A lotus seed heart alkaloid extract, its preparation method and application

Abstract

This invention discloses a lotus seed heart alkaloid extract, its preparation method, and its applications. The preparation method of the lotus seed heart alkaloid extract includes the following steps: 1) grinding dried lotus seed hearts into powder; 2) extracting the powder obtained in step 1) in an ethanol-water solution, removing the solvent from the resulting extract to obtain a total lotus seed heart extract; 3) separating and enriching the total lotus seed heart extract from step 2) using a D001 cation exchange resin to obtain the lotus seed heart alkaloid extract. The extraction method of this invention establishes key process parameters; the transfer rate and content of total alkaloids in the extract can reach over 50%, effectively reducing ionizing radiation-induced damage to human umbilical vein endothelial cells, improving ionizing radiation-induced hematopoietic system damage-related indicators to varying degrees, and protecting against ionizing radiation-induced intestinal damage. The lotus seed heart alkaloid extract of this invention can be applied to the prevention and / or treatment of diseases related to ionizing radiation damage.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a lotus seed heart alkaloid extract, its preparation method and application. Background Technology

[0002] Ionizing radiation is a product of nuclear energy and nuclear technology development, mainly including particle radiation (alpha particles, beta particles, neutrons, and protons) and high-energy electromagnetic waves (gamma rays and X-rays, etc.). As a clearly defined physical carcinogen, its biological effects are closely related to the type of radiation, radiation dose, and individual sensitivity. Improper use can cause systemic pathological changes, with radiation-sensitive target organs such as bone marrow and intestines prone to acute tissue damage, which can lead to acute radiation sickness characterized by dysfunction of the hematopoietic and digestive systems. Radiotherapy is one of the important treatment methods for pelvic malignancies (such as bladder cancer and cervical cancer). While killing tumor cells, it also causes radiation damage to adjacent normal intestinal tissues, inducing local inflammatory reactions, and in severe cases, may lead to complications such as radiation enteritis. Natural products have abundant antioxidant and anti-inflammatory activities and are gradually becoming an important direction for drug research in this field; however, no related drugs have been approved for marketing to date.

[0003] Lotus seed heart (a traditional Chinese medicine) Nelumbinis Plumula ) is the lotus (Nelumbo nucifera), a plant of the Nymphaeaceae family. Nelumbo nucifera The dried young leaves and radicle of mature seeds of *Gastrodia elata* (Garcinia natans) are bitter and cold in nature. First recorded in *Food and Medicine Materia Medica*, it was later included in *Compendium of Materia Medica* and *New Compilation of Materia Medica*. Currently, it is not only included in the *Chinese Pharmacopoeia*, but also approved by the Ministry of Health of my country as a food and medicine of the same origin, possessing the effects of calming the mind and spirit, harmonizing the heart and kidneys, and astringing essence and stopping bleeding. Isoquinoline alkaloids are a class of chemical components abundant in the lotus seed heart, mainly including benzylisoquinoline alkaloids (such as methyl lotusine, lotusine, isolivine, etc.) and apophene alkaloids (such as lotus leaf alkaloids, etc.). Among them, methyl lotusine is relatively abundant (9.367–12.067 mg / g) and is used in the Chinese Pharmacopoeia to control the quality of lotus seed heart medicinal materials.

[0004] The extraction of alkaloids from lotus seed heart is usually carried out by solvent extraction. Assisted heating reflux, ultrasonic, microwave or ultra-high pressure techniques can improve the extraction rate of these components. Orthogonal design / response surface methodology was used to optimize the extraction process, including solvent concentration, extraction time, and material-liquid ratio [Yang Chao, Tian Wenyue, Chen Guowei, Huang Jiajin, Wang Likang, Ding Lijun, Zheng Junxia. Research progress on extraction, separation, activity evaluation and preparation of alkaloids from lotus seed heart. Chinese Journal of Traditional Chinese Medicine, 2021, 39(09):142-144.]. Zhang Jixiang et al. investigated the separation and purification of lotus seed heart characteristic alkaloids using a combination of macroporous adsorption resin and cation exchange resin. They obtained lotus seed heart alkaloid extracts with contents of 33.1%, 34.5%, and 15.0% for lotus seed heart alkaloids, methyl lotus seed heart alkaloids, and isolivine heart alkaloids, respectively, with a sample yield of only 0.92% [Zhang Jixiang, Guo Jinsheng, Yang Jialing, Ou Lailiang. Study on separation and purification of total alkaloids from lotus seed heart by dual-column adsorption method. Ion Exchange and Adsorption, 2008, 24(6):512-517.]. In summary, current research mainly focuses on optimizing the extraction process of lotus seed heart extracts. The determination of key process parameters is primarily based on single-factor investigations, and the optimized key process parameters are basically fixed values. Furthermore, a systematic study on the enrichment process of lotus seed heart alkaloid components is still lacking, making it difficult to demonstrate the scientific nature and quality controllability of the lotus seed heart alkaloid extract preparation process, and also hindering the subsequent industrialization of the product. Summary of the Invention

[0005] The purpose of this invention is to provide a lotus seed heart alkaloid extract, its preparation method, and its applications. The lotus seed heart alkaloid extract of this invention is a brown solid powder, prepared by combining solvent extraction and cation exchange resin enrichment techniques. The preparation process is optimized using advanced quality by design (QbD) pharmaceutical concepts, optimizing key process parameters. Furthermore, the application of lotus seed heart alkaloid extract in diseases related to ionizing radiation damage has not been reported in the literature. This invention provides a candidate drug for the prevention and / or treatment of ionizing radiation damage or its induced diseases.

[0006] The present invention provides a method for preparing lotus seed heart alkaloid extract, comprising the following steps: 1) grinding dried lotus seed heart into powder; 2) Extract the powder obtained in step 1) in an ethanol-water solution, remove the solvent from the resulting extract, and obtain the total extract of lotus seed heart; 3) The total extract of lotus seed heart from step 2) is separated and enriched by D001 cation exchange resin to obtain lotus seed heart alkaloid extract.

[0007] In the above method, the particle size of the lotus seed heart powder can be 0~65 mesh, excluding 0.

