Traditional Chinese medicine effective component composition for intervening osteosarcoma angiogenesis

Abstract

The invention belongs to the technical field of medicines, and relates to a traditional Chinese medicine effective component composition for intervening osteosarcoma angiogenesis, which is composed of astragaloside and curcuma zedoary general flavone in a mass ratio of (1-4): 1. The effective component composition has a remarkable curative effect on tumor angiogenesis and regulation and control of osteosarcoma proliferation, growth and metastasis, all the components are natural effective monomer components, no hormone component or toxic or side effect exists, the composition is more easily absorbed by the human body, and no burden is caused to the liver and the kidney.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a composition of effective ingredients of traditional Chinese medicine for intervening in angiogenesis of osteosarcoma. Background Art

[0002] Osteosarcoma (OS) is a common primary malignant bone tumor originating from mesenchymal tissue, often affecting adolescents and children. Some patients with osteosarcoma develop lung metastases during diagnosis or treatment, and pulmonary metastasis is the leading cause of death. The development of osteosarcoma may be associated with Paget's disease, hereditary retinoblastoma, ionizing radiation, and alkylating agents. Modern medical treatments, including neoadjuvant chemotherapy, limb-sparing surgery, radiotherapy, and targeted therapy, are associated with rapid drug resistance, high tumor recurrence rates, and significant drug side effects. Therefore, the search for new treatments or medications is crucial.

[0003] Osteosarcoma relies on new blood vessels for vital nutritional support during its growth and spread. Inhibiting tumor angiogenesis can exert anti-tumor effects. Traditional Chinese medicine can mediate relevant signaling pathways, effectively inhibiting osteosarcoma angiogenesis and, consequently, inhibiting the malignant biological progression of osteosarcoma. Traditional Chinese medicine has no unified definition of osteosarcoma, classifying it under various categories, including "bone carbuncle," "stone carbuncle," "bone tumor," and "sarcoma." Qi deficiency and blood stasis are common throughout the pathogenesis of osteosarcoma. Qi-invigorating, blood-activating, and stasis-removing herbal remedies can inhibit excessive vascular growth and mitigate the progression of osteosarcoma.

[0004] Currently, Western medicine treatment for osteosarcoma primarily involves preoperative neoadjuvant chemotherapy with high-dose methotrexate, cisplatin, doxorubicin, and ifosfamide, combined with surgery and postoperative chemoradiotherapy. Patients with lung metastases can be treated with targeted anti-angiogenic drugs such as anlotinib. However, chemotherapy and targeted drugs have long treatment cycles and carry a high risk of adverse reactions such as bone marrow suppression, gastrointestinal reactions, hair loss, allergic reactions, and cardiac, hepato-renal, and hepatotoxicity, necessitating consistent monitoring of blood levels.

[0005] Guided by the holistic approach and syndrome differentiation and treatment principles of Traditional Chinese Medicine (TCM), the literature has reported studies on several TCM monomers, active ingredients, and compound formulas that intervene in angiogenesis in tumors such as lung cancer and liver cancer through signaling pathways. However, there is still a lack of unified guidelines for the TCM treatment of osteosarcoma, and there are relatively few studies reporting on TCM and their active ingredient combinations intervening in tumor angiogenesis and inhibiting osteosarcoma growth and metastasis. However, there are no reports on the compatibility and application of a combination containing the active ingredients of Astragalus and Curcuma to inhibit angiogenesis in osteosarcoma. Summary of the Invention

[0006] In response to the deficiencies in the prior art, the present invention relates to a Chinese medicine pair for intervening in tumor angiogenesis and treating osteosarcoma, and the medical use of the effective ingredient composition thereof, particularly the effects of different combinations of the anti-tumor active ingredients of the drug pair and the effective ingredient composition on anti-angiogenesis and inhibiting the growth and progression of osteosarcoma.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] The invention provides a traditional Chinese medicine effective ingredient composition for intervening in osteosarcoma angiogenesis. The effective ingredient composition consists of astragaloside IV and total flavonoids from Curcuma zedoariae. The mass ratio of the astragaloside IV to the total flavonoids from Curcuma zedoariae is 1-4:1.

[0009] Furthermore, the mass ratio of astragaloside IV to total flavonoids of Curcuma zedoaria is 2:1.

