight at 0.2kg. The lyophilized powder of the herbal composition used in this invention contained 96g of raw herb, resulting in an equivalent medium dose reference value of approximately 9.67 g / kg. Therefore, the NLP group was administered a low dose of 8g / kg dissolved in 4ml of physiological saline via gavage. The control group received the same dose of physiological saline once daily for 3 days. All groups were administered the same dose at the same time. All rats maintained a normal diet, and blood samples were collected from the heart one hour after the last administration on the third day. Blood samples were stored overnight at 4℃, centrifuged at 3000r / min for 15 minutes, and the supernatant was collected. Serum was sterilized using a 0.22µm filter membrane, inactivated in a 50℃ water bath for 30 minutes, and stored at -20℃.

[0064] 2. Cell culture and culture conditions

[0065] A172 and T98G glioblastoma cells were routinely cultured in DMEM medium containing 10% FBS, 100 μg / mL streptomycin, and 100 U / mL penicillin, and incubated overnight at 37°C in a 5% CO2 incubator.

[0066] The specific training and grouping are as follows: A172 cells: DMEM + 1% P / S + serum T98G cells: MEM + 1% P / S + serum Grouped by serum: the control group consisted of 10% control serum. The low-dose group consisted of 2.5% NLP-containing serum and 7.5% control serum. The medium-dose group consisted of 5% NLP-containing serum and 5% control serum. The high-dose group consisted of 10% NLP-containing serum. A172 and T98G cells were treated as controls and with drug-containing serum for 48 hours as described above.

[0067] Experimental Groups: A.Control B.NLP-L C.NLP-M D.NLP-H 3. CCK-8 test A172 and T98G cells were cultured for 48 hours after being added to different concentrations of serum according to their respective groups, and then cell viability was detected. The steps are as follows: 1) After culturing cells for 24 hours, digest and collect cells from each group, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend each group of cells in fresh complete culture medium. 2) Seed cell suspension (100 μL / well) into 96-well plates and pre-culture the plates at 37°C in a 5% CO2 incubator for a period of time; 3) After the cells have completely adhered to the cell wall, treat the cells with serum containing different drugs for 48 hours; 4) Add 10 μL of CCK-8 reaction solution to each well; 5) Incubate the culture plate in an incubator for 2 hours; 6) Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0068] 4. EDU testing

[0069] A172 and T98G cells were incubated with different concentrations of serum for 48 hours according to their respective groups, and cell proliferation was then detected. The steps are as follows: 1) Take cells in the logarithmic growth phase, using 5 × 10⁶ cells per well. 5 One cell was seeded in a 24-well plate. The treated climbing sheet was placed on the cell suspension and closely attached to the culture dish. The cells were cultured until the normal growth stage was reached. 2) Remove the supernatant culture medium, add different concentrations of drug serum to the cells according to the group, 500 μL per well, and incubate for 48 h; 3) Dilute the EDU solution with complete cell culture medium at a ratio of 500:1 to prepare an appropriate amount of 20 μM EDU culture medium; 4) Add an equal volume of 20 μM EDU medium preheated at 37℃ and incubate for 2 hours, then discard the medium; 5) Wash the cells three times with washing solution, 5 minutes each time; 6) Add 1 mL of fixative to each well and fix at room temperature for 15 min; 7) Remove the fixative and wash the cells three times with 1 mL of washing buffer per well for 3-5 min each time; 8) Remove the washing solution and incubate each well with 1 mL of permeabilizing solution at room temperature for 10 min; 9) Remove the permeabilizing solution and wash the cells in each well with 1 mL of washing buffer 1-2 times, 5 minutes each time.

[0070] 10) Add the prepared Click Additive Solution reaction solution to a 24-well plate, 100 μL per well. Gently shake the plate to ensure that the reaction mixture can evenly cover the sample. Incubate at room temperature in the dark for 30 min. 11) Remove the Click reaction solution and wash three times with washing solution, 5 minutes each time; 12) Counterstaining the nucleus: Add DAPI and incubate in the dark for 5 min to stain the nucleus. Wash three times with TBST for 5 min each time. 13) Mounting: Blot the liquid off the slide with absorbent paper, mount the slide with mounting solution containing anti-fluorescence quencher, and observe and acquire images under a fluorescence microscope.

