Application of alpha hederasaponin as sole active ingredient in preparation of medicine for treating and / or preventing myelosuppression

Abstract

The invention discloses an application of alpha hederasaponin as a unique active ingredient in preparation of a medicine for treating and / or preventing myelosuppression. The alpha hederasaponin has an improvement effect on mouse myelosuppression caused by cyclophosphamide, can increase the level of mouse peripheral blood leucocyte, inhibit bone marrow cell apoptosis, inhibit oxidative stress injury and inflammation and play a bone marrow protection role, and a scientific theoretical basis is provided for treatment of myelosuppression by the alpha hederasaponin.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology. More specifically, the present invention relates to the use of alphahederin as the sole active ingredient in the preparation of a medicament for treating and / or preventing myelosuppression. Background Art

[0002] Radiotherapy / chemotherapy, as an important means of tumor treatment, is widely used clinically. However, due to the non-selective destruction of normal tissue cells and tumor cells by radiation and chemotherapy drugs, radiotherapy / chemotherapy will cause a series of toxic and side effects in the body. Among them, myelosuppression is relatively common and is often the most common reason for the passive reduction or discontinuation of radiotherapy / chemotherapy. 80% of patients will experience myelosuppression during tumor radiotherapy / chemotherapy, resulting in leukopenia, and severe infections and bleeding often occur clinically. Therefore, during radiotherapy and chemotherapy, protecting the bone marrow hematopoietic function and increasing the number of peripheral white blood cells are of great significance for the treatment of tumors.

[0003] Pulsatilla root is the dried root of Pulsatilla chinensis (Bunge) Regel, a plant of the genus Pulsatilla in the family Ranunculaceae, and is a commonly used traditional Chinese medicine. Alphahederin is one of the active ingredients of Pulsatilla root, but the preventive and therapeutic effects of alphahederin on myelosuppression are not yet clear. Summary of the Invention

[0004] The present invention provides a use of alphahederin as the sole active ingredient in the preparation of a medicament for treating and / or preventing myelosuppression. Alphahederin has an improving effect on cyclophosphamide-induced myelosuppression in mice, can increase the level of peripheral blood white blood cells in mice, inhibit apoptosis of bone marrow cells, inhibit oxidative stress damage and inflammation, and play a role in protecting the bone marrow, providing a scientific theoretical basis for the treatment of myelosuppression with alphahederin.

[0005] To achieve these and other advantages in accordance with the present invention, there is provided a use of alphahederin as the sole active ingredient in the preparation of a medicament for treating and / or preventing myelosuppression.

[0006] Preferably, the myelosuppression is caused by tumor radiotherapy / chemotherapy.

[0007] Preferably, the medicament contains a therapeutically effective amount of alphahederin and a pharmaceutically acceptable carrier.

[0008] Preferably, the medicament contains a therapeutically effective amount of the hydrochloride, perchlorate, mesylate, phosphate, citrate or sulfate of alphahederin and a pharmaceutically acceptable carrier.

[0009] Preferably, pharmaceutically acceptable carriers include diluents, solubilizers, cosolvents, disintegrants, dispersants, lubricants, flavoring agents, antioxidants, binders, absorbents, wetting agents, buffers, and cross-linking agents.

[0010] Preferably, the drug is formulated into a pharmaceutically acceptable dosage form.

[0011] Preferably, the dosage forms include pills, tablets, powders, capsules, granules, powders, dripping pills, drops, sprays, injections, suspensions, ointments, gels, and suppositories.

[0012] Preferably, the drug upregulates the levels of peripheral blood cells.

[0013] Preferably, the drug upregulates the levels of hematopoietic factors.

[0014] Preferably, the dosage of alpha-hederin is not less than 0.5 mg / kg•d.