[0010] Furthermore, the preparation method of the total flavonoids of Curcuma zedoaria is as follows: (1) screening and washing Curcuma zedoaria leaves, drying at 60°C to constant weight, and crushing through a 40-mesh sieve to obtain Curcuma zedoaria leaf powder; (2) taking the Curcuma zedoaria leaf powder, wrapping it with filter paper, and placing it in a reflux device, adding 10 times the weight ratio of petroleum ether at 75°C, refluxing and extracting at 70°C for 8 hours until the petroleum ether is colorless, and recovering the petroleum ether by rotary evaporation, adding 10 times the weight ratio of acetone, refluxing and extracting at 70°C for 8 hours until the acetone is colorless, and recovering it by rotary evaporation, and vacuum drying to obtain the total flavonoids of Curcuma zedoaria.

[0011] Furthermore, the preparation method of astragaloside IV is as follows: (1) accurately weighing astragalus powder and placing it in an extraction kettle, injecting a certain amount of entrainer into the kettle, and sealing it with a cover; placing the extraction kettle in a supercritical device, and performing extraction by adjusting the temperature, pressure, and flow rate of the CO2 fluid; (2) after the extraction is completed, collecting the extract in a test tube, and rotating the kettle at 3000 r·min -1 The extract was concentrated and evaporated at a speed of 1000 nm, and then dissolved and quantified with anhydrous ethanol; (3) the astragaloside IV extract was weighed using a precision electronic balance and placed in a beaker, 10 mL of deionized water was added, and it was slightly heated to fully dissolve; after complete dissolution, it was cooled to room temperature and slowly transferred to a separatory funnel, and extracted in batches with 60 mL of water-saturated n-butanol solution, and the n-butanol solution was retained; it was then fully washed twice with 40% ammonia water, 10 mL each time, and the ammonia water after washing was discarded; (4) the retained n-butanol solution was evaporated to dryness under reduced pressure, and the residue was further dissolved with 5 mL of pure water, and passed through a perforated adsorption resin column, first eluted with 50 mL of pure water, the eluted water was removed, and then eluted with 30 mL of 40% ethanol, the eluent was removed, and eluted with 60 mL of 70% ethanol, the eluent was collected, and evaporated to dryness to obtain astragaloside IV.

[0012] Furthermore, in step (1), the extraction pressure is 30 MPa, the extraction temperature is 40°C, the extraction time is 6 min, and the entrainer flow rate is 5 mL·min-1 .

[0013] The present invention provides a Chinese patent medicine comprising a Chinese medicinal active ingredient composition for intervening in osteosarcoma angiogenesis. The dosage form of the Chinese patent medicine is selected from one of powders, tablets, granules, capsules, solutions, emulsions or suspensions.

[0014] Furthermore, the dosage form of the Chinese patent medicine is tablets.

[0015] The present invention provides use of the above-mentioned traditional Chinese medicine effective component composition for intervening in osteosarcoma angiogenesis in preparing a drug for treating osteosarcoma.

[0016] The Yellow Emperor's Classic of Internal Medicine states, "When vital energy is present within, pathogenic factors cannot enter; where pathogenic factors gather, the qi must be weakened." Deficiency of vital energy is the underlying basis for the development of osteosarcoma. The main etiologies and pathogenesis of this disease include "cold pathogenic qi accumulating in the muscles and meridians," "kidney qi deficiency causing toxic pathogens to gather in the bones," "qi and blood stagnation in the bones," and "accumulation of heat and toxins." Ye Tianshi's "Clinical Guide" from the Qing Dynasty noted that "at the beginning of the disease, qi stagnates in the meridians; over time, blood damage enters the collaterals," and "the collaterals are where blood gathers; with prolonged illness, stasis and blockage occur." When vital energy is deficient within the body, external pathogens invade, and vital energy cannot overcome them. Over time, phlegm, blood stasis, and toxins become tangible pathogens that block the collaterals, transforming into cancerous toxins that stagnate in the muscles and bones, forming tumors. Osteosarcoma patients suffer from a deficiency of vital energy, unable to promote normal blood flow. Over time, blood stasis blocks the collaterals, causing them to flow unchecked, proliferate uncontrollably, and become hyperactive. This traditional Chinese medicine theory of "prolonged illness entering the collaterals leads to hyperactivity and change" aligns with the Western medical theory of persistent abnormal blood vessels in tumors. Hyperactive collateral vessels continuously provide nutrients to tumor cells, promoting the proliferation, invasion, and migration of osteosarcoma. Because Qi deficiency and blood stasis are present throughout the pathogenesis of osteosarcoma, the use of Qi-invigorating, blood-activating, and stasis-removing drugs to inhibit excessive vascular growth may slow the progression of osteosarcoma.