[0071] 5. Colony formation determination

[0072] A172 and T98G cells were treated with compound serum containing brain tumor powder for 24 hours according to their respective groups. Cells in the logarithmic growth phase were collected from each group and cell suspensions were prepared. After counting, the cells from each group were seeded into 6-well plates, 200 cells per well, ensuring even distribution. The cells were then incubated in a CO2 incubator for 7 days. After cloning, the cells were carefully washed twice with PBS. 1 mL of anhydrous methanol was added to each well for fixation for 15 minutes. The methanol was then removed, and the cells were stained with an appropriate amount of crystal violet for 20 minutes. The crystal violet was then removed, and excess staining solution was washed away with PBS. The cells were then allowed to dry at room temperature. The 6-well plates were placed on white paper, and photographs were taken, one photograph per well.

[0073] 6. Transwell's ability to detect intrusions and migrations.

[0074] 6.1 Cell migration

[0075] 1) The A172 and T98G cell lines were digested, centrifuged, counted, and diluted with DMEM basal medium to a concentration of 1×10⁻⁶ cells / mL. 5 Cell suspension of cells / mL; 2) Set up three replicates for each group of cells. Add 600 μL of DMEM complete medium containing 20% ​​FBS to the matching 24-well plate. Carefully place the chamber into the well to avoid generating air bubbles. 3) Add 200 μL of well-mixed cell suspension to the chamber and incubate the cells in an incubator for 24 hours; 4) Remove the chamber, wash off the culture medium with PBS, fix with anhydrous methanol at 4°C for 30 min, stain with crystal violet for 20 min, rinse with PBS to remove excess dye, and finally wipe the cells inside the chamber with a cotton swab; randomly select 12 fields of view to observe the cells and take pictures, use ImageJ software to calculate the number of cells, and use Graphad software to organize the results and plot them.

[0076] 6.2 Cell Invasion

[0077] 1) Transwell chamber preparation. 50 mg / L Matrigel was diluted 1:4, and 50 µL of the diluted solution was used to coat the upper surface of the bottom membrane of the Transwell chamber. The solution was placed at 37 °C for 4 hours to allow Matrigel to polymerize into a gel.

[0078] 2) Digest, centrifuge, and count the A172 and T98G cell lines, and dilute them with DMEM basal medium to 1×10⁻⁶. 5 Cell suspension of cells / mL; 3) Set up three replicates for each group of cells. Add 600 μL of DMEM complete medium containing 20% ​​FBS to the matching 24-well plate. Carefully place the chamber into the well to avoid generating air bubbles. 4) Add 200 μL of well-mixed cell suspension to the chamber and incubate the cells in an incubator for 24 hours; 5) Remove the chamber, wash off the culture medium with PBS, fix with anhydrous methanol at 4°C for 30 min, stain with crystal violet for 20 min, rinse with PBS to remove excess dye, and finally wipe the cells inside the chamber with a cotton swab. 6) Randomly select 12 fields of view to observe cells and take pictures. Use ImageJ software to calculate the number of cells, and use Graphad software to organize the results and plot them.

[0079] 7. ELISA test

[0080] Cells from each group were collected, and ROS levels in the cells were detected using ELISA.

[0081] 1) Preparation: Remove the reagent kit from the refrigerator and allow it to equilibrate to room temperature for 30 minutes.

[0082] 2) Solution preparation: Dilute the 20-fold concentrated washing solution with distilled water to a solution of the original concentration.

[0083] 3) Add standards and test samples: Take a sufficient number of enzyme-labeled plates, fix them on the frame, and set up standard wells, test sample wells and blank control wells respectively. Record the position of each well. Add 50 μL of standard to the standard wells; add 10 μL of test sample to the test sample wells, and then add 40 μL of sample diluent; do not add any to the blank control wells.

[0084] 4) Incubation: Incubate in a 37℃ water bath or constant temperature incubator for 30 minutes.

[0085] 5) Washing the plate: Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid, let stand for 1 minute, shake off the washing liquid, pat dry on absorbent paper, and repeat the washing process 4 times (or you can use a plate washer to operate according to the instruction manual).

[0086] 6) Add enzyme-labeled working solution: Add 50 μL of enzyme-labeled working solution to each well, and do not add it to the blank control wells.

[0087] 7) Incubation: Incubate in a 37℃ water bath or constant temperature incubator for 30 minutes.

[0088] 8) Washing the plate: Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid, let stand for 1 minute, shake off the washing liquid, pat dry on absorbent paper, and repeat the washing process 4 times (or you can use a plate washer to operate according to the instruction manual).

[0089] 9) Color development: Add 50 μL of color developer A solution to each well, then add 50 μL of color developer B solution, mix with a plate mixer for 30 seconds (or gently shake and mix by hand for 30 seconds), and develop color at 37°C in the dark for 15 minutes.

