阅读说明：本技术 一种治疗淋巴水肿的小分子化合物及其用途 (Small molecule compound for treating lymphedema and application thereof ) 是由 梁倩倩 王拥军 徐浩 侯彤 刘洋 王晓赟 杨燕萍 于 2019-08-27 设计创作，主要内容包括：本发明涉及一种治疗淋巴水肿的小分子化合物,其有效成分为三七皂苷R1(Notoginsenoside R1),作为治疗淋巴水肿的药物,三七皂苷R1的有效作用浓度为0.01-1000μM。三七皂苷R1与药物学上可接受的药物辅料混合形成散剂、膏剂、粉剂、针剂、水剂、肠溶缓释制剂或注射剂。本发明经动物实验证明,三七皂苷R1可降低C57小鼠的纤维组织厚度,改善淋巴水肿小鼠的淋巴循环。本发明还公开了三七皂苷R1在制备治疗淋巴水肿的药物或保健品中的用途。(The invention relates to a micromolecule compound for treating lymphedema, the active ingredient of the micromolecule compound is Notoginsenoside R1 (Notogenoside R1), and the effective action concentration of the Notoginsenoside R1 is 0.01-1000 mu M as a medicament for treating the lymphedema. The notoginsenoside R1 can be mixed with pharmaceutically acceptable pharmaceutical adjuvants to form powder, unguent, powder, injection, aqua, enteric sustained release preparation or injection. Animal experiments prove that the notoginsenoside R1 can reduce the thickness of fibrous tissues of a C57 mouse and improve the lymphatic circulation of a lymphedema mouse. The invention also discloses application of the notoginsenoside R1 in preparing a medicament or a health-care product for treating lymphedema.)

1. A small molecular compound for treating lymphedema is characterized in that the small molecular compound is notoginsenoside R1, and the effective action concentration is 0.01-1000 mu M.

2. The small molecule compound for treating lymphedema according to claim 1, wherein the notoginsenoside R1 is mixed with pharmaceutically acceptable pharmaceutical excipients to form powder, ointment, powder, injection, aqueous solution, enteric sustained release preparation or injection.

3. Use of the small molecule compound for treating lymphedema in claim 1 or 2 in preparing a medicament or health product for preventing and treating lymphedema.

4. The use as claimed in claim 3, wherein the effective concentration of notoginsenoside R1 is 0.01-1000 μ M.

Technical Field

The invention relates to a small molecular compound for treating lymphedema, and provides application of the compound in preparing various forms of medicines or health-care foods for preventing and treating lymphedema.

Background

Lymphedema (lymphedema) subcutaneous fibrous connective tissue hyperplasia and fat sclerosis are caused by repeated infection of the body surface of soft tissue fluid caused by the obstruction of lymphatic return in certain parts of the body. The treatment is very difficult, and a safe and effective therapeutic drug is still lacking up to now. Lymphedema is treated according to different principles according to the morning and evening of the disease. The most important is early treatment to prevent the development of lesions. Satisfactory results cannot be obtained in the treatment of advanced cases, and the primary lymphedema is mainly treated symptomatically and can be locally bandaged by an elastic bandage. The search for safe and effective therapeutic drugs becomes a major problem to be solved urgently in the research of lymphedema.

The effective component notoginsenoside R1 researched by the invention can stimulate lymphatic endothelial cells to generate VEGFC to promote lymphatic generation and improve lymphatic return, thereby relieving lymphedema.

Disclosure of Invention

In view of the above-mentioned deficiencies of the prior art, according to the embodiments of the present invention, it is desirable to provide a small molecule compound for treating lymphedema with a definite therapeutic effect, and to propose a medical use thereof.

According to the embodiment, the effective component of the small molecular compound for treating lymphedema provided by the technical scheme of the invention is notoginsenoside R1, and the concentration is 0.01-1000 mu M. Notoginsenoside R1 is effective component extracted from dried root and rhizome of Notoginseng radix, and can reduce fibroadipose thickness of tail tissue of mouse with lymphedema model and improve lymphatic return function.

According to one embodiment, the technical scheme of the invention is based on that the notoginsenoside R1 is used as a medicament for treating lymphedema, and the effective action concentration of the notoginsenoside R1 is 0.01-1000 mu M.

According to one embodiment, in the technical scheme of the invention, the notoginsenoside R1 is mixed with pharmaceutically acceptable pharmaceutical excipients to form powder, paste, powder, injection, aqua or injection.

According to one embodiment, notoginsenoside R1 as a therapeutic agent for lymphedema, which is a pharmaceutical or health food in various forms prepared as a main active ingredient, can be used for preventing and treating the aforementioned lymphedema. When in use, subcutaneous, intravenous injection or anorectal administration can be adopted; the injection can be selected from normal saline, glucose, stabilizer, antiseptic, suspending agent or emulsifier.