[0017] Astragalus and Curcuma are a commonly used herbal duo for invigorating qi, promoting blood circulation, and removing blood stasis in the treatment of malignant tumors. Their combination can be used to treat various tumors, including gastric and liver cancer. Zhu Liangchun, a renowned Chinese medicine expert, believes that astragalus can replenish the five internal organs, while Curcuma promotes qi circulation and dissolves blood stasis. Their combined use can invigorate qi, remove blood stasis, and treat malignant tumors.

[0018] The inventors used different compatibility ratios of astragaloside IV and total flavonoids of Curcuma zedoaria from Astragalus membranaceus and Curcuma zedoaria to prepare a composition to intervene in the angiogenesis of osteosarcoma cells.

[0019] This prescription is based on the traditional Chinese medicine theory that "long-term illness enters the collaterals, causing hyperactivity of the collaterals." It uses Astragalus as the main ingredient to replenish the deficiency of the five internal organs and restore the body's vital energy to effectively resist pathogens. It also uses Curcuma as the auxiliary ingredient to promote qi circulation, break up blood stasis, and restrain the hyperplasia of the collaterals. The combination of the two can replenish qi and remove blood stasis to intervene in tumor blood vessel growth. The main pathogenesis of osteosarcoma patients is deficiency of vital energy, and long-term illness enters the collaterals and causes blood stasis. The present invention has a good effect of replenishing qi, activating blood circulation, and removing blood stasis, regulating qi and blood in the collaterals, and inhibiting the growth and metastasis of osteosarcoma. The composition of the present invention uses the effective active ingredients of Astragalus and Curcuma in different proportions. The dosage is small and specific, safe and non-toxic, without incompatibility taboos or dietary taboos, and the dosage form is convenient. It has a definite therapeutic effect and is suitable for long-term use by patients, with a treatment cycle of 14 days.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The effective ingredient composition of the present invention targets tumor angiogenesis and has significant therapeutic effects on regulating the proliferation, growth and metastasis of osteosarcoma.

[0022] (2) The Chinese medicinal herbs Astragalus membranaceus, from which the effective ingredient composition of the present invention is derived, are sweet and warm, and nourish the vital energy; Curcuma zedoaria, which is bitter and warm, and breaks up blood and eliminates symptoms; the combination of the two herbs has both attacking and nourishing effects, thus avoiding the body's loss of qi and damage to yin or other adverse effects caused by the excessive dryness or pungentness of the herbs.

[0023] (3) All components of the active ingredient composition of the present invention are natural effective monomer components, without any hormone components and toxic side effects, and are more easily absorbed by the human body without causing any burden on the liver and kidneys. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Different ratios of Astragalus and Curcuma have the best effect on the general signs and tumor changes in subcutaneous tumor-bearing nude mice, with 2:1 being the best.

[0025] A. Changes in body mass of nude mice in each group; B. Tumor volume of nude mice in each group at the last intervention; C. Changes in tumor volume of nude mice in each group; D. Tumor mass of nude mice in each group at the last intervention.

[0026] Note: NC: model group; 1:1: Astragalus-Zhuozhu 1:1 group; 2:1: Astragalus-Zhuozhu 2:1 group; 4:1: Astragalus-Zhuozhu 4:1 group. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

[0028] The preparation method of total flavonoids of Curcuma zedoaria is as follows: (1) screening and washing Curcuma zedoaria leaves, drying at 60°C to constant weight, and crushing through a 40-mesh sieve to obtain Curcuma zedoaria leaf powder; (2) taking the Curcuma zedoaria leaf powder, wrapping it with filter paper, and placing it in a reflux device, adding 10 times the weight ratio of petroleum ether at 75°C, refluxing and extracting at 70°C for 8 hours until the petroleum ether is colorless, and recovering the petroleum ether by rotary evaporation, adding 10 times the weight ratio of acetone, refluxing and extracting at 70°C for 8 hours until the acetone is colorless, and recovering it by rotary evaporation, and vacuum drying to obtain total flavonoids of Curcuma zedoaria.

[0029] The preparation method of astragaloside IV is as follows: (1) accurately weighing astragalus powder and placing it in an extraction kettle, injecting a certain amount of entrainer into the kettle, and sealing the kettle; placing the extraction kettle in a supercritical device, and performing extraction by adjusting the temperature, pressure, and flow rate of the CO2 fluid; (2) after the extraction is completed, collecting the extract in a test tube, and rotating the kettle at 3000 r·min -1 The extract was concentrated and evaporated at a speed of 1000 nm, and then dissolved and quantified with anhydrous ethanol; (3) the astragaloside IV extract was weighed using a precision electronic balance and placed in a beaker, 10 mL of deionized water was added, and it was slightly heated to fully dissolve; after complete dissolution, it was cooled to room temperature and slowly transferred to a separatory funnel, and extracted in batches with 60 mL of water-saturated n-butanol solution, and the n-butanol solution was retained; it was then fully washed twice with 40% ammonia water, 10 mL each time, and the ammonia water after washing was discarded; (4) the retained n-butanol solution was evaporated to dryness under reduced pressure, and the residue was further dissolved with 5 mL of pure water, and passed through a perforated adsorption resin column, first eluted with 50 mL of pure water, the eluted water was removed, and then eluted with 30 mL of 40% ethanol, the eluent was removed, and eluted with 60 mL of 70% ethanol, the eluent was collected, and evaporated to dryness to obtain astragaloside IV.