[0090] 10) Termination: Remove the microplate and add 50 μL of stop solution to each well to terminate the reaction (the color will immediately change from blue to yellow).

[0091] 11) Measurement: Zero the blank well and measure the absorbance (OD value) of each well at a wavelength of 450nm within 15 minutes after termination.

[0092] 8. q-PCR detection

[0093] The expression of COX-2 and IL-6 in cells of each group was detected by q-PCR, and the steps are as follows: 8.1 RNA Extraction 1) Cell treatment. Add 1 mL of Trizol to a 6-well plate and place it in a clean bench at room temperature for 10 min; 2) Transfer the lysis buffer to a new 1.5 mL centrifuge tube, add 200 μL of chloroform, shake well, let stand at room temperature for 2 min, and centrifuge at 12000 rpm for 10 min at 4 °C. 3) Transfer the supernatant to a new 1.5 mL centrifuge tube, add 500 μL of isopropanol, mix well, let stand at room temperature for 15 min, centrifuge at 12000 rpm for 15 min at 4 °C, and discard the supernatant. 4) Add 1 mL of 75% anhydrous ethanol to rinse the precipitate, centrifuge at 12000 rpm for 5 min at 4℃, and discard the supernatant. 5) Add 1 mL of anhydrous ethanol, rinse the precipitate, centrifuge at 12000 rpm for 5 min at 4 °C, discard the supernatant, and dry at room temperature for 10 min. 6) Add 40 μL of DEPC water to dissolve the RNA and store it at -80℃ for later use.

[0094] 8.2 Reverse transcription reaction

[0095] Prepare the following reaction system for reverse transcription. Reaction conditions: 42℃, 30 min; 85℃, 10 min.

[0096]

[0097] 8.3 qPCR reaction

[0098] 1) Design primers

[0099] 2) Prepare the following reaction system for real-time quantitative PCR. Thoroughly mix the solutions in the tubes using a vortex mixer, and briefly centrifuge at low speed. Reaction conditions: 95℃, 3 min denaturation; 95℃, 12 s; 62℃, 40 s; 40 cycles.

[0100]

[0101] 3) Spotting: Add the mixed liquid from step (2) into the well plate, ensuring 3 replicates for each gene in each sample.

[0102] 4) PCR reaction: The Real-time PCR instrument used is the MX3000P Real-time Fluorescence Quantitative PCR Instrument, and the PCR program has been optimized.

[0103] Place the 8-tube strip that has been spotted in step (3) onto a Real-time PCR instrument for PCR reaction.

[0104] 5) Reaction conditions: 95℃, 3 min denaturation; 95℃, 12 s; 62℃, 40 s; 40 cycles.

[0105] 9. Data Processing

[0106] All data are presented as mean ± standard deviation (Mean ± SD). One-way ANOVA was used to compare differences between groups. All statistical analyses were performed using GraghPad 7.0 software. A p-value < 0.05 was considered statistically significant between groups, and different symbols were used to represent the differences.

[0107] II. Experimental Results

[0108] 1. CCK-8 detection

[0109] CCK-8 test results are as follows Figure 1 As shown, where p < 0.05, The values ​​indicate p<0.01 vs Control group; #p<0.05, ## indicates p<0.01 vs NLP-L group; & indicates p<0.05, && indicates p<0.01 vs NLP-M group. Compared with the control group, the cell viability decreased sequentially with the addition of low, medium and high doses of NLP serum, and the T98G cell line showed a more significant effect.

[0110] A172 cell viability: Control > NLP-L > NLP-M > NLP-H.

[0111] T98G cell viability: Control > NLP-L > NLP-M > NLP-H.

[0112] 2. EDU testing

[0113] EDU test results are as follows Figures 2-3 As shown, where, Figure 2 The results are for the A172 EDU test. Figure 3 The results of T98G EDU detection are as follows: Compared with the Control group, when low, medium and high doses of NLP serum were added in sequence, the cell fluorescence intensity of EDU detection gradually decreased and the cell proliferation capacity gradually decreased.

[0114] A172 cell proliferation capacity: Control > NLP-L > NLP-M > NLP-H.

[0115] T98G cell proliferation capacity: Control > NLP-L > NLP-M > NLP-H.

[0116] 3. Colony formation determination

[0117] Cell cloning results as follows Figure 4 As shown, where p<0.05, The values ​​indicate p<0.01 vs Control group; #p<0.05, ## indicate p<0.01 vs NLP-L group; & indicates p<0.05, && indicates p<0.01 vs NLP-M group; A172 cell cloning results showed that, compared with the Control group, there was no significant difference between the low-dose NLP serum group and the Control group. With the addition of medium and high doses of NLP serum, the number of A172 cells gradually decreased, and the cell cloning ability decreased sequentially.