The invention carries out animal experiments, wherein 30 clean C57 female mice with the age of about 7-8 weeks are randomly divided into a blank control group, a model group and a notoginsenoside R1 group (the latter is the medicine of the invention and is also called as a treatment group) according to the body weight, the notoginsenoside R1 group is intragastrically infused with the notoginsenoside R1 every day, the WT group and the model group are intragastrically infused with the same amount of normal saline every day, and the analysis result of the animal experiments shows that the notoginsenoside R1 can reduce the swelling degree of the tail of the mouse and improve the lymphatic return. In addition, the invention also carries out cell experiments, culturing lymphatic endothelial cells, adding notoginsenoside R1 with different concentrations into treatment groups, collecting cells after 24 hours, carrying out Q-PCR and western blot detection experiments, and detecting the expression change of VEGFC mRNA and protein of the lymphatic endothelial cells. In vitro cytological experimental study shows that the notoginsenoside R1 increases VEGFC mRNA expression, increases protein expression, promotes lymphatic vessel generation and improves lymphatic return function. Therefore, various forms of medicines or health foods prepared by using notoginsenoside R1 as main active ingredients can be used for preventing and treating the lymphedema diseases.

Drawings

FIG. 1 shows the structural formula of notoginsenoside R1.

FIG. 2 is a graph showing the measurement results of the tail circumference of a mouse.

FIG. 3 is a graph showing the results of detecting the signal intensity of lymphatic vessels.

Fig. 4 is a graph showing the results of detection of VEGFC protein expression by lymphatic endothelial cells.

Detailed Description

The invention is further illustrated with reference to the following figures and specific examples. These examples are to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever. After reading the description of the invention, one skilled in the art can make various changes and modifications to the invention, and such equivalent changes and modifications also fall into the scope of the invention defined by the claims.

In the following examples of the present invention, notoginsenoside R1 (shown in figure 1 in simplified structure) was extracted from dried root and rhizome of notoginseng by commercially available methods or by conventional extraction methods in the field of traditional Chinese medicine. M is a molar concentration, namely mol/L; μ M is micromoles per liter.

Examples of the experiments

30 cleaning grade C57 female mice with the weight of about 7-8 weeks are randomly divided into a model group and a notoginsenoside R1 group according to the weight, and 10 wild type mice (WT) with the weight similar to that of a littermate Control are taken as a Control group. The notoginsenoside R1 group is administered by intragastric administration with notoginsenoside R120 mg/kg/d every day, and the Control group and model group are administered by intragastric administration with equal amount of physiological saline every day for 4 weeks. The swelling degree of the tail molding area of the mouse was measured every three days. As shown in figure 2, notoginsenoside R1 can reduce the swelling degree of mouse tail and improve lymphatic return.

Detection of reflux function of lymphatic vessel by notoginsenoside R1

After 4 weeks of gastric perfusion, the mice are placed in an anesthesia machine, after isoflurane anesthesia, hair is removed from the tail and the buttocks, a 30-gauge needle is used for injecting 10 mu L ICG (0.1 mu g/mL, distilled water is dissolved, and the mixture is preserved in a dark place) dye into the tail tip of the mouse, a strong light signal can be observed under near-infrared irradiation, the tail tip gradually enters ischial lymph nodes through lymphatic vessels on the two sides of the tail, 1 hour is continuously observed and photographed every 1s, after 1 hour of injection, the signal intensity of the lymphatic vessels tends to be stable, 600 seconds of continuous observation are carried out, one Pulse is recorded at each adjacent signal peak and trough according to the number of times of fluctuation of the signal intensity of the lymphatic vessels, and the Pulse number (Pulse) within 500sec can be. The Signal intensity of the ICG in the picture (Signal, S) was obtained using ImageJ software. As shown in figure 3, the pulse number of the notoginsenoside R1 group in the lymphatic vessel unit time is obviously increased, and the notoginsenoside R1 obviously improves the clearance rate of the lymphatic vessel.

And (3) detecting the VEGF-C protein expression of lymphatic endothelial cells by using the notoginsenoside R1.

Protein extraction: LEC treatment for 24h to remove supernatant, precooling sterile PBS washing X3 → lysate 400 μ L (1% PMSF, 1% phosphatase inhibitor), standing on ice for 10min → cell scraping protein → 14000r centrifugation for 15min, extracting supernatant at 4 ℃ → BCA method for measuring protein concentration, 95 μ LA + B (50: 1), adding 5 μ L supernatant, shaking table at 37 ℃ for 30min → microplate reader (wavelength 562nm) for measuring concentration → protein quantification, adding 5X sample Buffer, and metal bath denaturation at 100 ℃ for 15 min.

Electrophoresis: preparing separation gel, concentrating gel → adding 500mL of electrophoretic solution → loading 25 μ L (15-hole comb) → upper gel 80V for 30min, and when the sample reaches the junction of the concentrating gel and the separation gel, turning to 100V until the sample finishes electrophoretic film transfer: soaking PVDF membrane in methanol for 5min, placing the gel and membrane in the membrane transferring solution, and cooling for 90 min and 70min while adding ice.

Incubation and color development: blocking with 5% BSA for 1h → shaking table incubation for one time overnight for one time, washing membrane for 5min X3 → incubation for two antibodies for 1h at room temperature → washing membrane for 5minX3 → color development.

As shown in fig. 4, the expression level of VEGFC protein in lymphatic endothelial cells increased with the increase in the concentration of notoginsenoside R1, showing concentration dependence, compared to the control group. The notoginsenoside R1 is suggested to increase VEGFC protein expression and promote generation of lymphatic vessels, thereby promoting lymphatic vessel reflux function and improving lymphedema degree.