[0008] In the above method, the volume percentage concentration of ethanol in the aqueous ethanol solution can be 75% to 90%. The ratio of the ethanol aqueous solution to the powder can be 10~20:1 mL / g.

[0009] In the above method, the extraction temperature can be 70~100℃, the time can be 1~2 h, and the extraction can be repeated 2~3 times.

[0010] In this invention, in step 2), the obtained extract is concentrated to dryness under reduced pressure to obtain the total extract of lotus seed heart; In step 3), the eluent obtained by enrichment is concentrated to dryness under reduced pressure to obtain lotus seed heart alkaloid extract.

[0011] In the above method, the separation and enrichment conditions are as follows: the ratio of the aqueous solution of the D001 cation exchange resin to the total extract of lotus seed heart can be 8~30 mL:1 g, and the pH value of the loading solution can be 2~4. The elution solvent may be an aqueous solution of ethanol containing 1% NH3·H2O, wherein the volume percentage concentration of ethanol in the aqueous solution may be 90%~95%; The flow rate of the elution solvent can be 3~5 BV / h, and the elution column volume can be 2~4 BV.

[0012] This invention provides a lotus seed heart alkaloid extract prepared by the above method.

[0013] In this invention, the total alkaloid transfer rate of the lotus seed heart alkaloid extract can reach more than 50%, preferably more than 60%. The total alkaloid content in the lotus seed heart alkaloid extract can be 50% to 75% by mass, preferably 60% to 75%.

[0014] The lotus seed heart alkaloid extract described in this invention is used in the preparation of drugs for the prevention and / or treatment of diseases caused by ionizing radiation.

[0015] In the above applications, the diseases caused by ionizing radiation are ionizing radiation damage or diseases induced by ionizing radiation.

[0016] In the above applications, the prevention and / or treatment of diseases caused by ionizing radiation manifests as protection against endothelial damage caused by ionizing radiation.

[0017] In the above applications, the prevention and / or treatment of diseases caused by ionizing radiation manifests as protection against damage to the hematopoietic system and intestines caused by ionizing radiation.

[0018] In the above applications, the prevention and / or treatment of diseases related to ionizing radiation damage manifests as at least one of the following: protection against ionizing radiation-induced endothelial dysfunction, bone marrow hematopoietic dysfunction, and protection against radiation enteritis, a complication of radiotherapy.

[0019] The present invention has the following beneficial effects: 1) The lotus seed heart alkaloid extract obtained by this invention is rich in alkaloid components, and the transfer rate and content of its total alkaloids can reach more than 50%, and can meet the expected efficacy. 2) This invention is the first to adopt the QbD concept, combining solvent extraction and cation exchange resin enrichment technology to develop an extraction method for lotus seed heart alkaloid extract, and establishes the design space for key process parameters; 3) Using ionizing radiation-induced cell or mouse injury models, the protective effects and characteristics of lotus seed heart alkaloid extract against ionizing radiation injury were determined. The main effects were that it could effectively improve the indicators related to ionizing radiation-induced hematopoietic system injury, regulate inflammatory pathways to mediate pyroptosis and reduce ionizing radiation-induced vascular endothelial cell injury, and significantly improve the survival rate of mice with ionizing radiation-induced intestinal injury. Lotus seed heart alkaloid extract may be a candidate drug for the prevention and / or treatment of radiation-related diseases. Attached Figure Description

[0020] Figure 1 The alkaloid extract of lotus seed heart at 6.5 Gy 60 Effects of Co γ-ray irradiation on peripheral blood WBC in mice.

[0021] Figure 2 The alkaloid extract of lotus seed heart at 6.5 Gy 60 Effects of Co γ-ray irradiation on peripheral blood RBCs in mice.

[0022] Figure 3 The alkaloid extract of lotus seed heart at 6.5 Gy 60 Effects of Co γ-ray irradiation on peripheral blood HGB in mice.

[0023] Figure 4 The alkaloid extract of lotus seed heart at 6.5 Gy 60 Effects of Co γ-ray irradiation on peripheral blood PLT in mice.

[0024] Figure 5 The alkaloid extract of lotus seed heart at 14.0 Gy 60 Effects of localized abdominal radiation with Co γ rays on mouse survival.

[0025] Figure 6 The alkaloid extract of lotus seed heart at 15.0 Gy 60 Effects of Co γ-ray radiation on HUVEC cell survival.

[0026] Figure 7 The alkaloid extract of lotus seed heart at 15.0 Gy 60 Effects of Co-γ ray radiation on intracellular ROS levels in HUVEC cells.

[0027] Figure 8 The alkaloid extract of lotus seed heart at 15.0 Gy 60 Effects of Co-γ ray radiation on LDH release in HUVEC cells.

[0028] Figure 9 The alkaloid extract of lotus seed heart at 15.0 Gy 60 Effects of Co γ-ray radiation on Caspase-1 expression in HUVEC cells.

[0029] Figure 10 The alkaloid extract of lotus seed heart at 15.0 Gy 60 Effects of Co γ-ray radiation on the expression of pyroptosis effector proteins in HUVEC cells.

[0030] Figure 11 The alkaloid extract of lotus seed heart at 15.0 Gy 60 Effects of Co γ-ray radiation on IL-18 release in HUVEC cells.

[0031] Figure 12 The alkaloid extract of lotus seed heart at 15.0 Gy 60 Effects of Co γ-ray radiation on IL-1β release in HUVEC cells. Detailed Implementation

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] Example 1: Preparation of lotus seed heart alkaloid extract ① After crushing the lotus seed heart, a traditional Chinese medicine, pass it through a No. 4 sieve (particle size ≤ 65 mesh).

[0035] ② Weigh 5.00 g of lotus seed heart powder from step ①, and use the ethanol heating reflux extraction method. The ethanol concentration (both are volume percentages of the aqueous solution, the same below) is 90%, the material-to-liquid ratio is 15:1 (mL / g), the extraction temperature is 100 ℃, the extraction time is 1.5 h, and the extraction is performed twice to obtain the total extract of lotus seed heart. The total alkaloid transfer rate is 92.95%.