[0118] A172 cell cloning ability: Control, NLP-L > NLP-M > NLP-H.

[0119] The T98G cell cloning results showed that, compared with the Control group, there was no significant difference between the low-dose NLP serum group and the Control group. When medium and high doses of NLP serum were added, the number of T98G cells gradually decreased, and the cell cloning ability decreased accordingly.

[0120] T98G cell cloning ability: Control, NLP-L > NLP-M > NLP-H.

[0121] 4. Transwell

[0122] Cell migration results as follows Figure 5 As shown, where, p<0.05, The values ​​indicate p<0.01 vs Control group; #p<0.05, ## indicate p<0.01 vs NLP-L group; & indicates p<0.05, && indicates p<0.01 vs NLP-M group; The migration results of A172 cells and T98G cells showed that, compared with the Control group, the intercellular space increased, the number of cells decreased, and the cell migration ability decreased sequentially with the addition of low, medium, and high doses of NLP serum.

[0123] A172 cell migration ability: Control > NLP-L > NLP-M > NLP-H.

[0124] T98G cell migration ability: Control > NLP-L > NLP-M > NLP-H.

[0125] Cell invasion results as follows Figure 6 As shown, p<0.05, The values ​​indicate p<0.01 vs Control group; #p<0.05, ## indicate p<0.01 vs NLP-L group; & indicates p<0.05, && indicates p<0.01 vs NLP-M group; The invasion results of A172 cells and T98G cells showed that, compared with the Control group, the intercellular space increased, the number of cells decreased, and the cell invasion ability decreased sequentially with the addition of low, medium, and high doses of NLP serum.

[0126] A172 cell invasion ability: Control > NLP-L > NLP-M > NLP-H.

[0127] T98G cell invasion ability: Control>NLP-L>NLP-M>NLP-H.

[0128] 5. ELISA test

[0129] ELISA test, such as Figure 7 As shown, where, p<0.05, The values ​​indicated p<0.01 in the control group; #p<0.05 and ## indicate p<0.01 in the NLP-L group; & indicate p<0.05 and && indicate p<0.01 in the NLP-M group; ELISA was used to detect cellular ROS levels. Results for A172 cells and T98G cells showed: Compared with the control group, the ROS level in cells gradually increased when low, medium and high doses of NLP serum were added sequentially.

[0130] A172 cell ROS levels: Control <NLP-L<NLP-M<NLP-H。

[0131] T98G cell ROS levels: Control <NLP-L<NLP-M<NLP-H。

[0132] 6. q-PCR detection

[0133] The expression of COX-2 and IL-6 mRNA in A172 cells was detected by q-PCR, and the results are as follows: Figure 8 As shown, where p<0.05, The values ​​indicated by # represent p<0.01 vs Control group; #p<0.05 and ## represent p<0.01 vs NLP-L group; & represent p<0.05 and && represent p<0.01 vs NLP-M group. Compared with the Control group, the expression levels of COX-2 and IL-6 mRNA in cells decreased sequentially after the addition of low, medium, and high doses of NLP serum.

[0134] COX-2 and IL-6 mRNA expression levels in A172 cells: Control > NLP-L > NLP-M > NLP-H.

[0135] q-PCR was used to detect the expression of COX-2 and IL-6 mRNA in T98G cells, and the results are as follows: Figure 9 As shown, where, p<0.05, The values ​​indicated by # represent p<0.01 vs Control group; #p<0.05 and ## represent p<0.01 vs NLP-L group; & represent p<0.05 and && represent p<0.01 vs NLP-M group. Compared with the Control group, the expression levels of COX-2 and IL-6 mRNA in cells decreased sequentially after the addition of low, medium, and high doses of NLP serum.

[0136] COX-2 and IL-6 mRNA expression levels in T98G cells: Control > NLP-L > NLP-M > NLP-H.

[0137] Example 3: Clinical trial observation of the inhibitory effect of the traditional Chinese medicine composition of the present invention on the growth of glioma.