[0030] In step (1), the extraction pressure is 30 MPa, the extraction temperature is 40° C., the extraction time is 6 min, and the entrainer flow rate is 5 mL·min -1 .

[0031] Pharmacological Efficacy Example Experimental Study on the Effect of the Composition of the Effective Components of Traditional Chinese Medicine on Osteosarcoma

[0032] 1. Experimental Methods

[0033] 1. Cell Culture

[0034] The 143B cell line was inoculated in DMEM medium containing 10% FBS and cultured statically in a cell culture incubator at 37°C and 5% CO2. The cell growth status was observed and the cell culture medium was replaced in time. When the cells filled 70%-80% of the culture flask, they were digested with 0.25% trypsin and passaged at a passage ratio of 1:2-1:4.

[0035] 2. Animal Modeling, Grouping, and Drug Administration

[0036] Twenty-four BALB / c-nu nude mice were selected and inoculated with 143B osteosarcoma cells to establish a subcutaneous osteosarcoma tumor model. The specific method was as follows: human 143B osteosarcoma cells in the logarithmic growth phase were taken and made into a cell density of 1×10 7 mL -1 Take 0.1 mL of cell suspension and inject it into the right armpit of the mouse.

[0037] Astragaloside IV-total flavonoids of Curcuma zedoaria were prepared in different ratios: astragaloside IV-total flavonoids of Curcuma zedoaria 1:1 (astragaloside IV 50g, total flavonoids of Curcuma zedoaria 50g) (1:1 group), 2:1 (astragaloside IV 66g, total flavonoids of Curcuma zedoaria 33g) (2:1 group), 4:1 (astragaloside IV 80g, total flavonoids of Curcuma zedoaria 20g) (4:1 group).

[0038] After the model is successfully established, the tumor volume reaches 50 mm 3 The nude mice were randomly divided into the model group (NC group), astragaloside IV-total flavonoids of Curcuma 1:1, 2:1, 4:1 groups, with 6 mice in each group. The model group was given normal saline by gavage, and the astragaloside IV-total flavonoids of Curcuma 1:1, 2:1, 4:1 groups were given an equivalent dose of 9.25 g·kg -1 ) were gavaged, and the gavage volume was 0.01 mL·g -1 , administered continuously for 25 days.

[0039] 3.Indicator detection

[0040] Starting from the day of administration, nude mice were weighed at the same time point every 5 days to measure tumor mass and volume. The tumor volume was calculated as 0.52×ab 2 , a is the long diameter of the tumor, b is the short diameter of the tumor.

[0041] 4. Statistical Analysis

[0042] The experimental data were analyzed using GraphPad Prism 9.0 statistical software, and the results were expressed as mean ± standard deviation (x - ±s), one-way ANOVA was used to analyze the differences among the groups, and P < 0.05 was considered statistically significant.

[0043] 2. Experimental Results

[0044] See the results Figure 1 .

[0045] 2.1 Body weight of nude mice

[0046] Before administration, the nude mice in each group showed normal weight gain and behavior, with no significant differences in tumor size between groups. Following continued administration, the nude mice in the 4:1 group gradually lost weight and their weight decreased, while the weight of the nude mice in the other groups steadily increased. At the final measurement, the weight of the nude mice in the 4:1 group was significantly lower than that in the NC group, and there were no significant differences in weight between the other groups.

[0047] 2.2 Tumor volume in nude mice

[0048] Compared with the model group, the tumor volume growth of the 1:1 group and the 2:1 group slowed down significantly, and there was no significant difference in the tumor volume growth of the 4:1 group. At the same time, the tumor volume of the 2:1 group was significantly smaller than that of the 1:1 group.

[0049] 2.3 Tumor mass in nude mice

[0050] The tumor mass of the 1:1 and 2:1 groups was significantly smaller than that of the NC and 4:1 groups, and the tumor mass of the 2:1 group was smaller than that of the 1:1 group, and the differences were statistically significant.

