Selenium-loaded traditional Chinese medicine preparation for reducing bone marrow toxicity and preparation method thereof

Abstract

The invention relates to the technical field of selenium-loaded traditional Chinese medicine preparations, in particular to a selenium-loaded traditional Chinese medicine preparation for reducing bone marrow toxicity and a preparation method thereof. Preparation raw materials of the selenium-loaded traditional Chinese medicine preparation comprise the following components in parts by weight: 2-3 parts of a modified starch carrier, 0.4-0.6 part of modified nano-selenium and 80-100 parts of deionized water, the selenium-loaded traditional Chinese medicine preparation prepared by the invention has a good anti-tumor effect and can effectively reduce bone marrow toxicity caused by chemotherapy.

Description

Technical Field

[0001] The invention relates to the technical field of selenium-loaded Chinese medicinal preparations, and in particular to a selenium-loaded Chinese medicinal preparation capable of reducing bone marrow toxicity and a preparation method thereof. Background Art

[0002] As one of the main means of tumor treatment, chemotherapy is widely used in the clinical treatment of tumors. However, due to its lack of specificity, it will also destroy normal cells while killing tumor cells. Under the premise of not affecting the efficacy, reducing the toxicity of chemotherapy drugs and reducing side effects are the conditions that ideal chemotherapy protective drugs should have. Studies have shown that selenium compounds can effectively prevent and protect against chemotherapy-induced nephrotoxicity, especially cisplatin nephrotoxicity; at the cellular level, 11-mercapto-alcohol-nanoselenium (Se@MUN) can effectively reverse cisplatin nephrotoxicity and improve the survival rate of HK-2 cells. Doxorubicin belongs to the anthraquinone antibiotics and is a broad-spectrum anti-tumor drug, but its clinical application is limited due to severe bone marrow and myocardial toxicity. Dexrazoxane is the only drug approved by the State Food and Drug Administration to reduce the incidence and severity of doxorubicin-induced cardiotoxicity. However, dexrazoxane can aggravate the bone marrow suppression caused by chemotherapy drugs, and its combined use with chemotherapy affects the anti-tumor effect. Long-term use can cause secondary malignant tumors. In order to treat chemotherapy-induced bone marrow toxicity, granulocyte colony-stimulating factor drugs are currently used clinically to promote the proliferation, differentiation and activation of hematopoietic cells. However, such drugs are expensive and can cause adverse reactions such as bone pain, fever, and leukopenia. Some drugs may also stimulate the growth of certain malignant tumors. Summary of the invention

[0003] In view of the deficiencies of the prior art, the present invention provides a selenium-loaded traditional Chinese medicine preparation for reducing bone marrow toxicity and a preparation method thereof.

[0004] The present invention is achieved through the following technical solutions: A selenium-loaded traditional Chinese medicine preparation for reducing bone marrow toxicity comprises the following components in parts by weight: 2-3 parts of modified starch carrier, 0.4-0.6 parts of modified nano-selenium, and 80-100 parts of deionized water.

[0005] Furthermore, the raw materials for preparing the modified starch carrier include the following components in parts by weight: 1-1.5 parts of ursolic acid, 2-3 parts of folic acid, and 20-30 parts of waxy corn starch.

[0006] Furthermore, the preparation method of the modified starch carrier comprises the following steps: (1) Under nitrogen protection, ursolic acid was dissolved in acetone / DMF (v / v=2:1), Jones reagent was added dropwise at 0°C, the temperature was raised to room temperature for 1 h after the addition was completed, the temperature was lowered to 0°C, isopropanol was added, the mixture was stirred at 300-400 rpm for 20-30 min, poured into ice water, centrifuged at 8000 rpm at 4°C for 15-20 min, the precipitate was washed with deionized water, and vacuum dried at 50°C to obtain modified ursolic acid; (2) adding waxy corn starch to deionized water, stirring at 600-800 rpm for 1.5-2 h to obtain starch milk, taking the modified ursolic acid obtained in step (1) and EDC and DMAP, adding them to dichloromethane and mixing them evenly, adding them to the starch milk, stirring at 300 r / min for 22-24 h, dialyzing with a dialysis bag with a molecular weight cutoff of 3 kDa for 24 h, and freeze-drying at -20°C to obtain ursolic acid-starch; (3) The ursolic acid-starch obtained in step (2) is added to deionized water, stirred at 400-500 rpm for 2 h to obtain an ursolic acid-starch dispersion, folic acid and DMSO are mixed, DCC and NHS are added, and stirring is continued at 200-300 rpm at room temperature for 10-12 h, filtered, the filter cake is washed with deionized water, added to the ursolic acid-starch dispersion, stirred at 40-50° C. in the dark for 5-6 h, and after the reaction is completed, dialyzed with a dialysis bag with a molecular weight cutoff of 3 kDa for 48 h, and the product in the dialysis bag is vacuum dried at 70-80° C. to obtain a modified starch carrier.

[0007] Furthermore, in step (1), the mass concentration of ursolic acid in acetone / DMF is 10-15 mg / mL.

[0008] Furthermore, in step (1), the ratio of ursolic acid to Jones reagent is 1 g:3-5 mL.

