CN116036106A

CN116036106A - Application of medicine for treating lymphedema

Info

Publication number: CN116036106A
Application number: CN202111265773.9A
Authority: CN
Inventors: 梁倩倩; 王拥军; 徐浩; 施杞; 鲍嘉敏; 李宁; 刘洋; 张洪
Original assignee: Longhua Hospital Affiliated to Shanghai University of TCM
Current assignee: Longhua Hospital Affiliated to Shanghai University of TCM
Priority date: 2021-10-28
Filing date: 2021-10-28
Publication date: 2023-05-02

Abstract

The invention provides application of ginsenoside Re, or pharmaceutically acceptable salt or extract thereof in preparing a composition for treating lymphedema, which is particularly suitable for subjects with TNF-alpha content lower than 1.5ng/mL. The invention discovers that ginsenoside Re can stimulate the expression of VEGF-C mRNA, increase the protein expression quantity and promote the lymphatic endothelial cell migration function. Therefore, various forms of medicines or health care products prepared by taking the ginsenoside Re as the main active ingredient are very suitable for treating lymphedema.

Description

Application of medicine for treating lymphedema

Technical Field

The invention relates to the field of medicines, in particular to application of ginsenoside Re in preparing a pharmaceutical composition or a health-care product composition for preventing and/or treating lymphedema.

Background

Lymphedema is a chronic, progressive and incurable disease caused by inadequate blood supply to the lymphatic system and abnormal accumulation of interstitial fluid. After repeated infection of soft tissue fluid on the body surface caused by blocked lymphatic fluid backflow of a patient, subcutaneous fibrous connective tissue hyperplasia and fat hardening can be caused, and the life quality of the patient is reduced. It is counted that 2 hundred million people worldwide suffer from lymphedema. Among them, acquired lymphedema is the most common consequence of trauma, obstruction, infection, radiation injury or surgical intervention of the lymphatic system. Lymphedema of the extremities can cause swelling, discomfort and impaired function, often with recurrent infections and ulcers of the skin, with devastating and disabling properties. Lymphedema patients often feel social shame and spelt due to dyskinesia and deformity of the limbs. Currently, the treatment of lymphedema involves only conservative physiotherapy and surgery methods, lacking effective drug treatment.

The lymphatic endothelial cells are a single-layer flat epithelial structure which forms the lymphatic vessel wall and is connected with each other in a discontinuous button shape, and almost no tight connection exists between the cells, and can be involved in physiological processes such as regulating the recycling of the lymphocytes, maintaining the homeostasis, regulating the immune response of an organism and the like. Lymphedema, inflammation, infectious and immunological diseases, fibrosis and tumor metastasis have all been associated with abnormalities in lymphatic endothelial cells. Therefore, the normal structure and function of the lymphatic endothelial cells are maintained, and the lymphatic function is maintained and the lymphatic circulation is protected. VEGF-C gene therapy has good application prospect on lymphedema caused by lymphangiogenesis deficiency or lymphangiogenesis obstruction.

Therefore, searching for safe and effective therapeutic drugs and realizing accurate administration becomes a major problem to be solved in the research of treating lymphedema.

Disclosure of Invention

The invention aims to provide an application of a small molecular compound for promoting lymphangiogenesis, which has definite curative effect and few side effects, in preparing a composition for preventing and/or treating lymphedema.

In a first aspect of the present invention, there is provided the use of ginsenoside Re, or a pharmaceutically acceptable salt or extract thereof, for the preparation of a composition for the prevention and/or treatment of lymphedema;

in another preferred embodiment, the lymphedema is suitable for subjects with a low level of TNF- α in the body, wherein a low level of TNF- α means a TNF- α level of less than 1.5ng/mL.

In another preferred embodiment, low levels of TNF- α means levels of TNF- α below 1.5ng/mL in vivo, e.g., 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 0.8ng/mL, 1ng/mL, 1.2ng/mL, 1.5ng/mL.

In another preferred embodiment, the low TNF- α content is less than 10pg/mL, such as 0.1pg/mL, 0.2pg/mL, 0.3pg/mL, 0.5pg/mL, 0.6pg/mL, 0.8pg/mL, 1pg/mL, 2pg/mL, 5pg/mL, 8pg/mL, 10pg/mL.

In another preferred embodiment, the composition does not include a cell growth factor or an inflammatory factor, such as TNF- α.

In another preferred embodiment, the lymphedema is selected from the group consisting of: primary lymphedema and secondary lymphedema.

In another preferred embodiment, the secondary lymphedema is selected from the group consisting of: infectious lymphedema, damaging lymphedema, malignant lymphedema, or a combination thereof.