[0036] Calculation of total alkaloid transfer rate:

[0037] In the formula: Tr is the total alkaloid transfer rate (%) in the extract to be tested; W1 is the content of total alkaloids in the extract to be tested obtained from lotus seed heart (mg / g); W0 is the content of total alkaloids in the crude extract of lotus seed heart obtained from lotus seed heart (mg / g). The crude extract of lotus seed heart was obtained according to the extraction method mentioned in the 2020 edition of the Chinese Pharmacopoeia (Part I) - Medicinal Materials and Processed Pieces - Lotus Seed Heart. The operation procedure is as follows: take 2 g of lotus seed heart powder, add 30 mL of methanol, sonicate for 30 min, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of methanol.

[0038] The total alkaloid content was determined using an acidic dye colorimetric method. The principle is as follows: Alkaloids can combine with hydrogen ions in a suitable pH solution to form alkaloid cations, which then form colored ion-pair complexes with acidic dyes. These complexes can be extracted with organic solvents. The absorbance of the organic layer after extraction is recorded using a UV spectrophotometer. The total alkaloid content in the organic layer is directly proportional to its absorbance value. Procedure: The total extract and crude extract of lotus seed heart were dissolved in methanol to obtain a 0.5 mg / mL test solution. 0.4 mL of the test solution was placed in a 50 mL separatory funnel, and 7.0 mL of potassium hydrogen phthalate buffer (pH 4.5) was added. After shaking to mix thoroughly, 4.5 mL of bromocresol green solution was added, and the mixture was thoroughly shaken. Then, 8.0 mL of dichloromethane was added sequentially, and the mixture was shaken to extract. After standing for 2 hours, the dichloromethane layer was collected for analysis. Using dichloromethane as a blank control, the absorbance was measured at 407 nm using a UV-2501PC UV-Vis spectrophotometer (SHIMADZU, Japan). Simultaneously, a methylnephrine standard solution (concentration range: 0.1–0.2 mg / mL) was prepared as a reference solution. The reaction, extraction, and absorbance values ​​of the organic layer were recorded according to the above method. The total alkaloid content in the test solution was calculated based on the absorbance value of the reference solution in the organic layer.

[0039] ③ Weigh 2.00 g of the total extract of lotus seed heart from step ②, and enrich the alkaloids of lotus seed heart using D001 resin. The loading ratio of the aqueous solution of D001 cation exchange resin to the total extract of lotus seed heart is 30:1 (mL / g), the pH of the loading solution is 4, the eluent is an aqueous solution of ethanol containing 1% NH3·H2O (ethanol volume percentage is 95%), the elution flow rate is 5 BV / h, and the elution column volume is 2.0 BV. The lotus seed heart alkaloid extract was enriched, and its total alkaloid transfer rate was 57.26%, and the total alkaloid content was 55.81%.

[0040] Example 2: Preparation of lotus seed heart alkaloid extract ① The Chinese medicinal material lotus plumule is pulverized and passed through a No. 4 sieve (particle size ≤ 65 mesh).

[0041] ② Weigh 5.00 g of the lotus plumule powder obtained in step ①. With an ethanol concentration of 85%, a solid-liquid ratio of 15:1 (mL / g), an extraction temperature of 100 °C, an extraction time of 1.5 h, and an extraction number of 3 times, the total extract of lotus plumule is obtained, and the transfer rate of total alkaloids is 88.56%.

[0042] ③ Weigh 2.00 g of the total extract of lotus plumule obtained in step ②. Use D001 resin to enrich the alkaloids in lotus plumule. According to the ratio of the aqueous solution of D001 cation exchange resin to the loading amount of the total extract of lotus plumule being 30:1 (mL / g), the pH of the loading solution being 2.3, the eluent being an ethanol aqueous solution containing 1% NH₃·H₂O (the volume percentage concentration of ethanol is 93%), the elution flow rate being 3 BV / h, and the volume of the elution column being 2.0 BV, the alkaloid extract of lotus plumule is enriched. Its total alkaloid transfer rate is 73.54%, and the total alkaloid content is 58.64%.

[0043] Example 3: Preparation of the alkaloid extract of lotus plumule ① The Chinese medicinal material lotus plumule is pulverized and passed through a No. 4 sieve (particle size ≤ 65 mesh).

[0044] ② Weigh 5.00 g of the lotus plumule powder obtained in step ①. With an ethanol concentration of 76%, a solid-liquid ratio of 15:1 (mL / g), an extraction temperature of 90 °C, an extraction time of 2.0 h, and an extraction number of 3 times, the total extract of lotus plumule is obtained, and the transfer rate of total alkaloids is 87.61%.

[0045] ③ Weigh 2.00 g of the total extract of lotus plumule obtained in step ②. Use D001 resin to enrich the alkaloids in lotus plumule. According to the ratio of the aqueous solution of D001 cation exchange resin to the loading amount of the total extract of lotus plumule being 10:1 (mL / g), the pH of the loading solution being 2.0, the eluent being an ethanol aqueous solution containing 1% NH₃•H₂O (the volume percentage concentration of ethanol is 95%), the elution flow rate being 3 BV / h, and the volume of the elution column being 2.0 BV, the alkaloid extract of lotus plumule is enriched. Its total alkaloid transfer rate is 67.44%, and the total alkaloid content is 62.55%.

[0046] Example 4: Effect of the alkaloid extract of lotus plumule on the injury of the peripheral hematopoietic system in mice caused by 6.5 Gy 60 Co γ-ray whole-body irradiation 4.1 Experimental animals Male C57BL / 6J mice, about 6 - 8 weeks old, weighing 21 - 23 g, were purchased from Huafukang Biotechnology Co., Ltd., a total of 40 mice. The experimental animal license number: SCXK (Beijing) 2019 - 0008.

[0047] 4.2 Experimental Methods 4.2.1 Animal grouping and administration Animal grouping: Male C57BL / 6J mice were randomly divided into four groups: normal control group (Control), irradiation control group (IR group), irradiation + 150 mg / kg lotus seed heart alkaloid extract group (IR + 150 mg / kg NPAE group), irradiation + 75 mg / kg lotus seed heart alkaloid extract group (IR + 75 mg / kg NPAE group), and irradiation + amifostine group (IR + WR-2721 group), with 8 mice in each group.