[0009] Furthermore, in step (1), the volume ratio of the Jones reagent to isopropanol is 1:10.

[0010] Furthermore, in step (2), the mass fraction of waxy corn starch in the starch milk is 20wt%.

[0011] Furthermore, in step (2), the mass ratio of the modified ursolic acid, EDC and DMAP is 1-2:0.5-1.1:0.3-0.6.

[0012] Furthermore, in step (2), the mass concentration of the modified ursolic acid in dichloromethane is 4-6 mg / mL.

[0013] Furthermore, in step (3), the mass concentration of folic acid in DMSO is 0.01-0.02 g / mL.

[0014] Furthermore, in step (3), the mass ratio of folic acid, DCC and NHS is 2-3:3-4.5:1.5-2.5.

[0015] Furthermore, in step (3), the mass concentration of ursolic acid-starch in deionized water is 0.04-0.06 g / mL.

[0016] Furthermore, the raw materials for preparing the modified nano-selenium include the following components in parts by weight: 3-4 parts of chlorogenic acid, 2-2.5 parts of NHS, 3.5-4.5 parts of EDC, 6-8 parts of doxorubicin hydrochloride, and 0.3-0.45 parts of sodium selenite.

[0017] Furthermore, the preparation method of the modified nano-selenium comprises the following steps: (a) Under nitrogen protection, chlorogenic acid was dissolved in 0.1 M pH 5.5 citric acid buffer, EDC and NHS were added, and the mixture was stirred at room temperature for 1 h. The pH value was adjusted to 7.4-7.6 with 0.2 M phosphate buffer to obtain a mixed solution. Doxorubicin hydrochloride and pH=8 phosphate buffer were mixed evenly and added to the mixed solution. The mixture was stirred at 200-300 rpm for 24 h at room temperature in the dark, and then allowed to stand at 4°C for 2-3 h. The mixture was centrifuged at 5000 rpm for 20-30 min. The precipitate was washed with deionized water and dried in vacuo to obtain chlorogenic acid-doxorubicin. (b) The chlorogenic acid-doxorubicin obtained in step (a) is mixed evenly with deionized water to obtain a chlorogenic acid-doxorubicin solution, sodium selenite is added to water to obtain a sodium selenite solution, the chlorogenic acid-ascorbic acid solution is mixed with the sodium selenite solution, magnetically stirred at 600-700 rpm for 30-40 min, centrifuged at 8000 rpm for 20 min, the precipitate is washed with deionized water, and freeze-dried at -20°C to obtain modified nano-selenium.

[0018] Furthermore, in step (a), the mass concentration of chlorogenic acid in citric acid buffer is 10-16 mg / mL.

[0019] Furthermore, in step (a), the mass concentration of doxorubicin hydrochloride in phosphate buffer is 1-1.6 mg / mL.

[0020] Furthermore, in step (b), the mass concentration of chlorogenic acid-doxorubicin in deionized water is 4-6 mg / mL.

[0021] Furthermore, in step (b), the molar concentration of sodium selenite in water is 50 mM.