In another preferred embodiment, the infectious lymphedema is selected from the group consisting of: lymphedema caused by parasitic infection, lymphedema caused by fungal infection, and lymphedema caused by bacterial infection.

In another preferred embodiment, the damaged lymphedema is selected from the group consisting of: lymphedema caused by surgery, lymphedema caused by radiotherapy, lymphedema caused by burns.

In another preferred embodiment, the malignant lymphedema comprises lymphedema caused by tumor compression.

In another preferred embodiment, the secondary lymphedema also includes lymphedema caused by systemic diseases and lymphedema caused by pregnancy.

In another preferred embodiment, the lymphedema is treatable by VEGF-C therapy.

In another preferred embodiment, the prevention and/or treatment of lymphedema comprises one or more features selected from the group consisting of:

(i) Promoting VEGF-C expression of lymphatic endothelial cells;

(ii) Stimulating secretion of VEGF-C by lymphatic endothelial cells;

(iii) Stimulating lymphangiogenesis;

(iv) Promoting lymphatic endothelial cell migration;

(v) Preventing the regeneration of lymphatic effusion;

(vi) Improving lymphatic return function.

In another preferred embodiment, the expression comprises protein and/or mRNA expression.

In another preferred embodiment, the composition contains ginsenoside Re, or a pharmaceutically acceptable salt thereof, or an extract thereof, at a concentration of 1. Mu.M or more, preferably 5. Mu.M or more, more preferably 10. Mu.M or more.

In another preferred embodiment, the composition contains 0.1 to 99wt%, preferably 0.5 to 95wt%, more preferably 1 to 90wt% of ginsenoside Re, or a pharmaceutically acceptable salt thereof, or an extract thereof.

In another preferred embodiment, the composition further comprises pharmaceutically or nutraceutically acceptable excipients and/or carriers.

In another preferred embodiment, the dosage form of the composition is selected from the group consisting of: solid, liquid or semi-solid formulations.

In another preferred embodiment, the dosage form of the composition is selected from the group consisting of: gel, patch, tablet, capsule, powder, ointment, powder, injection, aqua, enteric sustained-release preparation or injection.

In another preferred embodiment, the composition is in the form of an injection.

In another preferred embodiment, the composition is administered to a subject by: oral, transdermal, intravenous, intramuscular or anorectal administration.

In another preferred embodiment, the extract is extracted from dried roots and rhizomes of notoginseng or notoginseng powder.

In another preferred embodiment, the content of ginsenoside Re in the extract is more than or equal to 7wt%, wherein the content C1 is calculated according to the dry weight of the extract.

In another preferred embodiment, the content of ginsenoside Re in the extract is C1-10 wt%, preferably 20-30 wt%, 40wt%, 50wt%, 60wt%, 70wt%, 80wt%, 90wt%, 95wt%, based on the dry weight of the extract, wherein the content of ginsenoside Re is C1.

In another preferred embodiment, the content C2 of ginsenoside Rg1 in the extract is less than or equal to 20wt%, preferably less than or equal to 10wt%, 8wt%, 5wt%, 3wt%, 2wt%, 1wt%, wherein the content C2 is calculated by dry weight of the extract.

In a second aspect of the invention, there is provided a pharmaceutical composition for use in the treatment of lymphedema, the pharmaceutical composition comprising:

(a) Ginsenoside Re, or pharmaceutically acceptable salt thereof, or extract thereof;

(b) A pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition does not include other cell growth factors or inflammatory factors, such as TNF- α.

In another preferred embodiment, the pharmaceutical composition comprises:

(c) Cell growth factors and inflammatory factors.

In another preferred embodiment, the cell growth factor is selected from the group consisting of: VEGF-C, VEGF-D.

In another preferred embodiment, the inflammatory factor is selected from the group consisting of: TNF-alpha, IL-1 beta, IL-6, TG F-beta.

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: solid, liquid or semi-solid formulations.

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: gel, patch, tablet, capsule, powder, ointment, powder, injection, aqua, enteric sustained-release preparation or injection.

In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: oral dosage forms, transdermal injection dosage forms, intravenous injection dosage forms, intramuscular injection dosage forms.

In another preferred example, the injection solution of the injection contains one or more of physiological saline, glucose, a stabilizer, a preservative, a suspending agent or an emulsifying agent.

In a third aspect of the present invention, there is provided a nutraceutical composition for treating lymphedema, the nutraceutical composition comprising:

(b) A carrier acceptable in health care.