[0051] Compared with the model group, the tumor mass and volume of the astragaloside IV-Zhuzhu total flavonoids 2:1 group were the smallest. The effective component composition of the traditional Chinese medicine of the present invention significantly inhibited the growth of osteosarcoma and had a significant tumor inhibition effect.

[0052] The above embodiments are merely examples for clarity of explanation and are not intended to limit the embodiments. Those skilled in the art will appreciate that other variations or modifications may be made based on the above description, and any such variations or modifications are still within the scope of protection of the present invention.

Claims

1. A Chinese medicinal active ingredient composition for intervening in angiogenesis of osteosarcoma, characterized in that: The effective component composition consists of astragaloside IV and total flavonoids from Curcuma zedoaria.

2. The effective component composition of traditional Chinese medicine for intervening in angiogenesis of osteosarcoma according to claim 1, characterized in that: The mass ratio of astragaloside IV to total flavonoids of Curcuma zedoaria is 1-4:

1.

3. The effective component composition of traditional Chinese medicine for intervening in angiogenesis of osteosarcoma according to claim 2, characterized in that: The mass ratio of astragaloside IV to total flavonoids from Curcuma zedoaria is 2:

1.

4. The Chinese medicinal active ingredient composition for intervening in osteosarcoma angiogenesis according to claim 1, characterized in that: The preparation method of the total flavonoids of Curcuma zedoaria is as follows: (1) screening and cleaning the leaves of Curcuma zedoaria, drying them at 60° C. to constant weight, and crushing them through a 40-mesh sieve to obtain Curcuma zedoaria leaf powder; (2) taking the Curcuma zedoaria leaf powder, wrapping it with filter paper, and placing it in a reflux device; adding 10 times the weight ratio of petroleum ether at 75° C., refluxing and extracting it at 70° C. for 8 h until the petroleum ether is colorless, and recovering the petroleum ether by rotary evaporation; adding 10 times the weight ratio of acetone, refluxing and extracting it at 70° C. for 8 h until the acetone is colorless, and recovering it by rotary evaporation; and vacuum drying to obtain the total flavonoids of Curcuma zedoaria.

5. The effective component composition of traditional Chinese medicine for intervening in osteosarcoma angiogenesis according to claim 1, characterized in that: The preparation method of astragaloside IV is as follows: (1) accurately weighing astragalus powder and placing it in an extraction kettle, injecting a certain amount of entrainer into the kettle, and sealing the kettle with a cover; placing the extraction kettle in a supercritical device, and performing extraction by adjusting the temperature, pressure, and flow rate of CO2 fluid; (2) after the extraction is completed, collecting the extract in a test tube, and rotating the kettle at 3000 r·min -1 The extract was concentrated and evaporated at a speed of 1000 nm, and then dissolved and quantified with anhydrous ethanol; (3) the astragaloside IV extract was weighed using a precision electronic balance and placed in a beaker, 10 mL of deionized water was added, and it was slightly heated to fully dissolve; after complete dissolution, it was cooled to room temperature and slowly transferred to a separatory funnel, and extracted in batches with 60 mL of water-saturated n-butanol solution, and the n-butanol solution was retained; it was then fully washed twice with 40% ammonia water, 10 mL each time, and the ammonia water after washing was discarded; (4) the retained n-butanol solution was evaporated to dryness under reduced pressure, and the residue was further dissolved with 5 mL of pure water, and passed through a perforated adsorption resin column, first eluted with 50 mL of pure water, the eluted water was removed, and then eluted with 30 mL of 40% ethanol, the eluent was removed, and eluted with 60 mL of 70% ethanol, the eluent was collected, and evaporated to dryness to obtain astragaloside IV.

6. The effective component composition of traditional Chinese medicine for intervening in angiogenesis of osteosarcoma according to claim 5, characterized in that: In step (1), the extraction pressure is 30 MPa, the extraction temperature is 40° C., the extraction time is 6 min, and the entrainer flow rate is 5 mL·min -1 .

7. The Chinese patent medicine of the Chinese medicinal active ingredient composition for intervening in osteosarcoma angiogenesis according to claim 1, characterized in that: The dosage form of the Chinese patent medicine is selected from one of powder, tablet, granule, capsule, solution, emulsion or suspension.

8. The Chinese patent medicine of the Chinese medicinal active ingredient composition for intervening in osteosarcoma angiogenesis according to claim 7, characterized in that: The dosage form of the Chinese patent medicine is tablets.

9. Use of the composition of effective ingredients of traditional Chinese medicine for intervening in angiogenesis of osteosarcoma according to any one of claims 1 to 8 in the preparation of a drug for treating osteosarcoma.

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