Traditional Chinese medicine compound exosome for treating ovarian cancer as well as preparation method and application thereof

Abstract

The invention discloses a traditional Chinese medicine compound exosome for treating ovarian cancer and a preparation method and application thereof. The preparation method of the traditional Chinese medicine compound exosome comprises the following steps: (1) soaking the raw material medicines in the traditional Chinese medicine compound in deionized water, boiling with big fire, and decocting for 35-45 minutes; then adjusting to slow fire, and continuously decocting for 30-35 minutes to obtain the traditional Chinese medicine compound water decoction. (2) preparing the traditional Chinese medicine compound exosome from the traditional Chinese medicine compound water decoction by an ultracentrifugation method; wherein the traditional Chinese medicine compound comprises the following raw material medicines: astragalus membranaceus, bighead atractylodes rhizome, cinnamon, curcuma zedoary, fructus lycii, seaweed, pericarpium citri reticulatae viride, lindera aggregata, turtle shell, oldenlandia diffusa, scorpio, artemisia apiacea, arisaema cum bile, edible tulip and radix ranunculi ternati. The prepared traditional Chinese medicine compound exosome can be used for treating ovarian cancer, and the curative effect of the exosome is superior to that of a traditional traditional Chinese medicine compound administration mode.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and more specifically, to a traditional Chinese medicine compound exosome for treating ovarian cancer, and a preparation method and application thereof. Background Art

[0002] Ovarian cancer is the most lethal gynecological malignancy, killing 184,799 women worldwide each year. Among all ovarian cancer subtypes, high-grade serous ovarian cancer (HGSOC) is the most common phenotype, accounting for approximately 80% of all ovarian cancer deaths. Due to unclear symptoms, only 20% of ovarian cancer cases can be detected and successfully treated in the early stages. Most HGSOC progress to the late stage, and patient outcomes are poor. Even after treatment, HGSOC often recurs due to the development of chemotherapy resistance, with an overall 5-year survival probability of 31%. Therefore, developing effective treatments for patients with ovarian cancer is crucial to improving prognosis and overall survival.

[0003] Traditional Chinese medicine (TCM) has a long history and is widely used in the field of disease treatment. It contains a variety of active ingredients and exhibits unique advantages in the treatment of complex diseases. However, the clinical application of TCM has long faced many challenges. On the one hand, many active ingredients of TCM have the problem of low solubility, which makes it difficult for them to fully dissolve in solution, greatly limiting the release and absorption of the drug. On the other hand, poor stability is also a major problem for TCM. Some active ingredients are prone to chemical changes during storage or in the body environment, resulting in reduced efficacy. Coupled with the influence of the first-pass effect, after oral TCM is absorbed through the gastrointestinal tract, some drugs will be metabolized and inactivated when passing through the liver, resulting in a reduction in the amount of drugs entering the systemic circulation and low bioavailability, which in turn affects the therapeutic effect. These problems have seriously affected the clinical efficacy of TCM.

[0004] Exosomes, nanoscale membrane vesicles secreted by cells, typically range in diameter from 30 to 300 nm and possess unique biological properties. Their membrane structure is similar to that of cell membranes, consisting of a phospholipid bilayer. This gives them excellent biocompatibility, making them less susceptible to recognition and clearance by the immune system upon entry, allowing them to persist stably within the body. Exosomes also possess the ability to cross multiple biological barriers, such as the blood-brain barrier and the placenta, enabling precise delivery of their cargo to target cells. Furthermore, exosomes protect the bioactive molecules they carry, preventing degradation during transport and ensuring their full function. Against this backdrop, the development of exosome-based Traditional Chinese Medicine (TCM) compound formulas for the treatment of ovarian cancer is expected to combine the multi-target advantages of TCM compound formulas with the precise delivery characteristics of exosomes, providing a new and effective approach for the treatment of ovarian cancer. This approach holds broad clinical application prospects and significant research value. Summary of the Invention

[0005] One object of the present invention is to provide a method for preparing exosomes from a traditional Chinese medicine compound for treating ovarian cancer. The exosomes from the traditional Chinese medicine compound prepared by this preparation method can effectively treat ovarian cancer.