In a fourth aspect of the invention, there is provided a method of treating lymphedema comprising the steps of: administering to a subject in need thereof a safe and effective amount of ginsenoside Re, or a pharmaceutically acceptable salt thereof, or an extract thereof.

In another preferred embodiment, the subject is a subject having a low TNF- α content.

In another preferred embodiment, the TNF- α content is low as described in the first aspect of the invention.

In another preferred embodiment, the subject is a mammal, such as a human, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, and the like.

In another preferred embodiment, the effective application concentration of ginsenoside Re is 1-100. Mu.M, more preferably 1-50. Mu.M.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Fig. 1 shows a structural formula of ginsenoside Re.

FIG. 2 shows the effect of ginsenoside Re on VEGF-C protein expression.

FIG. 3 shows the effect of ginsenoside Re on VEGF-C mRNA expression.

FIG. 4 shows the effect of ginsenoside Re on the scoring of lymphatic endothelial cells.

FIG. 5 shows the effect of ginsenoside Rg1 on VEGF-C mRNA expression in the absence and presence of TNF-alpha stimulation.

FIG. 6 shows the effect of ginsenoside Rg1 on VEGF-C protein expression in the absence of TNF- α stimulation.

FIG. 7 shows the effect of ginsenoside Rg1 on VEGF-C protein expression in the presence of TNF- α stimulation.

FIG. 8 shows that ginsenoside Rg1 promotes tube formation (A) and migration (B) of lymphatic endothelial cells in the presence of TNF- α stimulation.

Detailed Description

Through extensive and intensive studies, the present inventors have provided an application of ginsenoside Re in treating lymphedema by a large number of screening and testing. The inventors of the present invention have unexpectedly found for the first time that, unlike ginsenoside Rg1, which has to exert an effect of promoting VEGF-C expression in lymphatic endothelial cells under TNF-alpha stimulation, ginsenoside Re can stimulate VEGF-C mRNA expression without TNF-alpha stimulation, increase its protein expression level, promote lymphatic endothelial cell migration and thus improve lymphedema, and has high safety, is not affected by TNF-alpha content, and is very suitable for preparing a composition for treating lymphedema in subjects with low TNF-alpha content. The present invention has been completed on the basis of this finding.

Terminology

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40 ℃, preferably 25±5 ℃.

As used herein, the term "plurality" or "plurality" refers to 2 or more, such as 2, 3, 4, 5 or 6, per species.

Active ingredient

The active ingredient of the invention is ginsenoside Re or pharmaceutically acceptable salt thereof.

According to one embodiment, the small molecular compound of the medicine for treating lymphedema provided by the technical scheme of the invention is ginsenoside Re, and the application concentration of the small molecular compound is 1-50 mu M.

Ginsenoside Re is one of the effective components extracted from dried root and rhizome of Notoginseng radix, and can promote lymphatic endothelial cells to generate VEGF-C, promote lymphatic vessel generation, and improve lymphedema.

In the present invention, the active ingredient may be in the form of a plant extract containing ginsenoside Re. The extract may be derived from dried root and rhizome of Panax notoginseng.

Preferably, the content of ginsenoside Re in the extract is C1-7wt% or more, 8-wt% or more, preferably 9-wt% or more, such as 10-20wt% or more, 30-wt%, 40-50wt%, 60-wt%, 70-wt%, 80-wt%, 85-wt%, 90-wt% or 95-wt%, based on the dry weight of the extract.

In the present invention, racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., separated enantiomers) of ginsenoside Re are all intended to be included within the scope of the present invention. When the active ingredients herein have a defined stereochemistry (denoted R or S, or indicated with dashed or wedge-shaped bonds), those compounds will be understood by those skilled in the art to be substantially free of other isomers (e.g., at least 80%,90%,95%,98%,99% and up to 100% free of other isomers).

The active ingredients of the present invention may contain acidic moieties, including but not limited to hydroxyl groups, which may form physiologically acceptable salts with various organic or inorganic bases. Typical base-forming salts include ammonium salts, alkali metal salts such as sodium, lithium, potassium salts, alkaline earth metal salts such as calcium, magnesium salts, and salts with organic bases (e.g., organic amines), such as benzathine, dicyclohexylamine, hydrabamine (salts with N, N-bis (dehydroabietyl) ethylenediamine), N-methyl-D-glucamine, N-methyl-D-glucamide, t-butylamine, and salts with amino acids such as arginine, lysine, and the like.

The active ingredients of the present invention can treat lymphedema in the absence of TNF-alpha stimulation.

As used herein, the term "pharmaceutically, hygienically acceptable" component refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.