[0048] Animal administration: Lotus seed heart alkaloid extract was uniformly suspended in physiological saline solution containing 0.5% carboxymethyl cellulose sodium (CMC-Na), and the positive control drug amifostine (designated WR-2721) was dissolved in physiological saline. Three days before irradiation and two days after irradiation, the IR+150 mg / kg NPAE group was administered lotus seed heart alkaloid extract by gavage at 150 mg / kg / day, and the IR+75 mg / kg NPAE group was administered it at 75 mg / kg / day. Groups C and IR were administered 0.5% CMC-Na physiological saline solution by gavage daily for a total of 5 times. The IR+WR-2721 group was administered amifostine physiological saline solution intraperitoneally at 150 mg / kg / day 30 minutes before irradiation, for a total of one injection.

[0049] 4.2.2 Animal Irradiation Mice were randomly fixed in irradiation boxes to... 60 The patient underwent a single whole-body irradiation with Co-γ rays at a dose of 6.5 Gy (dose rate of 64.24 R / min, irradiation time of 9 minutes and 53 seconds). The control group was placed in an irradiation box but was not irradiated.

[0050] 4.2.3 Detection of peripheral blood cells in irradiated mice One day before irradiation and at 1, 4, 7, 10, 14, 18, 22, and 28 days after irradiation, 20 μL of tail vein blood was collected from mice and diluted in mouse blood routine diluent. The peripheral hematopoietic system of each group of mice was detected and compared using a BC 5000 Vet animal blood cell analyzer (Shenzhen Mindray Animal Medical Technology Co., Ltd.).

[0051] 4.3 Experimental Results 4.3.1 The effect of lotus seed heart alkaloid extract on 6.5 Gy 60 Effects of Coγ-ray irradiation on mouse peripheral blood leukocytes (WBCs) like Figure 1 As shown, 6.5 Gy 60Coγ rays can induce a sharp decline in peripheral blood WBC levels in mice. The WBC count in each irradiation group dropped to the lowest point on day 4 after irradiation, and then slowly began to recover. Mice given lotus seed heart alkaloid extract by gavage showed a slight increase in WBC levels on days 10 and 14 after irradiation, but the difference was not statistically significant.

[0052] 4.3.2 The effect of lotus seed heart alkaloid extract on 6.5 Gy 60 Effects of Coγ-ray irradiation on mouse peripheral blood erythrocytes (RBCs) like Figure 2 As shown, 6.5 Gy 60 Coγ rays induced a slow decrease in peripheral blood RBC levels in mice. The RBC count in the irradiated control group reached its lowest point on day 14 post-irradiation, and then tended to recover. Gavage administration of lotus seed heart alkaloid extract induced a decrease in RBC levels on day 14 post-irradiation. p <0.001), 18 d ( p <0.001), 22 d ( p (<0.01) significantly increased the level of peripheral blood RBCs in irradiated mice, with an effect superior to the positive control drug amifostine.

[0053] 4.3.3 The effect of lotus seed heart alkaloid extract on 6.5 Gy 60 Effects of Co-γ ray irradiation on peripheral blood hemoglobin (HGB) in mice like Figure 3 As shown, 6.5 Gy 60 Coγ rays induced a slow decrease in peripheral blood hemoglobin (HGB) levels in mice. In the irradiated control group, HGB levels reached their lowest point on day 14 post-irradiation and then tended to rise again. Gavage administration of lotus seed heart alkaloid extract induced a decrease in HGB levels on day 14 post-irradiation. p <0.001), 18 d ( p (<0.001) significantly increased the level of HGB in the peripheral blood of irradiated mice, and its effect was superior to that of the positive control drug amifostine.

[0054] 4.3.4 The effect of lotus seed heart alkaloid extract on 6.5 Gy 60 Effects of Coγ-ray irradiation on peripheral blood platelets (PLT) in mice like Figure 4 As shown, 6.5 Gy 60 Coγ rays induced a slow decrease in peripheral blood PLT levels in mice. In the irradiated control group, the PLT count reached its lowest point on day 10 post-irradiation and then tended to rise. Gavage administration of lotus seed heart alkaloid extract induced a decrease in PLT levels on day 14 post-irradiation. p <0.001), 18 d ( p <0.001), 22 d ( p<0.05), 28 d ( p <0.01) significantly increased the peripheral blood PLT level in irradiated mice, and its effect was slightly better than that of the positive drug amifostine.

[0055] In summary, 6.5 Gy 60 Coγ-ray irradiation can induce damage to the hematopoietic system in mice, mainly manifested by a significant decrease in the levels of peripheral blood WBC, RBC, HGB, and PLT in mice. However, intragastric administration of the alkaloid extract from lotus seed core can significantly increase the levels of peripheral blood RBC, HGB, and PLT in irradiated mice and promote the recovery of their peripheral blood cells. Thus, it can be seen that the alkaloid extract from lotus seed core 60 has a good protective effect on the damage of the peripheral hematopoietic system in mice caused by Coγ-ray irradiation.

[0056] Example 5: Effect of the alkaloid extract from lotus seed core on the survival of mice with intestinal injury caused by local abdominal irradiation with 14.0 Gy 60 Coγ-ray 5.1 Experimental animals Male C57BL / 6J mice, about 6 - 8 weeks old, weighing 21 - 23 g, were purchased from Huafukang Biotechnology Co., Ltd., with a total of 40 mice. The experimental animal license number: SCXK (Beijing) 2019 - 0008.

[0057] 5.2 Experimental methods 5.2.1 Animal grouping and administration Animal grouping: Male C57BL / 6J mice were randomly divided into a normal control group (denoted as the Control group), an irradiation control group (denoted as the IR group), an irradiation + 120 mg / kg alkaloid extract from lotus seed core group (denoted as the IR + 120 mg / kg NPAE group), and an irradiation + amifostine group (denoted as the IR + WR - 2721 group), with 10 mice in each group.