[0022] Furthermore, the present invention also provides a method for preparing the selenium-loaded Chinese medicinal preparation for reducing bone marrow toxicity, comprising the following steps: adding modified nano-selenium into deionized water, ultrasonicating at 500 W for 20-30 min to obtain a modified nano-selenium dispersion, adding a modified starch carrier to the modified nano-selenium dispersion, stirring at 40-50° C. for 5-6 h, cooling to room temperature, centrifuging at 10,000 r / min for 3-5 min, and vacuum drying the precipitate at 35-45° C. to obtain a selenium-loaded Chinese medicinal preparation for reducing bone marrow toxicity.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention adopts folic acid and modified ursolic acid grafted starch to obtain a modified starch carrier, adopts chlorogenic acid-adriamycin to prepare modified nano-selenium, combines the modified nano-selenium with the modified starch carrier through molecular forces, and the prepared selenium-loaded Chinese medicine preparation for reducing bone marrow toxicity has targeting, good biocompatibility, low cytotoxicity, and can exert long-term effect. Ursolic acid is a natural terpenoid compound with anti-tumor, antioxidant and other activities. The present invention oxidizes the hydroxyl group of ursolic acid to a carbonyl group, which is beneficial to subsequent operations, and the starch has good biocompatibility, safety and non-toxicity. The carboxyl group of the modified ursolic acid is connected to the hydroxyl group of the starch through an ester bond, and the ursolic acid is grafted onto the molecular chain of corn starch, which effectively improves the stability of ursolic acid, prolongs its action time, increases its bioavailability, and improves biocompatibility. The present invention covalently grafts folic acid onto the molecular chain of corn starch. The folic acid receptor is a receptor commonly present in malignant tumors. It is highly or overexpressed on the surface of tumor cells, but relatively conservatively expressed on the surface of normal cells. It can produce extremely high affinity for folic acid. By utilizing the characteristics of specific binding of folic acid to the receptor, the concentration of the drug in the local lesion can be increased, the efficacy can be improved, and the toxic side effects can be reduced, so as to achieve the purpose of targeted treatment, improve the bioavailability of anticancer drugs, reduce drug resistance, and achieve the purpose of improving drug targeted release. The present invention connects chlorogenic acid and doxorubicin to obtain chlorogenic acid-doxorubicin. Chlorogenic acid is a natural polyphenol compound with reducing properties. It can also stabilize nano-selenium through hydrogen bonds. The chlorogenic acid-doxorubicin prepared by the present invention can be used as a reducing agent and a stabilizer for preparing nano-selenium. Nano-selenium has the characteristics of high activity, good bioavailability and low toxicity, can reduce the drug resistance of chemotherapy drugs, enhance the efficacy of anticancer drugs, has better performance than selenium compounds, and is safer than commonly used selenium compounds such as sodium selenite. The modified nano-selenium has strong stability, good dispersibility, and good antioxidant activity. The present invention modifies chlorogenic acid and doxorubicin on the surface of nano-selenium, effectively improving the stability and anti-tumor effect of modified nano-selenium; in addition, chlorogenic acid is an active ingredient of traditional Chinese medicine, which helps to improve the anti-tumor effect and reduce the side effects of chemotherapy drugs. The modified starch carrier prepared by the present invention is connected with folic acid and modified ursolic acid groups, and the surface of nano-selenium is modified with doxorubicin groups. The hydrophobic pteridine part of folic acid can interact hydrophobically with the benzene ring of doxorubicin through π-π stacking, and the hydrophobic group in ursolic acid can also interact hydrophobically with the benzene ring of doxorubicin. The modified starch carrier and the modified nano-selenium form a cage structure, and the modified nano-selenium is combined and loaded on the modified starch carrier, which can delay drug release and improve bioavailability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The anti-tumor effects of the selenium-loaded traditional Chinese medicine preparations described in Example 1 and Comparative Examples 1-3 of the present invention; Figure 2 The tumor cell inhibition effect of the selenium-loaded traditional Chinese medicine preparations described in Example 1 and Comparative Examples 1-3 of the present invention; Figure 3 The biological safety of the modified starch carriers described in Examples 1-3 and Comparative Examples 1-3 of the present invention; Figure 4 This is a transmission electron microscopy image of the modified nano-selenium described in Example 2 of the present invention; Figure 5 This is a stability test of the modified nano-selenium described in Examples 1-3 of the present invention and Comparative Example 3. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention are purchased through commercial channels.

[0027] Example 1: A selenium-loaded traditional Chinese medicine preparation for reducing bone marrow toxicity, wherein the raw materials for preparation include the following components in parts by weight: 3 parts of modified starch carrier, 0.6 parts of modified nano-selenium, and 100 parts of deionized water.

[0028] The raw materials for preparing the modified starch carrier include the following components in parts by weight: 1.5 parts of ursolic acid, 3 parts of folic acid, and 30 parts of waxy corn starch.

[0029] The preparation method of the modified starch carrier comprises the following steps: (1) Under nitrogen protection, 1.5 g of ursolic acid was dissolved in 100 mL of acetone / DMF (v / v=2:1), and 7.5 mL of Jones reagent was added dropwise at 0°C. After the addition was completed, the mixture was heated to room temperature for 1 h, cooled to 0°C, 75 mL of isopropanol was added, and the mixture was stirred at 400 rpm for 30 min. The mixture was poured into ice water and centrifuged at 8000 rpm for 20 min at 4°C. The precipitate was washed with deionized water and dried in a vacuum at 50°C to obtain modified ursolic acid. (2) 30 g of waxy corn starch was added to deionized water and stirred at 800 rpm for 2 h to obtain starch milk, wherein the mass fraction of waxy corn starch in the starch milk was 20 wt %. The modified ursolic acid obtained in step (1) was added to dichloromethane with EDC and DMAP and mixed evenly, wherein the mass concentration of modified ursolic acid in dichloromethane was 6 mg / mL, and the mass ratio of modified ursolic acid, EDC and DMAP was 2:1.1:0.6. The mixture was added to the starch milk and stirred at 300 r / min for 24 h. The mixture was dialyzed with a dialysis bag with a molecular weight cutoff of 3 kDa for 24 h, and freeze-dried at -20°C to obtain ursolic acid-starch. (3) The ursolic acid-starch obtained in step (2) was added to deionized water and stirred at 500 rpm for 2 h to obtain a 0.06 g / mL ursolic acid-starch dispersion. 3 g of folic acid was mixed with 150 mL of DMSO, and 4.5 g of DCC and 2.5 g of NHS were added. The mixture was stirred at 300 rpm for 12 h at room temperature. The mixture was filtered and the filter cake was washed with deionized water and added to the ursolic acid-starch dispersion. The mixture was stirred at 50°C in the dark for 6 h. After the reaction was completed, the mixture was dialyzed with a dialysis bag with a molecular weight cutoff of 3 kDa for 48 h. The product in the dialysis bag was vacuum dried at 80°C to obtain a modified starch carrier.

[0030] The raw materials for preparing modified nano-selenium include the following components in parts by weight: 4 parts of chlorogenic acid, 2.5 parts of NHS, 4.5 parts of EDC, 8 parts of doxorubicin hydrochloride, and 0.45 parts of sodium selenite.