[0015] The present invention has at least the following beneficial effects: Alpha-hederin can significantly increase the decreased white blood cells (WBC) and red blood cells (RBC) caused by CTX, improve the decreased thymus index and increased spleen index in mice caused by CTX, increase the levels of hematopoietic-related factors such as IL-1β, IL-6, TNF-α, and GM-CSF in bone marrow tissue, inhibit the levels of ROS and MDA in bone marrow tissue, and increase the SOD level; in addition, alpha-hederin can inhibit the expression of endoplasmic reticulum stress signaling pathway-related proteins XBP1, ATF4, Bip, IRE1α, and p53, inhibit the expression of apoptosis-related proteins Bcl-2, cleaved caspase-9, cleaved caspase-3, and γ-H2AX, and increase the expression of Bax protein, thereby exerting a protective effect on cyclophosphamide-induced bone marrow suppression in mice. This means that the use of alpha-hederin as an adjuvant treatment can enable patients to better maintain bone marrow hematopoietic function during radiotherapy and chemotherapy, reduce the fluctuation of peripheral blood cell counts, reduce the risk of infection and bleeding, enable radiotherapy and chemotherapy to proceed smoothly, improve the treatment compliance and quality of life of cancer patients, open up a new path for cancer treatment, and provide strong evidence for the application of traditional Chinese medicine in modern medicine, and is expected to promote the further development of the integrated traditional Chinese and Western medicine treatment of cancer.

[0016] Other advantages, objectives, and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. Description of the Drawings

[0017] Figure 1Effect of alpha hederin of the present invention on the blood routine of mice; Figure 2 Effect of alpha hederin of the present invention on the body weight and organ index of mice; Figure 3 Effect of alpha hederin of the present invention on hematopoietic cytokines in the bone marrow tissue of mice; Figure 4 Effect of alpha hederin of the present invention on the ROS level in the bone marrow tissue of mice; Figure 5 Effect of alpha hederin of the present invention on the SOD and MDA levels in the bone marrow tissue of mice; Figure 6 Effect of alpha hederin of the present invention on protein expression in the bone marrow tissue of mice; Figure 7 Effect of the main saponin components of Pulsatilla chinensis on CTX-induced bone marrow suppression in mice. Detailed implementation mode

[0018] The present invention will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can implement it according to the description in the specification.

[0019] It should be understood that the terms such as "having", "comprising" and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0020] It should be noted that the experimental methods described in the following implementation schemes are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified. Therefore, it should not be construed as a limitation to the present invention.

[0021] Use of alpha hederin as the sole active ingredient in the preparation of a drug for treating and / or preventing bone marrow suppression.

[0022] Preferably, the drug contains a therapeutically effective amount of alpha hederin and a pharmaceutically acceptable carrier. The drug can also be a hydrochloride, perchlorate, mesylate, phosphate, citrate or sulfate of a therapeutically effective amount of alpha hederin and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include diluents, solubilizers, cosolvents, disintegrants, dispersants, lubricants, flavoring agents, antioxidants, binders, absorbents, wetting agents, buffers, crosslinking agents.

[0023] The drug is made into a pharmaceutically acceptable dosage form, including but not limited to pills, tablets, powders, capsules, granules, powders, dripping pills, drops, sprays, injections, suspensions, ointments, gels, suppositories.

[0024] 1 Materials and Methods 1.1 Experimental Animals SPF-grade male C57BL / 6J mice, 6 - 8 weeks old, weighing 19 - 22 g, 48 in number, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. with the license number: SCXK(Beijing)2021 - 0006, and were raised in the Experimental Animal Center of Guangxi University of Chinese Medicine. The breeding environment was at room temperature of 24°C and humidity of 40% - 60%.

[0025] 1.2 Animal Grouping After 3 days of adaptive feeding of the mice, they were randomly divided into a blank control group (CON), a model control group (MOL), a low-dose alpha-hederin group (L), a medium-dose alpha-hederin group (M), a high-dose alpha-hederin group (H), and a positive drug group rhG-CSF (P), with 8 mice in each group.

[0026] 1.3 Model Establishment Except for the blank control group, on the 1st, 2nd, and 3rd days of the experiment, the other model establishment groups were intraperitoneally injected (i.P.) with cyclophosphamide CTX (80 mg / kg), and the effect of model establishment was evaluated by detecting blood routine on the 3rd day. When the WBC level of the model mice dropped below 30% of that of the blank control group mice, the model establishment was considered successful.