[0006] Another object of the present invention is to provide a use of the above-mentioned traditional Chinese medicine compound exosomes in the preparation of a drug for treating ovarian cancer.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing exosomes from a traditional Chinese medicine compound for treating ovarian cancer, the method comprising the following steps:

[0009] (1) Soaking the raw materials in the Chinese herbal compound in deionized water, then boiling over high heat for 35-45 minutes; then lowering the heat to low heat and continuing to boil for 30-35 minutes to obtain the Chinese herbal compound decoction;

[0010] (2) preparing the Chinese medicine compound exosomes by ultracentrifugation of the Chinese medicine compound decoction;

[0011] Among them, the Chinese medicine compound includes the following raw materials: astragalus, white atractylodes, cinnamon, zedoaria, wolfberry, seaweed, green peel, lindera strychnifolia, turtle shell, white flower snake tongue grass, scorpion, Artemisia annua, Arisaema concinnum, Trillium gracile and cat's claw.

[0012] Furthermore, the Chinese medicine compound includes the following raw materials in parts by weight: 30-40 parts of astragalus, 15-20 parts of white atractylodes, 6-9 parts of cinnamon, 9-12 parts of zedoaria, 15-20 parts of wolfberry, 15-25 parts of seaweed, 6-9 parts of green peel, 6-9 parts of lindera, 12-15 parts of turtle shell, 15-30 parts of oldenlandia diffusa, 3-5 parts of scorpion, 12-20 parts of artemisia annua, 9-12 parts of arisaema, 12-25 parts of trillium, and 15-30 parts of cat's claw.

[0013] Illustratively, the Chinese herbal compound of the present invention includes the following raw materials in parts by weight: 30 parts of Astragalus, 15 parts of Atractylodes, 6 parts of cinnamon, 9 parts of Curcuma, 15 parts of Lycium barbarum, 15 parts of Sargassum, 6 parts of Citrus aurantium, 6 parts of Linderae scutellariae, 12 parts of Carapax tricolor, 15 parts of Hedyotis diffusa, 5 parts of Scorpio, 12 parts of Artemisia annua, 12 parts of Arisaema cinerea, 12 parts of Trillium gracile, and 15 parts of Cat's Claw.

[0014] The Chinese medicine formula of the present invention invigorates Qi and promotes blood circulation, detoxifies and fights cancer, and clears the three burners, making it suitable for the treatment of ovarian cancer. Astragalus and Atractylodes macrocephala nourish the Qi of the upper and middle burners, while Linderae radix and Cinnamon bark nourish the lower burner, warming and nourishing the kidney yang and promoting the rise of Yang Qi, and are collectively the monarch herbs; Curcuma zedoaria dissolves blood stasis, Citrus aurantium soothes the liver and promotes Qi circulation, Sargassum, Herba Cat's Claws, Trillium gracile, and Arisaema cinerea resolve phlegm and resolve nodules, Carapax tricolor softens and resolves nodules, and Lycium barbarum nourishes the liver and kidneys and strengthens vital Qi, and are collectively the minister herbs; Artemisia annua and Hedyotis diffusa clear heat, detoxify, and fight cancer, and are collectively the adjuvant herbs; Scorpion enters the meridians, attacks toxins, resolves nodules, and clears the meridians to relieve pain, and is collectively the guiding herb. When used to treat ovarian cancer, the exosomes prepared from these herbs are superior to traditional Chinese medicine compound treatments.

[0015] Furthermore, in the above preparation method, in step (1), the amount of deionized water used is 5-8 times the total weight of the raw material.

[0016] Furthermore, in step (1), the mixture is soaked for 30-60 minutes.