[0031] The preparation method of modified nano-selenium comprises the following steps: (a) Under nitrogen protection, 4 g of chlorogenic acid was dissolved in 250 mL of 0.1 M pH 5.5 citric acid buffer, 4.5 g of EDC and 2.5 g of NHS were added, and the mixture was stirred at room temperature for 1 h. The pH value was adjusted to 7.6 with 0.2 M phosphate buffer to obtain a mixed solution. 8 g of doxorubicin hydrochloride and 5 L of pH=8 phosphate buffer were mixed evenly and added to the mixed solution. The mixture was stirred at 300 rpm for 24 h at room temperature in the dark, allowed to stand at 4°C for 3 h, and centrifuged at 5000 rpm for 30 min. The precipitate was washed with deionized water and dried in vacuo to obtain chlorogenic acid-doxorubicin. (b) The chlorogenic acid-doxorubicin obtained in step (a) was mixed evenly with deionized water to obtain a 6 mg / mL chlorogenic acid-doxorubicin solution, 0.45 g sodium selenite was added to the water to obtain a 50 mM sodium selenite solution, the chlorogenic acid-ascorbic acid solution was mixed with the sodium selenite solution, magnetically stirred at 700 rpm for 40 min, centrifuged at 8000 rpm for 20 min, the precipitate was washed with deionized water, and freeze-dried at -20°C to obtain modified nano-selenium.

[0032] This embodiment also provides a method for preparing the selenium-loaded Chinese medicine preparation for reducing bone marrow toxicity, comprising the following steps: adding 0.6 g of modified nano-selenium into 100 g of deionized water, ultrasonicating at 500 W for 30 min to obtain a modified nano-selenium dispersion, adding 3 g of a modified starch carrier to the modified nano-selenium dispersion, stirring at 50° C. for 6 h, cooling to room temperature, centrifuging at 10,000 r / min for 5 min, and vacuum drying the precipitate at 45° C. to obtain a selenium-loaded Chinese medicine preparation for reducing bone marrow toxicity.

[0033] Example 2: A selenium-loaded traditional Chinese medicine preparation for reducing bone marrow toxicity, wherein the raw materials for preparation include the following components in parts by weight: 2 parts of modified starch carrier, 0.4 parts of modified nano-selenium, and 80 parts of deionized water.

[0034] The raw materials for preparing the modified starch carrier include the following components in parts by weight: 1 part of ursolic acid, 2 parts of folic acid, and 20 parts of waxy corn starch.

[0035] The preparation method of the modified starch carrier comprises the following steps: (1) Under nitrogen protection, 1 g of ursolic acid was dissolved in 100 mL of acetone / DMF (v / v=2:1), and 3 mL of Jones reagent was added dropwise at 0°C. After the addition was completed, the mixture was heated to room temperature for 1 h, cooled to 0°C, 30 mL of isopropanol was added, and the mixture was stirred at 300 rpm for 20 min. The mixture was poured into ice water and centrifuged at 8000 rpm for 15 min at 4°C. The precipitate was washed with deionized water and dried in a vacuum at 50°C to obtain modified ursolic acid. (2) Add 20 g of waxy corn starch to deionized water and stir at 600 rpm for 1.5 h to obtain starch milk, wherein the mass fraction of waxy corn starch in the starch milk is 20 wt %. Take the modified ursolic acid obtained in step (1), EDC and DMAP, add them to dichloromethane and mix them evenly. The mass concentration of modified ursolic acid in dichloromethane is 4 mg / mL, and the mass ratio of modified ursolic acid, EDC and DMAP is 1:0.5:0.3. Add them to the starch milk and stir at 300 r / min for 22 h. Dilute with a dialysis bag with a molecular weight cutoff of 3 kDa for 24 h, and freeze-dry at -20°C to obtain ursolic acid-starch. (3) The ursolic acid-starch obtained in step (2) was added to deionized water and stirred at 400 rpm for 2 h to obtain a 0.04 g / mL ursolic acid-starch dispersion. 2 g folic acid (200 mL) was mixed with DMSO, and 3 g DCC and 1.5 g NHS were added. The mixture was stirred at 200 rpm for 10 h at room temperature. The filter cake was filtered and washed with deionized water. The mixture was added to the ursolic acid-starch dispersion and stirred at 40°C in the dark for 5 h. After the reaction was completed, the mixture was dialyzed with a dialysis bag with a molecular weight cutoff of 3 kDa for 48 h. The product in the dialysis bag was vacuum dried at 70°C to obtain a modified starch carrier.

[0036] The raw materials for preparing modified nano-selenium include the following components in parts by weight: 3 parts of chlorogenic acid, 2 parts of NHS, 3.5 parts of EDC, 6 parts of doxorubicin hydrochloride, and 0.3 parts of sodium selenite.