[0058] Animal administration: The alkaloid extract from lotus seed core was evenly suspended in a physiological saline solution containing 0.5% CMC - Na, and the positive drug WR - 2721 was dissolved in physiological saline. Three days before irradiation and two days after irradiation, the IR + 120 mg / kg NPAE group was intragastrically administered the alkaloid extract from lotus seed core at 120 mg / kg / d, the Control group and the IR group were intragastrically administered a physiological saline solution containing 0.5% CMC - Na according to body weight daily, for a total of 5 times; the IR + WR - 2721 group was intraperitoneally injected with an amifostine physiological saline solution at 150 mg / kg / d 30 minutes before irradiation, for a total of 1 time.

[0059] 5.2.2 Animal irradiation Mice were anesthetized by intraperitoneal injection of sodium pentobarbital according to their body weight. They were then randomly fixed to acrylic glass plates with tape. Lead bricks were used to partially shield the mice's heads and tails, exposing the abdomen (from the pubis to the xiphoid process). 60 The abdomen was irradiated with a single dose of Co gamma rays at a dose of 14.0 Gy (dose rate of 62.95 R / min, irradiation time of 21 minutes and 58 seconds). The control group was anesthetized and fixed in the same manner but were not irradiated.

[0060] 5.2.3 Survival status of irradiated mice The survival and living conditions of mice were observed and recorded daily for 30 days after irradiation. The survival rate and average survival time of mice 30 days after irradiation were calculated, and the mouse survival curve (Kaplan-Meier) was plotted.

[0061] 5.3 Experimental Results 14.0 Gy 60 Localized abdominal radiation with Coγ rays can cause death in mice. Survival rates of mice in each group 30 days after irradiation were observed (e.g., Figure 5 As shown in the figure, the survival rates of mice 30 days after irradiation in the normal control group, irradiation control group, irradiation + 120 mg / kg lotus seed heart alkaloid extract group, and irradiation + amifostine group were 100%, 10%, 70%, and 50%, respectively. These results indicate that gavage administration of lotus seed heart alkaloid extract significantly improved the survival rate of mice 30 days after irradiation, and the effect was superior to that of the positive control drug amifostine. Therefore, lotus seed heart alkaloid extract has the effect of... 60 Coγ-ray localized abdominal radiation has a good protective effect against intestinal damage induced by Coγ-rays in mice.

[0062] Example 6: The effect of lotus seed heart alkaloid extract on 15.0 Gy 60 Effects of Co-γ ray radiation on human umbilical vein endothelial cells (HUVECs) 6.1 Experimental Cells and Reagents Human umbilical vein endothelial (HUVEC) cells were purchased from the Cell Resource Center of the School of Basic Medical Sciences, Peking Union Medical College.

[0063] Fetal bovine serum was purchased from Gibco, USA; low-glucose DMEM medium and CCK-8 enhanced detection kit were purchased from Dalian Meilun Biotechnology Co., Ltd.; double antibodies (penicillin-streptomycin), trypsin, and phosphate-buffered saline (PBS) were all purchased from Zhongke Maichen Technology Co., Ltd.

[0064] 6.2 Experimental Methods 6.2.1 60 Establishment of a Co-γ ray radiation-induced HUVEC cell damage model HUVEC cells were cultured in 96-well or 6-well plates to...60 A radiation-induced HUVEC cell damage model was established by single-use Co-γ irradiation of cell culture plates, with a radiation dose of 15.0 Gy.

[0065] 6.2.2 60 Detection of HUVEC cell viability under Co-γ irradiation CCK-8 assay principle: CCK-8 reagent is oxidized and reduced by intracellular dehydrogenases to produce a water-soluble orange-yellow formazan. The amount of formazan produced is directly proportional to the number of viable cells. The absorbance (OD) value is measured at 450 nm and compared to indicate the relative level of cell viability. Procedure: HUVEC cells are cultured in low-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrin antibiotics. HUVEC cells are then cultured at a density of 5 × 10⁶ cells / year. 3 Cells / well were seeded in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. A blank control group (Control group), an irradiation control group (IR group), and an irradiation + different concentrations of lotus seed heart alkaloid extract treatment group (IR+NPAE group) were set up. The lotus seed heart alkaloid extract was diluted to different concentrations with culture medium and added to the cells. The cells were incubated for another 2 h, and then... 60 HUVEC cells were irradiated with Co γ rays (dose rate 88.98 R / min, irradiation time 17 min 12 s). After irradiation, the cells were cultured in an incubator for 84 h. The culture medium was then discarded, and 100 μL of serum-free medium containing 10% CCK-8 was added to each well. The cells were incubated at 37℃ for another 30 min. The OD value at 450 nm was measured using a MULTISKAN SkyHigh microplate reader (Thermo, USA). Cell viability (%) = (absorbance of treatment group - background absorbance) / (absorbance of blank group - background absorbance) × 100%.

[0066] 6.2.3 60 Detection of reactive oxygen species (ROS) generation in HUVEC cells induced by Co gamma-ray radiation Dihydroethidium (DHE) fluorescent probe detection principle: DHE is a commonly used superoxide anion fluorescent detection probe. Under the action of superoxide anions in living cells, DHE can be dehydrogenated to generate ethidium bromide. Ethidium bromide then binds to intracellular RNA or DNA to produce red fluorescence. Therefore, the stronger the red fluorescence, the higher the ROS level. Procedure: HUVEC cells are prepared at 2 × 10⁻⁶ cells / year. 5One sample per well was inoculated into a 6-well plate and incubated at 37°C with 5% CO2 and saturated humidity for 24 h. Control, IR, and IR+NPAE groups were established. NPAE stock solution was diluted with culture medium and added to the corresponding wells. The Control and IR groups were supplemented with 100 μL of DMSO medium of equal concentration. Incubation continued. Two h after drug administration, [the following was observed]... 60 HUVEC cell culture plates were irradiated with Co γ rays (dose rate 90.83 R / min, irradiation time 16 min 50 s). After irradiation, the cell culture plates were placed in a 37℃, 5% CO2 incubator for 48 h. The culture medium in the wells was discarded, and the plates were rinsed twice with serum-free medium. Then, serum-free medium diluted with Hoechst 33342 fluorescent probe (1:200 dilution, ImmunoChemistryTechnologies, USA) and DHE fluorescent probe (1:3000 dilution, Shanghai Bosheng Biotechnology Co., Ltd.) were added. The plates were incubated at 37℃ for 20 min. The fluorescence intensity of each group of cells was observed and photographed at an excitation wavelength of 530 nm (red fluorescence, DHE) and 360 nm (blue fluorescence, Hoechst 33342) using a CKX53 fluorescence inverted microscope (Olmypus, Japan). The images were then used for quantitative analysis of fluorescence intensity using ImageJ software.