[0017] Furthermore, in step (2), the specific steps of preparing the Chinese medicine compound exosomes by ultracentrifugation include:

[0018] a. The herbal compound decoction prepared in step (1) was transferred to a centrifuge tube and centrifuged at 4°C, 2000 × g for 30 min;

[0019] b. Transfer the supernatant to a new centrifuge tube and centrifuge at 10,000 × g at 4°C for 45 min.

[0020] c. Take the supernatant, filter it through a 0.45 μm filter membrane, and collect the filtrate;

[0021] d. Transfer the filtrate to a new centrifuge tube and ultracentrifuge at 100,000 × g at 4°C for 70 min.

[0022] e. Remove the supernatant, resuspend in 10 mL of pre-chilled 1× PBS, and ultracentrifuge again at 100,000 × g at 4°C for 70 min.

[0023] f. Remove the supernatant and resuspend in 100 μL of pre-chilled 1× PBS.

[0024] In a second aspect, the present invention provides a traditional Chinese medicine compound exosome for treating ovarian cancer prepared by the above preparation method.

[0025] In a third aspect, the present invention also claims to protect the use of the traditional Chinese medicine compound exosomes prepared by the present invention in the preparation of drugs for treating ovarian cancer.

[0026] The beneficial effects of the present invention are as follows:

[0027] Pharmacodynamic experiments have shown that the exosomes of the traditional Chinese medicine compound of the present invention can significantly inhibit tumor growth in ovarian cancer-bearing mice and are suitable for traditional Chinese medicine treatment of ovarian cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figure shows the changes in fluorescence intensity of tumor burden in mice after 3 weeks of in vivo imaging; compared with the simple tumor-bearing group (model group), *P<0.05. DETAILED DESCRIPTION

[0029] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0030] Example 1 In vitro test

[0031] 1. Experimental Materials

[0032] (1) Experimental cells: ID8-Luc, purchased from Shanghai Fuheng Biotechnology Co., Ltd.

[0033] (2) Experimental animals: C57BL / 6 mice, weighing (20±2) g, 6-8 weeks old, female, SPF grade, purchased from Spefoc Biotechnology Co., Ltd.

[0034] (3) Experimental drugs:

[0035] Prescription No. 1 (comparative prescription): Astragalus 15g, Atractylodes 9g, Cinnamon 5g, Curcuma 6g, Polygonum multiflorum fruit 9g, Citrus aurantium 9g, Trillium 6g, Arisaema 6g, Artemisia annua 9g, Solanum nigrum 12g, Lithospermum officinale 12g, Scorpion 3g.

[0036] Recipe No. 2 (the Chinese medicine compound of the present invention): Astragalus 30g, Atractylodes macrocephala 15g, cinnamon bark 6g, Curcuma 9g, Lycium barbarum 15g, Sargassum 15g, Citrus aurantium 6g, Linderae 6g, Carapace of Turtle 12g, Hedyotis diffusa 15g, Scorpio 5g, Artemisia annua 12g, Arisaema consanguineum 12g, Trillium gracile 12g, and Cat's Claw 15g.

[0037] Solution for Recipe No. 1 and No. 2:

[0038] Recipe No. 1:

[0039] Main medicine: Astragalus, Atractylodes, nourish the qi of the lungs and spleen.

[0040] Assistant drugs: Curcuma zedoaria and Lithospermum officinale can break up blood and remove blood stasis; Citrus aurantium can soothe the liver and promote qi circulation; Artemisia annua and Solanum nigrum can clear away heat, detoxify and fight cancer; Trillium gracile and Arisaema can resolve phlegm and disperse nodules; Scorpion can attack toxins, disperse nodules, dredge meridians and relieve pain.

[0041] Auxiliary drug: Red safflower seeds promote blood circulation and diuresis.

[0042] Assistant drug: Cinnamon enters the kidney meridian, warms and nourishes kidney yang, and helps the yang energy rise.

[0043] No. 2 party:

[0044] Main medicine: Astragalus and Atractylodes macrocephala nourish the Qi of the upper and middle Jiao, while Linderae odoratum and cinnamon nourish the lower Jiao, warming and nourishing the kidney Yang and helping the Yang Qi to rise.