[0037] The preparation method of modified nano-selenium comprises the following steps: (a) Under nitrogen protection, 3 g of chlorogenic acid was dissolved in 300 mL of 0.1 M pH 5.5 citric acid buffer, 3.5 g of EDC and 2 g of NHS were added, and the mixture was stirred at room temperature for 1 h. The pH value was adjusted to 7.4 with 0.2 M phosphate buffer to obtain a mixed solution. 6 g of doxorubicin hydrochloride and 6 L of phosphate buffer at pH = 8 were mixed evenly and added to the mixed solution. The mixture was stirred at 200 rpm for 24 h at room temperature in the dark, allowed to stand at 4 ° C for 2 h, and centrifuged at 5000 rpm for 20 min. The precipitate was washed with deionized water and dried in vacuo to obtain chlorogenic acid-doxorubicin. (b) The chlorogenic acid-doxorubicin obtained in step (a) was mixed evenly with deionized water to obtain a 4 mg / mL chlorogenic acid-doxorubicin solution, 0.3 g sodium selenite was added to the water to obtain a 50 mM sodium selenite solution, the chlorogenic acid-ascorbic acid solution was mixed with the sodium selenite solution, magnetic stirring was performed at 600 rpm for 30 min, and centrifugation was performed at 8000 rpm for 20 min. The precipitate was washed with deionized water and freeze-dried at -20°C to obtain modified nano-selenium.

[0038] This embodiment also provides a method for preparing the selenium-loaded Chinese medicine preparation for reducing bone marrow toxicity, comprising the following steps: adding 0.4 g of modified nano-selenium to 80 g of deionized water, ultrasonicating at 500 W for 20 min to obtain a modified nano-selenium dispersion, adding 2 g of a modified starch carrier to the modified nano-selenium dispersion, stirring at 40° C. for 5 h, cooling to room temperature, centrifuging at 10,000 r / min for 3 min, and vacuum drying the precipitate at 35° C. to obtain a selenium-loaded Chinese medicine preparation for reducing bone marrow toxicity.

[0039] Example 3: A selenium-loaded traditional Chinese medicine preparation for reducing bone marrow toxicity, wherein the raw materials for preparation include the following components in parts by weight: 2.5 parts of modified starch carrier, 0.5 parts of modified nano-selenium, and 95 parts of deionized water.

[0040] The raw materials for preparing the modified starch carrier include the following components in parts by weight: 1.4 parts of ursolic acid, 2.5 parts of folic acid, and 25 parts of waxy corn starch.

[0041] The preparation method of the modified starch carrier comprises the following steps: (1) Under nitrogen protection, 1.4 g of ursolic acid was dissolved in 100 mL of acetone / DMF (v / v=2:1), and 4.9 mL of Jones reagent was added dropwise at 0°C. After the addition was completed, the temperature was raised to room temperature for reaction for 1 h, then the temperature was lowered to 0°C, 49 mL of isopropanol was added, and the mixture was stirred at 350 rpm for 25 min. The mixture was poured into ice water, and centrifuged at 8000 rpm at 4°C for 18 min. The precipitate was washed with deionized water and dried in a vacuum at 50°C to obtain modified ursolic acid. (2) 25 g of waxy corn starch was added to deionized water and stirred at 700 rpm for 1.8 h to obtain starch milk, wherein the mass fraction of waxy corn starch in the starch milk was 20 wt %. The modified ursolic acid obtained in step (1) was added to dichloromethane with EDC and DMAP and mixed evenly, wherein the mass concentration of the modified ursolic acid in dichloromethane was 5 mg / mL, and the mass ratio of the modified ursolic acid, EDC and DMAP was 1.5:0.8:0.5. The mixture was added to the starch milk and stirred at 300 r / min for 23 h. The mixture was dialyzed with a dialysis bag with a molecular weight cutoff of 3 kDa for 24 h, and freeze-dried at -20°C to obtain ursolic acid-starch. (3) The ursolic acid-starch obtained in step (2) was added to deionized water and stirred at 450 rpm for 2 h to obtain a 0.05 g / mL ursolic acid-starch dispersion. 2.5 g folic acid was mixed with 200 mL DMSO, and 4 g DCC and 2 g NHS were added. The mixture was stirred at 250 rpm for 11 h at room temperature. The filter cake was filtered and washed with deionized water. The filter cake was added to the ursolic acid-starch dispersion and stirred at 45 °C in the dark for 5.5 h. After the reaction was completed, the mixture was dialyzed with a dialysis bag with a molecular weight cutoff of 3 kDa for 48 h. The product in the dialysis bag was vacuum dried at 75 °C to obtain a modified starch carrier.

[0042] The raw materials for preparing modified nano-selenium include the following components in parts by weight: 3.5 parts of chlorogenic acid, 2.3 parts of NHS, 4 parts of EDC, 7 parts of doxorubicin hydrochloride, and 0.4 parts of sodium selenite.

[0043] The preparation method of modified nano-selenium comprises the following steps: (a) Under nitrogen protection, 3.5 g of chlorogenic acid was dissolved in 250 mL of 0.1 M pH 5.5 citric acid buffer, 4 g of EDC and 2.3 g of NHS were added, and the mixture was stirred at room temperature for 1 h. The pH value was adjusted to 7.5 with 0.2 M phosphate buffer to obtain a mixed solution. 7 g of doxorubicin hydrochloride and 5 L of phosphate buffer at pH = 8 were mixed evenly and added to the mixed solution. The mixture was stirred at 250 rpm for 24 h at room temperature in the dark, allowed to stand at 4 ° C for 2.5 h, and centrifuged at 5000 rpm for 25 min. The precipitate was washed with deionized water and dried in vacuo to obtain chlorogenic acid-doxorubicin. (b) The chlorogenic acid-doxorubicin obtained in step (a) was mixed evenly with deionized water to obtain a 5 mg / mL chlorogenic acid-doxorubicin solution, 0.4 g sodium selenite was added to the water to obtain a 50 mM sodium selenite solution, the chlorogenic acid-ascorbic acid solution was mixed with the sodium selenite solution, magnetically stirred at 650 rpm for 35 min, centrifuged at 8000 rpm for 20 min, the precipitate was washed with deionized water, and freeze-dried at -20°C to obtain modified nano-selenium.