[0067] 6.2.4 60 Detection of lactate dehydrogenase (LDH) release in HUVEC cell supernatant induced by Co gamma ray radiation LDH Detection Kit Principle: When cells are damaged or die, they release LDH into the culture medium. LDH catalyzes the formation of pyruvate and NADH from lactate. NADH, via an electron carrier, reduces the LDH detection reagent (water-soluble tetrazolium salt WST) to water-soluble orange WST formazan. The absorbance of WST formazan is directly proportional to the LDH content. Detection is performed at 490 nm, and the OD value is compared to reflect the relative levels of dead / damaged cells. Procedure: HUVEC cells are cultured at 5 × 10⁻⁶ cells / year. 3 One sample per well was inoculated into a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. Control, IR, and IR+NPAE groups were set up, and the drug solution was added sequentially. Incubation was continued for 2 h, followed by... 60HUVEC cell culture plates were irradiated with Co γ rays (dose rate 89.85 R / min, irradiation time 17 min 01 sec), and the cells were cultured for another 96 h after irradiation. 100 μL of cell culture supernatant from each group was aspirated into a new 96-well plate. Following the LDH detection kit procedure, 100 μL of the detection working solution was added to the cell culture supernatant. The plates were cultured in the dark at room temperature (25℃) for 30 min, and then 10 μL of stop solution was added to each well. The OD value was measured at 490 nm using a microplate reader, and the LDH release rate was calculated.

[0068] In the formula: A is the OD value of the cell supernatant in each drug administration group; B is the OD value of the cell supernatant in the IR group; C is the OD value of the cell-free blank culture medium.

[0069] 6.2.5 60 Detection of the pyroptosis effect of Co gamma-ray radiation on HUVEC cells (1) Detection of the expression level of Caspase-1, a marker of pyroptosis HUVEC cells were administered at 2×10 5 Inoculate one cell per well into a 6-well plate and incubate at 37°C with 5% CO2 and saturated humidity for 24 h. Set up Control, IR, and IR+NPAE groups, add drug solution according to group, and continue incubation for 2 h, then proceed with... 60 HUVEC cell culture plates were irradiated with Co γ rays (dose rate 89.66 R / min, irradiation time 17 minutes 04 seconds), and cultured for another 72 hours after irradiation. Cells from each group were digested with trypsin and collected in 1.5 mL EP tubes. The cells were washed twice with PBS. Following the instructions of the FAM-FLICA Caspase-1 Assay kit (ImmunoChemistry Technologies, USA), 50 μL of FLICA Caspase-1 reagent was diluted 150-fold to obtain FLICA Caspase-1 working solution. 200 μL of FLICA Caspase-1 working solution was added to each cell tube, and the cells were incubated at 37°C for 60 min. Then, the cells were centrifuged at 1200 rpm / min for 5 min at 4°C, and the supernatant was discarded. The cells were washed once with buffer wash, centrifuged again, and the supernatant was discarded. 300 μL of propidium iodide (PI) staining solution was added to each cell tube, and the cells were incubated at room temperature in the dark for 5 min. FLICA Caspase-1 (excitation wavelength 488 nm, emission wavelength 510 nm) and PI (excitation wavelength 488 nm, emission wavelength 580 nm) were detected using a FACSAria II flow cytometer (BD Bioscience, USA).

[0070] (2) Detection of expression levels of pyroptosis marker proteins HUVEC cells were administered at 2×10 5 One sample per well was inoculated into a 6-well plate and incubated at 37°C with 5% CO2 and saturated humidity for 24 h. Control, IR, and IR+NPAE groups were set up, and the drug solution was added sequentially. Incubation was continued for 2 h, followed by... 60 HUVEC cell culture plates were irradiated with Co γ rays (dose rate 87.81 R / min, irradiation time 17 minutes 25 seconds), and the cells were cultured for another 72 hours after irradiation.

[0071] ① Extraction and concentration determination of total cellular protein: Cells were digested with trypsin and collected in 1.5 mL EP tubes. The tubes were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. 200 μL of protein lysis buffer (containing 10% protease inhibitor) was added, and the mixture was thoroughly mixed by pipetting. The cells were then vortexed for 15 s on ice for 30 min, with vortexing for 15 s every 5 min during lysis. The cells were centrifuged at 12000 rpm for 15 min, and the supernatant was collected.

[0072] Following the procedure outlined in the BCA protein concentration assay kit (Jiangsu Kangwei Century Biotechnology Co., Ltd.), 12 μL of sample was diluted with 48 μL of PBS (1:5 dilution), and 25 μL of each solution was added to the wells of a labeled 96-well plate. Two replicates were performed for each sample. 200 μL of BCA working solution (BCA-A:BCA-B = 50:1) was added to each well, and the mixture was thoroughly mixed. The 96-well plate was then capped and incubated at 37°C for 30 min. After cooling to room temperature, the absorbance was measured at 540 nm, and the protein concentration was calculated. The protein concentration was adjusted to the same level using PBS. 160 μL of protein sample was taken, and 40 μL of loading buffer (5×) was added. After thorough mixing, the sample was boiled in water for 10 min to denature the protein. After returning to room temperature, the sample was stored at -20°C.

[0073] ② Gel preparation: In this experiment, according to the predicted molecular weight and standard molecular weight gradient of the target protein, SDS-PAGE gels with concentrations of 7.5%, 10%, and 12.5% ​​were prepared according to the instructions of the one-step PAGE gel rapid preparation kit (Shanghai Yamei Biomedical Technology Co., Ltd.). After solidification and stabilization, the comb teeth were removed and the gels were ready for electrophoresis.