[0045] Assistant drugs: Curcuma zedoaria breaks up blood and removes blood stasis; Citrus aurantium soothes the liver and promotes qi circulation; Sargassum, Cat's Claw, Trillium gracile, and Arisaema consanguineum resolve phlegm and disperse nodules; Tortoise shell softens and disperses nodules; Lycium barbarum nourishes the liver and kidneys and strengthens vital energy.

[0046] Auxiliary drugs: Artemisia annua and Houttuynia cordata clear away heat, detoxify and fight cancer.

[0047] Ingredients: Scorpion can attack poison, disperse stagnation, dredge meridians and relieve pain.

[0048] (4) Preparation of exosomes from recipe No. 1 and recipe No. 2:

[0049] The Chinese herbal medicine slices in the formula were soaked in 1L deionized water for 30 minutes, boiled over high heat, decocted for 40 minutes, and then adjusted to low heat and continued to decoct for 30 minutes to produce about 250 ml of Yiqi Huoxue Jiedu Recipe (abbreviated as YQHX) water decoction.

[0050] Exosomes were prepared by ultracentrifugation: a. Transfer the Yiqi Huoxue Jiedu Recipe (YQHX) decoction to a new centrifuge tube and centrifuge at 2000 × g for 30 min at 4°C. b. Transfer the supernatant to a new centrifuge tube and centrifuge at 10,000 × g for 45 min at 4°C to remove larger vesicles. c. Filter the supernatant through a 0.45 μm filter membrane to collect the filtrate. d. Transfer the filtrate to a new centrifuge tube and ultracentrifuge at 100,000 × g for 70 min at 4°C in an ultracentrifuge rotor. e. Remove the supernatant and resuspend the suspension in 100 mL of pre-chilled 1× PBS (Phosphate buffered saline). Select an ultracentrifuge rotor and ultracentrifuge again at 100,000 × g for 70 min at 4°C. f. Remove the supernatant and resuspend the suspension in 100 μL of pre-chilled 1× PBS. Exosomes of Formula 1 and Formula 2 were prepared respectively, and the resuspended concentration was 7.5*10 10 pcs / ml.

[0051] (5) Preparation of the water decoction of Formula No. 2: Soak the Chinese herbal medicine slices of Formula No. 2 in 1 L of pure water for 30 minutes, bring to a boil over high heat, and simmer for 40 minutes. Then, reduce the heat to a low heat and continue simmering for 30 minutes to produce approximately 250 ml of the water decoction of YQHX Formula No. 2.

[0052] 2. Experimental Grouping

[0053] Grouping, modeling and drug administration

[0054] 1) The simple tumor-bearing group received intraperitoneal injection of tumor cells and oral administration of normal saline;

[0055] 2) YQHX No. 1 exosome group: intraperitoneal injection of tumor cells + oral administration of YQHX No. 1 exosomes;

[0056] 3) YQHX No. 2 exosome group: intraperitoneal injection of tumor cells + oral administration of YQHX No. 2 exosomes;

[0057] 4) YQHX No. 2 decoction group: tumor cells were injected intraperitoneally and YQHX No. 2 decoction was administered orally.

[0058] 3. Animal Modeling

[0059] All four groups of mice were injected with tumor cells into the peritoneal cavity to establish the model. The operation sequence was as follows: after 7 days of adaptive culture of mice, ID8-Luc cells in the logarithmic growth phase were taken and resuspended in sterile PBS (pH 7.4) to a volume of 1×10 7 The suspension was placed on ice and 500 μL of ID8-Luc cell suspension was injected into the peritoneal cavity of each mouse to simulate the peritoneal spread of epithelial ovarian cancer. Each mouse was injected with 5*10 6 Drug administration began 2 weeks after model establishment.

[0060] IV. Model Evaluation

[0061] The fluorescence intensity of tumor burden displayed by small animal in vivo imaging equipment was used as the model evaluation indicator.