[0044] This embodiment also provides a method for preparing the selenium-loaded Chinese medicine preparation for reducing bone marrow toxicity, comprising the following steps: adding 0.5 g of modified nano-selenium to 95 g of deionized water, ultrasonicating at 500 W for 25 min to obtain a modified nano-selenium dispersion, adding 2.5 g of modified starch carrier to the modified nano-selenium dispersion, stirring at 45° C. for 5.5 h, cooling to room temperature, centrifuging at 10,000 r / min for 4 min, and vacuum drying the precipitate at 40° C. to obtain a selenium-loaded Chinese medicine preparation for reducing bone marrow toxicity.

[0045] The only difference between Comparative Example 1 and Example 1 is that folic acid is not added, that is, step (3) is not performed; The difference between Comparative Example 2 and Example 1 is that ursolic acid is not added, that is, steps (1)-(2) are not performed, and the ursolic acid-starch in step (3) is replaced by waxy corn starch; The only difference between Comparative Example 3 and Example 1 is that ferulic acid is used instead of chlorogenic acid.

[0046] Experimental Example 1: 6-8 week old male BALB / c mice were taken and after 1 week of adaptive feeding, 5 mice were randomly selected and each mouse was subcutaneously injected with 10 6 Caco-2 cells were collected and the subcutaneous transplanted tumors were removed surgically 7 days later and minced into 1 mm 3 The size is reserved. The mice were anesthetized with 1.5% isoflurane, and the peritoneum of the mice was cut open with scissors (wounds of about 1 cm). The free cecum of the mice was gently pulled out of the body with forceps, and the vascular-rich area at the junction of the cecum and the colon was found. A tumor mass was attached here with Histoacryl tissue glue. After 10 seconds, the previously pulled-out intestinal segment was put back into the abdomen and sutured with 0-4 surgical sutures. After all mice woke up, they were put back into the SPF-level barrier for breeding. If they were observed for three days without any harm, the colon cancer model was successfully established. Three days after the operation, the mice were randomly divided into 8 groups to start the experiment, with 5 mice in each group. On the first day, the selenium-loaded Chinese medicine preparations of Examples 1-3 and Comparative Examples 1-3 were gavaged and administered. At the same time, a control group was set up. The control group was intraperitoneally injected with doxorubicin, and the blank group was injected with PBS. The dosage of doxorubicin was 2 mg / kg. After that, the injection was given once every 3 days, for a total of 3 injections. The tumor volume was measured after 14 days. The volume is calculated by the length and width of the tumor: V=(length×width 2 ) / 2. The result is Figure 1 shown.

[0047] Figure 1 The results showed that the selenium-loaded Chinese medicine preparations used in Examples 1-3 and Comparative Examples 1-3 had an inhibitory effect on tumor growth compared with the PBS blank group, proving that the selenium-loaded Chinese medicine preparations had anti-tumor ability. The group treated with doxorubicin alone could observe a significant reduction in mouse tumors, and doxorubicin had a good anti-tumor effect. The Example 1-3 group showed a stronger inhibitory effect on tumor growth relative to Comparative Examples 1-3. Comparative Example 1 did not add folic acid, and the drug loading and targeting decreased. Comparative Example 2 did not add ursolic acid, the drug loading decreased, and the inhibitory effect on tumors decreased; Comparative Example 3 did not add chlorogenic acid, and the anti-tumor effect decreased. The selenium-loaded Chinese medicine preparation of the present invention can achieve a tumor-killing effect through the joint action of ursolic acid and chlorogenic acid, the active ingredients of traditional Chinese medicine, nano-selenium and doxorubicin.

[0048] Experimental Example 2: Take 8-week-old SPF-grade C57BL / 6 mice, adaptively feed for 1 week, colon cancer modeling, and randomly divide them into 7 groups, 5 in each group, named AG group, among which AD group is gavaged with selenium-loaded Chinese medicine preparations of Example 1 and Comparative Examples 1-3, E group is intraperitoneally injected with doxorubicin, F group is intraperitoneally injected with doxorubicin and dextromethamine, G group is intraperitoneally injected with PBS, and the dosage of doxorubicin in each group is 4 mg / kg, and the dosage of dextromethamine is 50 mg / kg. The mice were given once a week for 3 consecutive weeks. Blood was collected from the eye sockets 24 hours after the end of the administration to obtain whole blood. 100 μL of whole blood was routinely tested for peripheral blood red blood cell, white blood cell, and platelet cell counts. The mice were killed by dislocation of the neck, and the femurs were stripped. The complete right femur of the mice was repeatedly rinsed with 3% glacial acetic acid solution to make the bone marrow cells in 10 mL of 3% glacial acetic acid solution, fully mixed, filtered and dispersed, and bone marrow nucleated cells were counted. The results are shown in Table 1.