[0074] ③ Protein sample loading and electrophoresis: Assemble the electrophoresis equipment and check for leaks. After passing the inspection, prepare the electrophoresis buffer and add it to the mark. Load the sample at a protein amount of 20 μg / g, and add 5 μL of protein molecule marker to both sides of the sample. First, use a constant voltage current of 80V for electrophoresis. After 20 minutes, when the sample enters the separation layer, adjust the voltage to a constant voltage of 120V until the target protein is in the appropriate position.

[0075] ④ Transfer and Blocking: Prepare transfer buffer in advance and pre-cool at -20℃. Remove the gel from the electrophoresis tank, trim off the non-protein portion, and cut the PVDF membrane to the gel size. Activate the membrane by soaking it in anhydrous methanol for 30 seconds, then soak it in transfer buffer. After assembling the clamps and removing air, place the membrane in the pre-cooled transfer buffer, and keep the electrophoresis tank on ice. Perform transfer at a constant current of 200 mA, adjusting the transfer time to 70-90 min depending on the protein molecular weight. After electrotransfer, wash the membrane with TBST to remove any remaining transfer buffer, then block the membrane in rapid blocking buffer on a shaker at room temperature for 30 min.

[0076] ⑤ Primary antibody incubation: After blocking, carefully remove the PVDF membrane, cut the appropriate membrane according to the protein molecule marker, and place it in the labeled antibody incubation box. Dilute the pro-caspase-1 antibody (Abcam, USA, catalog number: ab179515) and GSDMD antibody (Abcam, USA, catalog number: ab219800) at a ratio of 1:1000 to obtain the primary antibody solution, and then add it into the incubation box until it covers the surface of the PVDF membrane. Incubate overnight at 4°C on a shaker.

[0077] ⑥ Secondary antibody incubation: Recover the primary antibody solution, wash the membrane three times with TBST, each time for 10 min on a shaker at room temperature. Dilute the GAPDH antibody (Cell Signaling Technology, USA, catalog number: 2118S) at a ratio of 1:5000 to obtain the secondary antibody solution, then add it to the antibody incubation box, incubate slowly on a shaker at room temperature for 90 min, then place the membrane in TBST and wash it three times, each time for 10 min on a shaker at room temperature.

[0078] ⑦ Development and analysis: Prepare the chemiluminescence working solution (SuperSingular) according to the ratio of solution A to solution B of 1:1. TM West PicoPLUS luminescent substrate (Thermo Fisher Scientific, USA) was dropped onto the membrane and soaked for 1 min. The substrate was then developed and photographed using the ChemiDoc chemiluminescence imaging system (Bio-Rad, USA). The images were analyzed for grayscale values ​​using ImageJ software.

[0079] (3) Detection of the release of pyroptosis markers interleukin-18 (IL-18) and interleukin-1β (IL-1β) in cell culture supernatant ELISA kit principle: Specific antibodies are coated onto an enzyme-linked immunosorbent assay (ELISA) plate. Standards or samples are added to the plate, and the analyte in the sample is captured by the antibodies on the plate. Detection antibodies are then added, forming an antibody-antigen-antibody complex. Enzymes and substrates are added, and a color reaction occurs between the enzyme and the substrate. The OD value is detected at 450 nm, indicating the relative level of the analyte. Procedure: HUVEC cells are cultured at 5 × 10⁻⁶ cells / year. 3 One sample per well was inoculated into a 96-well plate and incubated at 37°C with 5% CO2 for 24 h. Control, IR, and IR+NPAE groups were set up. NPAE stock solution was diluted with culture medium and added to the corresponding wells. The Control and IR groups were supplemented with an equal concentration of DMSO. Three parallel control wells were set up. Incubation continued for 2 h after drug administration. 60 HUVEC cell culture plates were irradiated with Co γ rays (IL-18: dose rate 90.60 R / min, irradiation time 16 min 53 s; IL-1β: dose rate 87.81 R / min, irradiation time 17 min 25 s). After irradiation, the cell culture plates were placed in a 37℃, 5% CO2 incubator and cultured for 96 h. After the culture was completed, the supernatant was carefully aspirated and the samples were stored at -80℃. The levels of IL-18 and IL-1β in the cell supernatant were detected according to the instructions of the provided human IL-18 ELISA kit (Beijing Dakwei Biotechnology Co., Ltd.) and human IL-1β ELISA kit (Jiangsu Enzyme Immunoassay Co., Ltd.).

[0080] 6.3 Experimental Results 6.3.1 The effects of lotus seed heart alkaloid extract on 60 Effects of Co-γ-ray radiation on HUVEC cell survival The results show (e.g.) Figure 6 As shown), via 60 After Co γ-ray irradiation, the cell survival rate in the IR group decreased to 48.59% ( p <0.0001), 5–20 μg / mL lotus seed heart alkaloid extract can significantly increase the survival rate of irradiated cells to 76.38% (20 μg / mL, p <0.0001), 73.91% (10 μg / mL, p <0.0001), 72.73% (5 μg / mL, p <0.0001), indicating that the lotus seed heart alkaloid extract has an effect on... 60 Co γ-ray radiation has a good protective effect against HUVEC cell damage induced by radiation.

[0081] 6.3.2 The effects of lotus seed heart alkaloid extract on... 60Effects of Co-γ-ray radiation on ROS levels in HUVEC cells The results show (e.g.) Figure 7 As shown), via 60 Following Co-γ irradiation, the level of red fluorescence in cells of the IR group significantly increased, indicating an increase in intracellular ROS levels after irradiation. The 5–20 μg / mL lotus seed heart alkaloid extract treatment group significantly reduced the level of red fluorescence in irradiated cells, indicating that the lotus seed heart alkaloid extract can effectively alleviate [the condition]. 60 Co-γ ray radiation leads to the accumulation of ROS in HUVEC cells.