[0062] In vivo imaging: The growth of tumor tissue in each group of mice was observed using small animal in vivo imaging equipment before administration (administration started 2 weeks after modeling) and 3 weeks after administration.

[0063] 5. Medication Treatment

[0064] Two weeks after modeling, mice were randomly regrouped (n=6 per group, ear-tagged) and began receiving medication at a standard dose of 20 μl / g body weight. For details on medication intervention, see the experimental groupings.

[0065] 1) Simple tumor-bearing group: PBS was administered orally according to body weight, 20 μl / g, once a day for 21 consecutive days;

[0066] 2) YQHX No. 1 exosome group: Exosomes from YQHX No. 1 were administered orally based on body weight, 20 μl / g, once a day for 21 consecutive days;

[0067] 3) YQHX No. 2 exosome group: Exosomes from YQHX No. 2 were administered orally based on body weight, 20 μl / g, once a day for 21 consecutive days;

[0068] 4) YQHX No. 2 decoction group: The YQHX No. 2 decoction was administered by gavage according to body weight, 20 μl / g, once a day for 21 consecutive days.

[0069] 6. Efficacy Evaluation

[0070] After administration, the general condition of the mice was closely observed, including mental state, coat color, activity, diet, and urination and defecation.

[0071] In vivo imaging observation to measure tumor burden: The growth of tumor tissue in each group of mice was observed using small animal in vivo imaging equipment 1 day before and 3 weeks after administration.

[0072] VII. Experimental Results

[0073] 1. Efficacy evaluation

[0074] According to the fluorescence intensity of tumor load in vivo imaging: after 21 days of intervention, the exosome group of YQHX No. 2 formula can inhibit tumor growth, P < 0.05. The exosomes of YQHX No. 1 formula and the water decoction of YQHX No. 2 formula can inhibit tumor growth, but there is no statistical significance (P > 0.05). Figure 1 .

[0075] 8. Discussion

[0076] In summary, exosomes from YQHX No. 2 formula effectively inhibited tumor growth, with superior efficacy compared to the decoction of YQHX No. 2 formula and the exosomes from YQHX No. 1 formula. Furthermore, the differences in efficacy between mice treated with exosomes from YQHX No. 2 formula were smaller than those treated with the decoction of YQHX No. 2 formula, suggesting that the therapeutic efficacy of exosomes from YQHX No. 2 formula is more stable than that of the decoction of YQHX No. 2 formula.

[0077] In addition, the present invention also greatly improves the efficacy of the drug by changing the composition of the Chinese medicine formula:

[0078] YQHX No. 2 is a modified version of QYHX No. 1. The exosomes prepared by YQHX No. 2 have a more significant anti-tumor effect than those prepared by YQHX No. 1. Compared with YQHX No. 1, the differences of YQHX No. 2 are as follows: (1) Addition and subtraction of drugs: 3 Chinese herbs, namely, Fructus Polygoni Multiflori, Solanum nigrum, and Lithospermum officinale, are removed, and 6 Chinese herbs, namely, Linderae scutellariae, Carapax Trionycis, Hedyotis diffusa, Herba Cat's Claws, Seaweed, and Fructus Lycii, are added. (2) Addition and subtraction of dosage: 8 Chinese herbs are added with dosage changes: Atractylodes macrocephala is increased from 9g to 15g, Cinnamon bark is increased from 5g to 6g, Curcuma zedoaria is increased from 6g to 9g, Citrus aurantium is reduced from 9g to 6g, Scorpion is increased from 3g to 5g, Artemisia annua is increased from 9g to 12g, Arisaema consanguineum is increased from 6g to 12g, and Trillium gracile is increased from 6g to 12g. It can be seen that the exosomes prepared using YQHX No. 2 formula are more effective in treating ovarian cancer.