[0138] Case 1: Male, 46 years old. On November 14, 2024, a right temporoparietal lobe biopsy was performed. Pathology: glioma, WHO grade 4, negative for methylation in gene testing. After completing concurrent chemoradiotherapy, he is currently continuing temozolomide 360mg chemotherapy (6 cycles). In February 2025, he began taking the traditional Chinese medicine composition of this invention, Brain Tumor Powder (composition formula: Tianlong 3g, Centipede 3g, Sparganium 9g, Curcuma zedoaria 9g, Hedyotis diffusa 30g, Acorus tatarinowii 12g, Taraxacum mongolicum 15g, Poria cocos 12g, Atractylodes macrocephala 12g), decocted in water and taken twice daily, 150mL each time. The total treatment duration was 120 days, during which no other treatments were administered. Figure 10 As shown, the enhanced MRI of the head performed on July 20, 2025, compared with the enhanced MRI of the head performed on May 30, 2025, shows that the tumor area has shrunk.

[0139] Case 2: A 66-year-old male was found to have a mass in the right basal ganglia and paraventricular region in November 2023. On November 15, 2023, a biopsy of the intracranial mass was performed, and pathology revealed a glioblastoma (WHO grade 4). He completed concurrent chemoradiotherapy, but no gene testing was performed. He received six cycles of bevacizumab (discontinued in March 2024), followed by long-term high-dose temozolomide chemotherapy. On August 29, 2024, a follow-up head MRI showed an increase in tumor size compared to May 28, 2024. In September 2024, he began treatment with the traditional Chinese medicine composition of this invention, Brain Tumor Powder (composition formula: Tianlong 3g, Centipede 3g, Sparganium 12g, Curcuma zedoaria 12g, Hedyotis diffusa 30g, Acorus tatarinowii 12g, Taraxacum mongolicum 12g, Poria cocos 12g, Atractylodes macrocephala 12g), decocted in water and taken orally twice daily, 120mL each time, for a total treatment duration of 210 days. No other treatments were administered during this period. Figure 11 As shown, on October 31, 2024, April 10, 2025, and July 10, 2025, the tumor shrank or at least did not progress in each follow-up cranial MRI.

[0140] The embodiments described above are merely preferred embodiments of the present invention, and not all feasible embodiments of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A traditional Chinese medicine composition for treating brain tumors, characterized in that, The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 3-5 parts of Tianlong (a type of herb), 3-5 parts of centipede, 6-12 parts of Sanleng (another type of herb), 6-12 parts of Ezhu (another type of herb), 15-60 parts of Baihuasheshecao (another type of herb), 9-12 parts of Shichangpu (another type of herb), 10-20 parts of Pugongying (another type of herb), 9-12 parts of Fuling (another type of herb), and 9-12 parts of Baizhu (another type of herb).

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 3-5 parts of Tianlong (a type of medicinal herb), 3-5 parts of centipede, 6-12 parts of Sanleng (another type of medicinal herb), 6-12 parts of Ezhu (another type of medicinal herb), 20-40 parts of Baihuasheshecao (another type of medicinal herb), 9-12 parts of Shichangpu (another type of medicinal herb), 10-20 parts of Pugongying (another type of medicinal herb), 9-12 parts of Fuling (another type of medicinal herb), and 9-12 parts of Baizhu (another type of medicinal herb).

3. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 3 parts of Tianlong (a type of medicinal herb), 3 parts of centipede, 9 parts of Sanleng (another type of medicinal herb), 9 parts of Ezhu (another type of medicinal herb), 30 parts of Baihuasheshecao (another type of medicinal herb), 12 parts of Shichangpu (another type of medicinal herb), 15 parts of Pugongying (another type of medicinal herb), 12 parts of Fuling (another type of medicinal herb), and 12 parts of Baizhu (another type of medicinal herb).

4. The traditional Chinese medicine composition according to any one of claims 1-3, characterized in that, The traditional Chinese medicine composition also includes, by weight, parts of raw materials: Licorice root 3-9 parts, dried tangerine peel 3-9 parts, and roasted hawthorn, malt, and barley sprouts 9-15 parts.

5. A method for preparing the traditional Chinese medicine composition according to any one of claims 1-4, characterized in that, The preparation method includes: Each raw material of the traditional Chinese medicine composition is pulverized and sieved to obtain raw material powder. A predetermined weight of each raw material powder is weighed and mixed to obtain the traditional Chinese medicine composition.

6. A pharmaceutical composition for treating brain tumors, characterized in that, It is prepared from the composition of any one of claims 1-4.

7. The pharmaceutical composition according to claim 6, characterized in that, The brain tumors include pituitary tumors, brain metastases, and gliomas.

8. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition further includes one or more pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the pharmaceutical composition includes one or more of the following: pills, capsules, tablets, powders, solutions, or suspensions.

10. The use of the traditional Chinese medicine composition according to any one of claims 1-4, the preparation method according to claim 5, and the pharmaceutical composition according to any one of claims 6-9 in the preparation of a medicament for treating brain tumors.

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