[0027] 1.4 Drug Administration On the 3rd day of the experiment, the alpha-hederin administration groups were intraperitoneally injected with alpha-hederin (1 mg / kg), alpha-hederin (3 mg / kg), and alpha-hederin (5 mg / kg) respectively, the model control group was given an equal volume of normal saline, and the positive drug group was subcutaneously injected with rhG-CSF (40 μg / kg), once a day for 7 consecutive days.

[0028] 1.5 Measurement Indexes After the drug administration ended on the 9th day, the mice were fasted for 12 hours and samples were taken on the 10th day. (1) After the experiment, the mice in each group had their eyes enucleated to collect blood, and blood routine indexes such as peripheral blood WBC and RBC were detected by blood routine; (2) The thymus and spleen of the mice were taken and weighed respectively to calculate the organ index; (3) The femurs and tibias of the mice were ground with lysis buffer and normal saline, and the levels of hematopoiesis-related factors in the bone marrow tissue of the mice were detected by ELISA, and the expression levels of proteins related to the endoplasmic reticulum stress and apoptosis signaling pathways in the bone marrow tissue were detected by western blotting; (4) One mouse from each group was left to take bone marrow cells for flow cytometry to detect the ROS level.

[0029] 1.6 Statistical Analysis Data analysis was performed using GraphPad Prism 7.0 software. One-way ANOVA was used for comparison among multiple groups, and t-test was used for comparison between two groups. A P value < 0.05 was considered statistically significant.

[0030] 2 Experimental results 2.1 Analysis of the general status of mice Except for the blank control group, after modeling, mice in each modeling group showed varying degrees of decreased appetite, weight loss, and reduced strength. The alpha-hederin administration groups and the positive drug group could improve the conditions of decreased appetite and weight loss in mice.

[0031] 2.2 Effects of alpha-hederin on the blood routine of mice; The blood routine results are as Figure 1 shown (A: WBC level; B: RBC level; C: PLT level, ##P < 0.01 vs control; *P < 0.05 vs model; ***P < 0.001 vs model). CTX could significantly inhibit the levels of WBC and RBC, and there was no obvious change in the platelet (PLT) level. However, the medium-dose group, high-dose group of alpha-hederin, and the positive drug group could significantly increase the levels of WBC and RBC ( Figure 2 A, 2B), but had no obvious effect on the number of platelets ( Figure 2 C). The results showed that alpha-hederin had the effect of increasing white blood cells.

[0032] 2.3 Effects of alpha-hederin on the body weight and organ indices of mice The results of body weight and organ indices are as Figure 2 shown (A: Changes in mouse body weight; B: Mouse thymus index; C: Mouse spleen index, P < 0.001 vs control; **P < 0.01 vs model; ***P < 0.001 vs model). After CTX modeling, the body weight of mice decreased significantly, while the medium-dose group, high-dose group of alpha-hederin, and the positive drug group could significantly improve the body weight reduction caused by CTX ( Figure 3 A). CTX could significantly decrease the thymus index, while the medium-dose group, high-dose group of alpha-hederin, and the positive drug group could significantly increase the thymus index of mice ( Figure 3 B). CTX could increase the spleen index of mice, while the medium-dose group, high-dose group of alpha-hederin, and the positive drug group could significantly inhibit the spleen index of mice ( Figure 3C). The results showed that alphahederin could improve the immune function of mice, enhance the physical fitness of mice, and at the same time, significantly improve the increase in spleen index caused by CTX-induced compensatory hematopoiesis of the spleen and alleviate the bone marrow suppression symptoms of mice.

[0033] 2.4 Effect of alphahederin on hematopoietic cytokines in bone marrow tissue The ELISA results were as Figure 3 shown (A: IL-1β level in mouse bone marrow tissue; B: IL-6 level in mouse bone marrow tissue; C: TNF-α level in mouse bone marrow tissue; D: GM-CSF level in mouse bone marrow tissue, P < 0.001 vs control; *P < 0.05 vs model; **P < 0.01 vs model; ***P < 0.001 vs model). After CTX modeling, the levels of IL-1β, IL-6, TNF-α and GM-CSF in mouse bone marrow tissue were significantly decreased, while each alphahederin administration group and the positive drug group could significantly increase the expression of hematopoietic-related cytokines such as IL-1β, IL-6, TNF-α and GM-CSF in bone marrow tissue ( Figure 3 A - D), thereby improving the bone marrow hematopoietic microenvironment, promoting the proliferation, differentiation and chemotaxis of blood cells, and alleviating the bone marrow suppression symptoms caused by CTX.