[0079] The present invention greatly improves the efficacy of traditional Chinese medicine by preparing the traditional Chinese medicine compound into the form of exosomes before administering the drug:

[0080] Experimental results show that compared to the YQHX No. 2 traditional Chinese medicine decoction, the exosomes from YQHX No. 2 formula exhibited superior and more stable tumor-suppressing effects. Furthermore, the exosomes from YQHX No. 2 formula are extracted from the YQHX No. 2 traditional Chinese medicine decoction through ultracentrifugation and other methods, resulting in higher purity and a simpler composition than traditional Chinese medicine decoctions. Therefore, once the drug enters the body, it can more effectively target the disease. Furthermore, with the same oral administration volume, the exosome concentration can be more easily adjusted. While maintaining the same administration volume, higher concentrations can be achieved, something that cannot be achieved with traditional Chinese medicine decoctions concentrated through prolonged decoction. Therefore, it is more suitable for high-concentration, precise drug delivery.

[0081] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for preparing exosomes from a traditional Chinese medicine compound for treating ovarian cancer, characterized in that: The preparation method comprises the following steps: (1) Soaking the raw materials in the Chinese herbal compound in deionized water, then boiling over high heat for 35-45 minutes; then lowering the heat to low heat and continuing to boil for 30-35 minutes to obtain the Chinese herbal compound decoction; (2) preparing the Chinese medicine compound exosomes by ultracentrifugation of the Chinese medicine compound decoction; Among them, the Chinese medicine compound includes the following raw materials: astragalus, white atractylodes, cinnamon, zedoaria, wolfberry, seaweed, green peel, lindera strychnifolia, turtle shell, white flower snake tongue grass, scorpion, Artemisia annua, Arisaema concinnum, Trillium gracile and cat's claw.

2. The preparation method according to claim 1, characterized in that The traditional Chinese medicine compound comprises the following raw materials in parts by weight: 30-40 parts of astragalus, 15-20 parts of white atractylodes, 6-9 parts of cinnamon bark, 9-12 parts of zedoaria, 15-20 parts of wolfberry, 15-25 parts of seaweed, 6-9 parts of green peel, 6-9 parts of lindera striata, 12-15 parts of turtle shell, 15-30 parts of oldenlandia diffusa, 3-5 parts of scorpion, 12-20 parts of artemisia annua, 9-12 parts of arisaema, 12-25 parts of trillium, and 15-30 parts of cat's claw.

3. The preparation method according to claim 1, characterized in that The traditional Chinese medicine compound comprises the following raw materials in parts by weight: 30 parts of astragalus, 15 parts of white atractylodes, 6 parts of cinnamon bark, 9 parts of zedoaria, 15 parts of wolfberry, 15 parts of seaweed, 6 parts of green peel, 6 parts of lindera striata, 12 parts of turtle shell, 15 parts of oldenlandia diffusa, 5 parts of scorpion, 12 parts of artemisia annua, 12 parts of arisaema, 12 parts of trillium, and 15 parts of cat's claw.

4. The preparation method according to claim 1, characterized in that In step (1), the amount of deionized water used is 5-8 times the total weight of the raw material.

5. The preparation method according to claim 1, characterized in that In step (1), soak for 30-60 minutes.

6. The preparation method according to claim 1, characterized in that In step (2), the specific steps of preparing the Chinese medicine compound exosomes by ultracentrifugation include: a. The herbal compound decoction prepared in step (1) was transferred to a centrifuge tube and centrifuged at 4°C, 2000 × g for 30 min; b. Transfer the supernatant to a new centrifuge tube and centrifuge at 10,000 × g at 4°C for 45 min. c. Take the supernatant, filter it through a 0.45 μm filter membrane, and collect the filtrate; d. Transfer the filtrate to a new centrifuge tube and ultracentrifuge at 100,000 × g at 4°C for 70 min. e. Remove the supernatant, resuspend in 10 mL of pre-chilled 1× PBS, and ultracentrifuge again at 100,000 × g at 4°C for 70 min. f. Remove the supernatant and resuspend in 100 μL of pre-chilled 1× PBS.

7. Exosomes from a traditional Chinese medicine compound for treating ovarian cancer obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the traditional Chinese medicine compound exosomes according to claim 7 in the preparation of a drug for treating ovarian cancer.

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