Use of total alkaloids of platanus acerifolia in preparation of medicine for reversing oral mucositis caused by cancer treatment and prepared medicine

Abstract

The application discloses application of total alkaloids in plumula nelumbinis in preparation of a medicine for treating oral mucositis caused by cancer treatment and the prepared medicine. The main active components of the total alkaloids in plumula nelumbinis can effectively weaken damage of a chemotherapy drug to normal human epidermal cells, and can enhance an antitumor effect of the chemotherapy drug, which not only widens the application range of the total alkaloids in plumula nelumbinis in the medical field, but also provides a brand-new treatment medicine for cancer patients with oral mucositis, and therefore has important clinical significance.

Description

Application of total alkaloids from lotus seed hearts in the preparation of drugs to reverse oral mucositis caused by cancer treatment and the prepared drugs Technical Field

[0001] This invention relates to the pharmaceutical use of a compound and the preparation of a drug, specifically to the application of total alkaloids from lotus seed heart in the preparation of a drug for reversing oral mucositis caused by cancer treatment and the preparation of the drug. Background Technology

[0002] Chemotherapy-induced stomatitis is a significant toxic side effect of systemic chemotherapy and head and neck radiotherapy, manifesting as erosion and ulceration of the oral mucosa and subsequent bacterial infection. The incidence of chemotherapy-induced stomatitis is approximately 20% to 40% in cancer patients receiving chemotherapy. However, for head and neck cancer patients receiving both chemotherapy and radiotherapy, this proportion rises to over 90%. This type of stomatitis not only causes significant pain and affects patients' daily eating habits, but also often leads to changes in cancer treatment plans, increases the risk of various bacterial and fungal infections, and ultimately impacts patient prognosis.

[0003] The pathogenesis of oral mucositis involves the excessive production of reactive oxygen species and the upregulation of pro-inflammatory cytokines. Although it is a common side effect of cancer treatment, there is currently no universally accepted standard regimen for the prevention or treatment of oral mucositis.

[0004] Lotus seed heart is the dried young leaf and radicle of the mature seed of *Nelumbo nucifera* Gaertn., a plant in the Nymphaeaceae family. It is bitter and cold in nature, and enters the heart and kidney meridians. It has the functions of clearing the heart and calming the mind, harmonizing the heart and kidneys, astringing essence and stopping bleeding. It is used to treat heat entering the pericardium, delirium, heart-kidney disharmony, insomnia, seminal emission, and hematemesis due to blood heat. Modern research shows that the main active components of lotus seed heart are alkaloids, including methyl lotusinine, isoliensinine, and lotusinine. Although lotus seed heart alkaloids have been reported to have antiarrhythmic, anticancer, antioxidant, and anti-insomnia activities, there are no reports of total lotus seed heart alkaloids having a protective effect against chemotherapy-induced oral mucositis. Summary of the Invention

[0005] Purpose of the invention: This invention aims to provide the application of total alkaloids from lotus seed heart in the preparation of a drug to reverse oral mucositis caused by cancer treatment. The active components of total alkaloids from lotus seed heart can effectively reverse the toxicity of chemotherapy drugs to human immortalized epidermal cells and are beneficial to the anti-tumor efficacy of chemotherapy drugs. This invention also aims to provide a drug to reverse oral mucositis caused by cancer treatment.

[0006] Technical solution: The total alkaloids from lotus seed hearts described in this invention can be used in the preparation of drugs to reverse oral mucositis caused by cancer treatment.

[0007] Furthermore, the total alkaloids in the lotus seed heart mainly include liensinine, isoliensinine, methyl liensinine, nuciferine, pronuciferine, lotusine, methylcorypalline, and demethyl-coclaurine. It also contains trace amounts of flavonoids such as galoteolin, hyperin, and rutin. Methyl liensinine and isoliensinine are the most active components, and their structures are as follows:

[0008]

[0009] Furthermore, the cancer treatment includes chemotherapy and / or radiotherapy, and even further, the chemotherapy drugs include at least one of 5-fluorouracil, methotrexate, docetaxel, doxorubicin, cyclophosphamide, platinum-based drugs, gemcitabine, and irinotecan hydrochloride.

[0010] This invention also provides a medicament for reversing oral mucositis caused by cancer treatment, comprising total alkaloids from lotus seed heart and / or various pharmaceutically acceptable and widely used carriers known in the art, such as diluents, binders, absorbents, disintegrants, dispersants, humectants, solubilizers, buffers, surfactants, etc.

[0011] Furthermore, the drug can be formulated into various dosage forms such as solids, liquids, and gases, and can be administered orally, by injection, nasal cavity, oral mucosa, skin, intraperitoneal cavity, or rectum. Oral dosage forms include nebulizers, powders, tablets, granules, capsules, solutions, emulsions, gels, or suspensions. Injection administration includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, or intracavitary injection.