[0034] 2.5 Effect of alphahederin on ROS level in bone marrow tissue The flow cytometry results were as Figure 4 shown (A: Flow cytometry detection of ROS level in mouse bone marrow tissue; B: Statistical chart of flow cytometry ROS fluorescence value, P < 0.001 vs control; ***P < 0.001 vs model). CTX could significantly increase the ROS level in bone marrow tissue, and each alphahederin administration group could significantly reduce the increase in ROS in bone marrow tissue caused by CTX ( Figure 5 A, 5B). Alphahederin has antioxidant effects.

[0035] 2.6 Effect of alphahederin on SOD and MDA levels in bone marrow tissue The ELISA results were as Figure 5 shown (A: Flow cytometry detection of SOD level in mouse bone marrow tissue; B: Flow cytometry detection of MDA level in mouse bone marrow tissue, P < 0.001 vs control; **P < 0.01 vs model; ***P < 0.001 vs model). CTX could significantly reduce the SOD level in bone marrow tissue and increase the MDA level in bone marrow tissue, and each alphahederin administration group could significantly increase the SOD level (Figure 5 A), reducing the MDA level ( Figure 5 B). The above results further indicate that alpha-hederin exerts a protective effect on CTX-induced myelosuppression by inhibiting the oxidative stress signaling pathway.

[0036] 2.7 Effect of alpha-hederin on protein expression in bone marrow tissue The results of Western blotting are as Figure 6 shown (A: Detection of the expression levels of apoptosis signaling pathway proteins in mouse bone marrow tissue by Western blotting; B: Detection of the expression levels of endoplasmic reticulum stress signaling pathway proteins in mouse bone marrow tissue by Western blotting). CTX can inhibit the expression of Bax protein and increase Bcl-2, cleaved caspase-9, cleaved caspase-3, and γH2AX, while each alpha-hederin administration group can reverse the expression of apoptosis signaling pathway proteins ( Figure 6 A). In addition, CTX can increase the expression of XBP1, ATF4, Bip, IRE1α, and P53 proteins, while each alpha-hederin administration group can inhibit the expression of these endoplasmic reticulum stress signaling pathway-related proteins ( Figure 6 B). The above results suggest that alpha-hederin may exert a protective effect on CTX-induced inhibition of bone marrow cells by regulating the endoplasmic reticulum stress signaling pathway and the apoptosis signaling pathway.

[0037] 3 Effects of the main saponin components of Pulsatilla chinensis on CTX-induced myelosuppression in mice The modeling and drug administration were repeated using the method in 1, the indexes were measured, and the key saponin components in Pulsatilla chinensis were compared. The results of blood routine are as Figure 7 shown (A: WBC level; B: Thymus index; C: RBC level, ##P < 0.01 vs control, P < 0.001 vs control; *P < 0.05 vs model; **P < 0.01 vs model; ***P < 0.001 vs model). CTX can significantly inhibit the levels of WBC and RBC and significantly reduce the thymus index. Alpha-hederin, positive control drug (P), pulchinenoside B4 (B4), pulchinenoside (B5), and sanguiin H6 (DY2) can significantly increase the RBC level ( Figure 7 C), but except for alpha-hederin, other saponins have no obvious effect on increasing WBC ( Figure 7 A), and except for alpha-hederin, other saponins have no obvious effect on increasing the thymus index ( Figure 7B). The results showed that compared with other saponins, alphahederin had a significant effect on increasing white blood cells, and at the same time could increase the thymus index, thus playing an immunomodulatory role, which other saponins did not have.