[0082] 6.3.3 The effects of lotus seed heart alkaloid extract on... 60 Effect of Co-γ ray radiation on LDH levels in HUVEC cell supernatant The results show (e.g.) Figure 8 As shown), via 60 Following Co-γ irradiation, the LDH level in the cell supernatant of the IR group significantly increased, indicating that radiation can induce pyroptosis and release large amounts of LDH; the 5–20 μg / mL lotus seed heart alkaloid extract treatment group significantly reduced the LDH level in the supernatant of irradiated cells, indicating that lotus seed heart alkaloid extract can effectively alleviate LDH levels. 60 Co gamma ray radiation induces pyroptosis in HUVEC cells.

[0083] 6.3.4 The effects of lotus seed heart alkaloid extract on 60 Effects of Co-γ-ray radiation on pyroptosis damage in HUVEC cells (1) Expression level of Caspase-1, a marker of pyroptosis The results show (e.g.) Figure 9 As shown), via 60 Following Co γ-ray irradiation, the expression levels of Caspase-1 and PI in the IR group cells were significantly increased, indicating that radiation could induce pyroptosis, with the pyroptosis rate increasing to 31.52 ± 6.35% (pyroptosis rate = Q2 + Q3). Treatment with 5–20 μg / mL lotus seed heart alkaloid extract significantly reduced the expression levels of Caspase-1 and PI in irradiated cells, and 20 and 10 μg / mL lotus seed heart alkaloid extract significantly reduced the post-irradiation pyroptosis rate to 14.22 ± 2.53%, respectively. p <0.001) and 20.32 ± 3.82% ( p <0.05), indicating that lotus seed heart alkaloid extract can effectively protect... 60 Co gamma ray radiation induces pyroptosis in HUVEC cells.

[0084] (2) Expression levels of pyroptosis effector proteins The results show (e.g.) Figure 10 As shown), via 60 Following Co-γ irradiation, the expression of the pyroptosis effector protein GSDMD-N was significantly upregulated in the IR group, while the expression of pro-caspase-1 and GSDMD was significantly downregulated. Lotus seed heart alkaloid extract (5–20 μg / mL) could reverse the expression of radiation-induced pyroptosis effector proteins, upregulate the expression of pro-caspase-1 and GSDMD in irradiated cells, and downregulate the expression of GSDMD-N, indicating that lotus seed heart alkaloid extract can improve... 60 Co γ-ray radiation induces abnormal expression of pyroptosis effector proteins in HUVEC cells.

[0085] (3) Release of pyroptosis marker IL-18 from cell supernatant The results show (e.g.) Figure 11 As shown), via 60 Following Co-γ irradiation, the release of IL-18 in the cell supernatant of the IR group was significantly increased, indicating that radiation can induce pyroptosis and release large amounts of IL-18. The administration of 5–20 μg / mL lotus seed heart alkaloid extract significantly reduced the release of IL-18 in the irradiated cell supernatant, indicating that lotus seed heart alkaloid extract can effectively alleviate... 60 Co gamma ray radiation induces pyroptosis in HUVEC cells.

[0086] (3) Release of pyroptosis marker IL-1β from cell supernatant The results show (e.g.) Figure 12 As shown), via 60 Following Co γ-ray irradiation, the release of IL-1β in the cell supernatant of the IR group was significantly increased, indicating that radiation can induce pyroptosis and release large amounts of IL-1β. The administration of 5–20 μg / mL lotus seed heart alkaloid extract significantly reduced the release of IL-1β in the irradiated cell supernatant, indicating that lotus seed heart alkaloid extract can effectively alleviate... 60 Co gamma ray radiation induces pyroptosis in HUVEC cells.

Claims

1. A method for preparing a lotus seed heart alkaloid extract, comprising the following steps: 1) grinding dried lotus seed hearts into powder; 2) Extract the powder obtained in step 1) in an ethanol-water solution, remove the solvent from the resulting extract, and obtain the total extract of lotus seed heart; 3) The total extract of lotus seed heart from step 2) is separated and enriched by D001 cation exchange resin to obtain lotus seed heart alkaloid extract.

2. The method according to claim 1, characterized in that, The lotus seed heart is ground into powder with a particle size of 0~65 mesh, excluding 0; The volume percentage concentration of ethanol in the ethanol-water solution is 75%~90%; The ratio of the ethanol aqueous solution to the powder is 10~20:1 mL / g; The extraction temperature is 70~100℃; The extraction time is 1-2 hours, and the extraction is repeated 2-3 times.

3. The method according to any one of claims 1-2, characterized in that, The separation and enrichment conditions are as follows: the ratio of the aqueous solution of the D001 cation exchange resin to the total extract of lotus seed heart is 8~30 mL:1 g, and the pH of the loading solution is 2~4. The elution solvent is an aqueous ethanol solution containing 1% NH3·H2O, wherein the volume percentage concentration of ethanol in the aqueous ethanol solution is 90%~95%; the flow rate of the elution solvent is 3~5 BV / h, and the elution column volume is 2~4 BV.

4. The lotus seed heart alkaloid extract prepared by any one of claims 1-3.

5. The lotus seed heart alkaloid extract according to claim 4, characterized in that, The total alkaloid content of the lotus seed heart alkaloid extract is 50%~75% by mass.

6. The use of the lotus seed heart alkaloid extract according to claim 4 or 5 in the preparation of a medicament for the prevention and / or treatment of diseases caused by ionizing radiation.

7. The application according to claim 6, characterized in that, The diseases caused by ionizing radiation are ionizing radiation damage or diseases induced by ionizing radiation.

8. The application according to claim 6 or 7, characterized in that, The prevention and / or treatment of diseases caused by ionizing radiation manifests as protection against endothelial damage caused by ionizing radiation.

9. The application according to claim 6 or 7, characterized in that, The prevention and / or treatment of diseases caused by ionizing radiation refers to protection against damage to the hematopoietic system and intestines caused by ionizing radiation.

10. The application according to claim 6 or 7, characterized in that, The prevention and / or treatment of diseases caused by ionizing radiation refers to at least one of the following: protection against ionizing radiation-induced endothelial dysfunction, bone marrow hematopoietic dysfunction, and protection against radiation enteritis, a complication of radiotherapy.

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