[0012] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The main active components of total alkaloids from lotus seed heart can effectively weaken the damage of chemotherapy drugs to normal human epidermal (HaCaT) cells, while enhancing the anti-tumor efficacy of chemotherapy drugs. This not only broadens the scope of application of total alkaloids from lotus seed heart in the pharmaceutical field, but also provides a new treatment drug for cancer patients suffering from oral mucositis caused by cancer treatment, thus having important clinical significance. Attached Figure Description

[0013] Figure 1 shows the toxicity experiment of 5-FU on HaCaT cells;

[0014] Figure 2 shows the toxicity experiment of methylnephrine on HaCaT cells;

[0015] Figure 3 shows the toxicity experiment of isolithocarmine on HaCaT cells;

[0016] Figure 4 shows the toxicity experiment of lotus seed alkaloids on HaCaT cells;

[0017] Figure 5 shows the effect of pretreatment with methylnephrine and isonephrine on the killing effect of 5-FU on HaCaT cells.

[0018] Figure 6 shows the effect of lotus seed alkaloid pretreatment on 5-FU killing HaCaT cells;

[0019] Figure 7 shows the toxicity experiment of 5-FU on 4T1 cells;

[0020] Figure 8 shows the effect of methyl lotusine pretreatment on the killing effect of 5-FU on 4T1 cells;

[0021] Figure 9 shows the effect of isolithocarmine pretreatment on the killing effect of 5-FU on 4T1 cells.

[0022] Figure 10 shows the effect of lotus seed alkaloid pretreatment on 5-FU killing of 4T1 cells. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0024] 1. Experimental Materials and Instruments

[0025] Compounds: Methylnepenone, purity ≥98%; Isolipenone, purity ≥98%; Nepenone, purity ≥98%; Nepenone, purity ≥98%, all purchased from Wuhan Tianzhi Biotechnology Co., Ltd. Before use, the compound powder was prepared into a 20 mM stock solution using dimethyl sulfoxide; 5-FU, purity ≥98%, purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Before use, the compound powder was prepared into a 400 mM stock solution using dimethyl sulfoxide; all stock solutions were stored at -20℃. Before use, the solutions were diluted to the required concentration using the appropriate cell culture medium.

[0026] Cell lines: Human immortalized epithelial cells (HaCaT) were selected to mimic oral mucosal epithelial cells and were purchased from Servicebio; mouse breast cancer cells (4T1) were purchased from Procell (Wuhan).

[0027] Cell culture reagents: DMEM medium, fetal bovine serum (FBS), and penicillin-streptomycin mixture (100×) were all purchased from Servicebio. Complete culture medium was prepared with a mixture of 10% FBS and 1% penicillin-streptomycin.

[0028] The relevant indicator detection kit is Cell Counting Kit-8 (CCK8), purchased from APExBIO. Store at -20℃ and dissolve at room temperature before use.

[0029] Experimental instruments: VS-1300L-U clean bench (Suzhou Antai Air Technology Co., Ltd.), carbon dioxide incubator (Thermo Scientific), Invitrogen hemocytometer, inverted fluorescence microscope (Leica, Germany), BioTek microplate reader, etc.

[0030] 2. Experimental Methods

[0031] Cell viability assay: Cell viability was detected using a CCK8 assay kit, and the OD value at 450 nM was measured using a microplate reader. Cell viability (%) = [(experimental wells - blank wells) / (control wells - blank wells)] × 100%; graphs were plotted using Graphpad Prism 8.0 software.

[0032] Cytotoxicity analysis was performed: the cytotoxicity of 5-FU to HaCaT cells and the safe concentrations of the main active components of lotus seed heart alkaloids, methyl lotus seed heart alkaloid, isolivine heart alkaloid, and lotus seed heart alkaloid in HaCaT cells were determined by CCK8 assay. Specifically, HaCaT cells (1.5 × 10⁻⁶) were... 4 Cells were seeded per well into 96-well plates and incubated for 24 h. Then, cells were treated with different concentrations of methylcaenine, isolienine, and caenine (0.1-20 μM) for 24 h, or with or without 5-FU (0.1-50 μM) for 24 h and 48 h. The supernatant was discarded, and the cells were washed once with phosphate-buffered saline (PBS). CCK8 reagent was then added, and the cells were incubated for another 0.5-1.5 h. The absorbance at 450 nm was measured using a microplate reader. Cell viability was expressed as a percentage of the control (untreated cells), and the cytotoxicity of 5-FU, methylcaenine, isolienine, and caenine was determined separately. Cells with a viability greater than 90% were considered non-cytotoxic.

[0033] The effects of the main components of total alkaloids in lotus seed heart—methyllotusinine, isolinusinine, and lobeline—on the killing effect of 5-FU on HaCaT cells were investigated: HaCaT cells (1.5 × 10⁻⁶) were... 4 Cells were seeded at 100 cells / well in 96-well plates and incubated for 24 h. Then, within safe limits, cells were pretreated with different concentrations of methylcaenine, isolienine, and caenine for 24 h. The supernatant was removed, and the cells were treated with 5-FU for 48 h, followed by supernatant removal. Cells were washed once with PBS, and then CCK8 reagent was added, followed by incubation for 0.5–1.5 h. The absorbance at 450 nm was measured using a microplate reader. Blank control group, positive control group, 5-FU treatment alone group, and drug pretreatment + 5-FU treatment group were set up.