[0049] Table 1:

[0050] The results in Table 1 show that the selenium-loaded Chinese herbal preparations of Example 1 and Comparative Examples 1-3 have a protective effect on the bone marrow suppression effect of doxorubicin, while dexrazoxane has no protective effect on the bone marrow toxicity of doxorubicin and may aggravate the bone marrow suppression effect of doxorubicin. Among them, the effect of reducing bone toxicity in Example 1 is the most significant, indicating that the selenium-loaded Chinese herbal preparation of the present invention has targeting, can enter the tumor site, reduces the in vivo diffusion of free doxorubicin, reduces the damage to normal cells, can significantly inhibit the bone marrow toxicity of doxorubicin, reduces the toxic and side effects of chemotherapy, and has high biological safety.

[0051] Experimental Example 3: Caco-2 cells in the logarithmic growth phase were cultured at 5×10 4The selenium-loaded Chinese medicine preparations obtained in Example 1 and Comparative Examples 1-3 and free doxorubicin hydrochloride were inoculated into a 96-well plate at a concentration of 10, 100, 10, and 15 μg / mL and cultured for 24 h. After the culture was completed, the culture medium was aspirated and the selenium-loaded Chinese medicine preparations obtained in Example 1 and Comparative Examples 1-3 and free doxorubicin hydrochloride were diluted with fresh culture medium to a doxorubicin content of 10, 100, and 15 μg / mL. 3 and 10 4 μg / mL, 100 μL was added to the Caco-2 cell culture medium and continued to be cultured for 24 h. The control group used free doxorubicin, and the blank group added the same volume of fresh culture medium. After 24 h of drug treatment, the supernatant was discarded, and 100 μL of 0.5 mg / mL MTT solution was added. The cells were incubated at 37°C for 4 h, the culture medium was carefully aspirated, and 100 μL of DMSO was added. The cells were shaken for 10 min, and the absorbance of each well sample at 490 nm was measured with an enzyme marker. Cell survival rate (%) = (OD drug group / OD blank group) × 100%. The results are shown in Figure 2 shown.

[0052] Figure 2 The results showed that at low dosage concentrations, the control group had a better inhibitory effect on Caco-2 cells than Example 1 and Comparative Examples 1-3. 3 When the dosage is 2.5 μg / mL, the dosage concentration is continued to increase, the inhibitory effect of the control group is basically stable, and the inhibitory effect of Example 1 and Comparative Example 1-3 groups is significantly enhanced. This is because relative to the administration of selenium-loaded Chinese medicine preparations, free doxorubicin hydrochloride molecules are better able to enter Caco-2 cells, and therefore approach saturation faster. Under the treatment conditions of selenium-loaded Chinese medicine preparations, doxorubicin hydrochloride and other active ingredients need to be released from the carrier and absorbed by cells, which is an ongoing process. Therefore, as the dosage concentration increases, the inhibitory effect is more significant.

[0053] Experimental Example 4: Select HUVEC cells in the logarithmic growth phase with good growth status to prepare cell suspension and count them. The experimental group and the control group were 1×10 5 The cells were inoculated into 96-well plates at a concentration of 100 μL / mL. The blank group was replaced with 100 μL of culture medium instead of cell suspension and placed in a 37°C, 5% CO 2 The cells were cultured in a cell culture incubator for 2 h, the culture medium of each well was aspirated, and 100 μL of the modified starch carrier solution of Example 1-3 prepared with fresh culture medium at 10 mg / mL was added. The control group and the blank group were added with the same volume of culture medium, and the culture was continued for 48 h. The culture medium in the well plate was discarded, and 100 μL of CCK8 solution diluted 10 times with fresh culture medium was added to each well. After incubation in a cell culture incubator for 3 h in the dark, the absorbance at 450 nm was measured with an ELISA instrument, and the cell viability of each group was calculated. Cell viability was calculated according to the following formula: Cell viability (%) = [(OD给药组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )]×100%. The result is Figure 3 shown. Figure 3 The results show that the modified starch carriers of Examples 1-3 and Comparative Examples 1-3 have low toxicity to normal cells and have relatively good biocompatibility.

[0054] Experimental Example 5: The modified nano-selenium prepared in Example 2 was analyzed by transmission electron microscopy. The results are as follows: Figure 4 As shown in the figure, the modified nano-selenium is uniformly dispersed in spheres. The nano-selenium is successfully prepared. The loading of doxorubicin does not affect the dispersibility and morphology of selenium nanoparticles. The modified nano-selenium has a small particle size and has the potential to penetrate the cell membrane. The modified nano-selenium prepared in Examples 1-3 and Comparative Example 3 was dispersed in ultrapure water and allowed to stand for 30 days. The particle size increase rate before and after standing was calculated. The results are shown in the figure. Figure 5 As shown, Figure 5 The results show that the particle size change of the modified nano-selenium in Examples 1-3 is smaller than that in Comparative Example 3, indicating that the modified nano-selenium of the present invention has better stability.