[0038] From a chemical structure perspective, saponin compounds are usually composed of steroidal or triterpenoid aglycones and sugar chains, and their biological activities are often affected by the number, type, and connection mode of sugar chains. Alphahederin belongs to triterpenoid saponins, has a unique parent nucleus structure, and shows a special conformation at the glycosylation site, making it have significant effects in immunomodulation and anti-apoptosis. Taking ginsenosides as an example, compared with alphahederin, ginsenosides are mainly derived from plants of the genus Panax in the Araliaceae family (such as Panax ginseng), and their typical structure is centered on dammarane-type or oleanane-type and contains various different sugar group modifications. These structural characteristics determine that the mechanism of action of ginsenosides in immunomodulation, anti-fatigue, anti-inflammatory, etc. is different from that of alphahederin. Specifically, ginsenosides affect the function of immune cells by regulating signal pathways such as PI3K / Akt, NF-κB, and MAPK, while alphahederin is more inclined to directly improve the bone marrow suppression state by inhibiting the expression of apoptosis-related proteins (such as Caspase-3, Bax / Bcl-2). In summary, although alphahederin and ginsenosides both belong to saponin compounds, due to the differences in the core parent nucleus structure and sugar chain modification, there are significant differences in their mechanism of action and pharmacological activities. Therefore, in bone marrow transplantation or bone marrow protection research, it is not possible to simply replace the function of alphahederin through the modification of saponin structure, but it is necessary to explore its potential drug application value based on in-depth research on molecular mechanisms.

[0039] 4 Conclusions Compared with other saponins, alphahederin can significantly increase the decrease in white blood cells (WBC) and red blood cells (RBC) caused by CTX, improve the decrease in thymus index and the increase in spleen index in mice caused by CTX, increase the levels of hematopoiesis-related factors such as IL-1β, IL-6, TNF-α, and GM-CSF in bone marrow tissue, thereby improving the bone marrow hematopoietic microenvironment, promoting the proliferation, differentiation, and chemotaxis of blood cells, and alleviating the bone marrow suppression symptoms caused by CTX.

[0040] Alpha hederin exerts an anti - myelosuppressive effect by inhibiting the levels of ROS and MDA in bone marrow tissue, increasing the level of SOD, and regulating the level of oxidative stress. In addition, alpha hederin can inhibit the expression of endoplasmic reticulum stress signaling pathway - related proteins XBP1, ATF4, Bip, IRE1α, and p53, inhibit the expression of apoptosis - related proteins Bcl - 2, cleaved caspase - 9, cleaved caspase - 3, γ - H2AX, and increase the expression of Bax protein, thereby exerting a protective effect on cyclophosphamide - induced myelosuppression in mice.

[0041] The number of devices and the processing scale described here are used to simplify the description of the present invention. Applications, modifications, and variations of the present invention will be obvious to those skilled in the art.

[0042] Although the embodiments of the present invention have been disclosed as above, it is not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the illustrated and described examples here.

Claims

1. Use of alpha-hederin as the only active ingredient in the preparation of a medicament for treating and / or preventing myelosuppression.

2. The use according to claim 1, characterized in that, Myelosuppression is caused by tumor radiotherapy / chemotherapy.

3. The use according to claim 1, characterized in that, The medicament contains a therapeutically effective amount of alpha-hederin and a pharmaceutically acceptable carrier.

4. The use according to claim 3, characterized in that, The medicament contains a therapeutically effective amount of the hydrochloride, perchlorate, mesylate, phosphate, citrate or sulfate of alpha-hederin and a pharmaceutically acceptable carrier.

5. The use according to claim 3 or 4, characterized in that, Pharmaceutically acceptable carriers include diluents, solubilizers, cosolvents, disintegrants, dispersants, lubricants, flavoring agents, antioxidants, binders, absorbents, wetting agents, buffers, cross-linking agents.

6. The use according to claim 5, wherein, The medicament is made into a pharmaceutically acceptable dosage form.

7. The use according to claim 6, wherein The dosage forms include pills, tablets, powders, capsules, granules, powders, dripping pills, drops, sprays, injections, suspensions, ointments, gels, suppositories.

8. The use according to claim 1, characterized in that, The medicament up-regulates the level of peripheral blood cells.

9. The use according to claim 1, characterized in that, The medicament up-regulates the level of hematopoietic factors.

10. The use according to claim 1, wherein, The administration dose of alpha-hederin is not less than 0.5 mg / kg•d.

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