[0034] This study investigated the effects of the main components of total alkaloids in lotus seed heart—methyllotusinine, isolinusinine, and lotusinine—on the cytotoxic effects of 5-FU on 4T1 cells.

[0035] (1) First, the toxicity of 5-FU to 4T1 cells was determined: 4T1 cells (0.8 × 10⁻⁶) were subjected to an experiment. 4 Cells were seeded into 96-well plates (number of cells per well) and incubated for 24 hours. The cells were then treated with 5-FU (0.1-50 μM) for 48 hours. The supernatant was discarded, and the cells were washed once with PBS. The cells were then incubated with CCK8 reagent for another 0.5-1 hours. The absorbance at 450 nm was measured using a microplate reader.

[0036] (2) Then, the effects of the main components of lotus seed heart alkaloids, methyl lotusine, isolivineine, and lotusine on the killing effect of 5-FU on 4T1 cells were determined: 4T1 cells (0.8×10⁻⁶) were... 4 Cells were seeded per well into 96-well plates and incubated for 24 h. Then, cells were pretreated with methylcaenine, isolienine, and caenine (2, 10, and 50 μM) for 24 h. After removing the supernatant, cells were treated with 5-FU for 48 h. The supernatant was discarded, and cells were washed once with PBS. Cells were then incubated with CCK8 reagent for 0.5–1 h, and absorbance was measured at 450 nm using a microplate reader. A blank control group, a positive control group, a 5-FU-only treatment group, and a drug pretreatment + 5-FU treatment group were set up.

[0037] 3. Experimental Results

[0038] The toxicity of 5-FU, methylcaenine, isolienine, and caenine to HaCaT cells was investigated: At a concentration of 1 μM and an action time of 48 h, the cell viability of HaCaT cells was 58.6% (Figure 1), so a concentration of 1 μM was chosen for subsequent experiments. Methylcaenine, isolienine, and caenine showed virtually no toxicity to HaCaT cells at concentrations below 5 μM (Figures 2-4), so concentrations of 2 μM and 5 μM were used for subsequent experiments.

[0039] Effects of the main components of lotus seed heart alkaloids—methyllotus seed heart alkaloids, isolidinine, and lotus seed heart alkaloids—on the killing effect of 5-FU on HaCaT cells: With 5-FU alone, the average cell viability was 52.6%. After prophylactic administration of 2 μM and 5 μM methyllotus seed heart alkaloids, the average cell viability was 59.1% and 63.3%, respectively. After prophylactic administration of 2 μM and 5 μM isolidinine, the average cell viability was 56.6% and 64.4%, respectively. It is evident that 5 μM methyllotus seed heart alkaloids and isolidinine significantly improved cell viability compared to the 5-FU alone treatment group (Figure 5). In the lotus seed heart alkaloid experiment, with 5-FU alone, the average cell viability was 51.4%. After prophylactic administration of 2 μM and 5 μM lotus seed heart alkaloids, the average cell viability was 52.4% and 54.6%, respectively. It is evident that there was no significant difference in cell viability between the 5 μM lotus seed heart alkaloids and the 5-FU alone treatment group (Figure 6). (*P<0.05,**P<0.01,***P<0.001,****P<0.0001)

[0040] Effects of the main components of total alkaloids in lotus seed heart, namely methyl lotusine, isolivineine, and lotusine, on the killing effect of 5-FU on 4T1 cells:

[0041] (1) First, the toxicity of 5-FU to 4T1 cells was determined. The results showed that when the concentration of 5-FU was 1 μM, the cell viability was 49.7% after 48 h of treatment. Therefore, the concentration of 5-FU was 1 μM in the following experiments (Figure 7).

[0042] (2) Effects of the main components of total alkaloids in lotus seed heart, methyl lotusine, isolivineine, and lotusine, on the killing effect of 5-FU on 4T1 cells. The results showed that methyl lotusine, isolivineine, and lotusine did not affect the killing effect of 5-FU on 4T1 cells; on the contrary, they were beneficial to the killing effect of 5-FU on 4T1 cells (Figures 8-10). (#P<0.05,##P<0.01,###P<0.001,####P<0.0001).

Claims

1. The application of isolidinine or methylolidinine in the preparation of drugs to reverse oral mucositis caused by cancer treatment, characterized in that, The cancer treatment is chemotherapy, the chemotherapy drug is 5-fluorouracil, and the cancer is breast cancer.

2. The application of isolidinine or methylolidinine according to claim 1 in the preparation of a drug for reversing oral mucositis caused by cancer treatment, characterized in that, The isolithocarmine or methylolithocarmine can effectively reverse the toxicity of chemotherapy drugs on human immortalized epidermal cells.

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