[0055] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

Claims

1. A selenium-loaded Chinese medicine preparation for reducing bone marrow toxicity, characterized in that: The raw materials for preparation include the following components in parts by weight: 2-3 parts of modified starch carrier, 0.4-0.6 parts of modified nano-selenium, and 80-100 parts of deionized water; The raw materials for preparing the modified starch carrier include the following components in parts by weight: 1-1.5 parts of ursolic acid, 2-3 parts of folic acid, and 20-30 parts of waxy corn starch; The preparation method of the modified starch carrier comprises the following steps: (1) Under nitrogen protection, ursolic acid was dissolved in acetone / DMF, Jones reagent was added dropwise at 0°C, the temperature was raised to room temperature for reaction after the addition was completed, the temperature was lowered to 0°C, isopropanol was added, the mixture was stirred, poured into ice water, centrifuged, the precipitate was washed with deionized water, and vacuum dried to obtain modified ursolic acid; (2) adding waxy corn starch to deionized water and stirring to obtain starch milk, taking the modified ursolic acid obtained in step (1) and EDC and DMAP and adding them to dichloromethane, mixing them evenly, adding them to the starch milk, stirring, dialyzing, and freeze-drying to obtain ursolic acid-starch; (3) adding the ursolic acid-starch obtained in step (2) into deionized water and stirring to obtain an ursolic acid-starch dispersion, mixing folic acid and DMSO, adding DCC and NHS, stirring continuously at room temperature, filtering with suction, washing the filter cake with deionized water, adding it into the ursolic acid-starch dispersion, stirring to react in the dark, dialyzing after the reaction is completed, and vacuum drying to obtain a modified starch carrier; The raw materials for preparing the modified nano-selenium include the following components in parts by weight: 3-4 parts of chlorogenic acid, 2-2.5 parts of NHS, 3.5-4.5 parts of EDC, 6-8 parts of doxorubicin hydrochloride, and 0.3-0.45 parts of sodium selenite; The preparation method of the modified nano-selenium comprises the following steps: (a) Under nitrogen protection, chlorogenic acid is dissolved in citric acid buffer, EDC and NHS are added, stirred at room temperature, and the pH value is adjusted with phosphate buffer to obtain a mixed solution, doxorubicin hydrochloride and phosphate buffer are mixed evenly, added to the mixed solution, stirred for reaction, allowed to stand, centrifuged, the precipitate is washed, and vacuum dried to obtain chlorogenic acid-doxorubicin; (b) The chlorogenic acid-doxorubicin obtained in step (a) is mixed evenly with deionized water to obtain a chlorogenic acid-doxorubicin solution, sodium selenite is added to water to obtain a sodium selenite solution, the chlorogenic acid-ascorbic acid solution is mixed with the sodium selenite solution, magnetic stirring is performed, centrifugation is performed, the precipitate is washed with deionized water, and freeze-dried to obtain modified nano-selenium.

2. The selenium-loaded Chinese medicine preparation for reducing bone marrow toxicity according to claim 1, characterized in that: In step (1), the mass concentration of ursolic acid in acetone / DMF is 10-15 mg / mL; the dosage ratio of ursolic acid to Jones reagent is 1 g:3-5 mL; and the volume ratio of Jones reagent to isopropanol is 1:

10.

3. The selenium-loaded Chinese medicine preparation for reducing bone marrow toxicity according to claim 2, characterized in that: In step (2), the mass fraction of waxy corn starch in the starch milk is 20wt%; the mass ratio of modified ursolic acid, EDC and DMAP is 1-2:0.5-1.1:0.3-0.6; and the mass concentration of modified ursolic acid in dichloromethane is 4-6 mg / mL.

4. The selenium-loaded Chinese medicine preparation for reducing bone marrow toxicity according to claim 3, characterized in that: In step (3), the mass concentration of folic acid in DMSO is 0.01-0.02 g / mL; the mass ratio of folic acid, DCC and NHS is 2-3:3-4.5:1.5-2.5; and the mass concentration of ursolic acid-starch in deionized water is 0.04-0.06 g / mL.

5. The selenium-loaded Chinese medicine preparation for reducing bone marrow toxicity according to claim 4, characterized in that: In step (a), the mass concentration of chlorogenic acid in citric acid buffer is 10-16 mg / mL; the mass concentration of doxorubicin hydrochloride in phosphate buffer is 1-1.6 mg / mL.

6. The selenium-loaded Chinese medicinal preparation for reducing bone marrow toxicity according to claim 5, characterized in that: In step (b), the mass concentration of chlorogenic acid-doxorubicin in deionized water is 4-6 mg / mL; the molar concentration of sodium selenite in water is 50 mM.

7. A method for preparing a selenium-loaded Chinese medicinal preparation for reducing bone marrow toxicity as claimed in any one of claims 1 to 6, characterized in that: The following steps are involved: The modified nano-selenium is added into deionized water, ultrasonicated to obtain the modified nano-selenium dispersion, the modified starch carrier is added into the modified nano-selenium dispersion, stirred, cooled, centrifuged and vacuum dried to obtain the selenium-loaded traditional Chinese medicine preparation for reducing bone marrow toxicity.

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