As used herein, the term "effective amount" refers to an amount that is functional or active in and acceptable to a human and/or animal. It will be appreciated by those of ordinary skill in the art that the "effective amount" may vary depending on the form of the pharmaceutical composition, nutraceutical composition, the adjuvant used, the severity of the disease, and the combination with other drugs, nutraceuticals, or foods.

As used herein, "promoting", "accelerating" includes accelerating the rate of lymphedema regression, shortening the edema regression time, and does not necessarily require a 100% cure. In some embodiments, the active ingredients of the present invention increase the rate of edema resolution by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80% compared to the absence of the active ingredient of the present invention.

Lymphedema (lymphedema)

Lymphedema is a chronic, progressive and incurable disease caused by inadequate blood supply to the lymphatic system and abnormal accumulation of interstitial fluid. After repeated infection of soft tissue fluid on the body surface caused by blocked lymphatic fluid backflow of a patient, subcutaneous fibrous connective tissue hyperplasia and fat hardening can be caused, and the life quality of the patient is reduced.

In the present invention, there is no particular limitation on the kind of lymphedema, including lymphedema of various types or sites. For example, primary lymphedema, secondary lymphedema.

In the present invention, subjects suffering from lymphedema are mainly those with low levels of TNF-alpha.

In another preferred embodiment, low levels of TNF- α means levels of TNF- α below 0.5-1.5ng/mL, such as 0.5ng/mL, 0.8ng/mL, 1ng/mL, 1.2ng/mL, 1.5ng/mL.

In another preferred embodiment, the low TNF- α content means that the TNF- α content is less than 10pg/mL, such as 0.1pg/mL, 0.2pg/mL, 0.3pg/mL, 0.5pg/mL, 0.6pg/mL, 0.8pg/mL, 1pg/mL, 2pg/mL, 5pg/mL, 8pg/mL, 10pg/mL.

Use of the same

According to one embodiment, the invention provides a ginsenoside Re-based medicament for treating lymphedema, and the effective acting concentration is 1-50 mu M.

According to one embodiment, in the technical scheme of the invention, ginsenoside Re is mixed with pharmaceutically acceptable pharmaceutical auxiliary materials to form powder, paste, powder, injection, water or injection.

According to one embodiment, various forms of compositions prepared by taking ginsenoside Re as a main active ingredient of a lymphedema treatment drug can be used for preventing and/or treating lymphedema. Particularly lymphedema in patients with low levels of TNF-alpha, and no additional TNF-alpha is required in the composition.

Can be used by subcutaneous, intravenous injection or anorectal administration; the injection can be prepared from physiological saline, glucose, stabilizer, antiseptic, suspending agent or emulsifying agent.

In vitro researches are carried out, after ginsenoside Re (0, 2.5, 5, 10, 25 and 50 mu M) with different concentrations are respectively interfered with lymphatic endothelial cells for 24 hours, the cells are recovered to extract total protein and mRNA, RT-qPCR and Western blot detection are carried out, and the expression changes of related VEGF-C mRNA and protein are detected. Experimental tube formation experiments show that ginsenoside Re can stimulate VEGF-C mRNA expression, increase protein expression amount and promote lymphatic endothelial cell migration. Therefore, various forms of medicines or health care products prepared by taking ginsenoside Re as a main active ingredient can be used for treating lymphedema.

Compositions and methods of administration

The composition of the present invention comprises ginsenoside Re, or pharmaceutically acceptable salts thereof, as an active ingredient. The active ingredient of the invention may be in the form of a plant extract containing ginsenoside Re.

The active ingredient of the invention not only can promote the generation of VEGF-C by lymphatic endothelial cells, but also can promote the generation of lymphatic vessels, and is very suitable for preparing a composition for treating lymphedema.

The composition of the present invention comprises the ginsenoside Re or the pharmaceutically acceptable salt thereof of the present invention in a safe and effective amount range, and pharmaceutically or hygienically acceptable excipients or carriers.

Typically, the compositions of the present invention contain 1-2000mg of the active ingredient/agent of the present invention, more preferably 10-500mg of the active ingredient/agent of the present invention. Preferably, the "one dose" is a capsule or tablet.

"pharmaceutically or hygienically acceptable carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatibility" as used herein means that the components of the composition are capable of blending with and between the active ingredients of the present invention without significantly reducing the efficacy of the active ingredients. Examples of pharmaceutically or hygienically acceptable carrier moieties are cellulose and derivatives thereof (e.g. sodium carboxymethylcellulose, sodium ethylcellulose)Cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g.

) Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water and the like.

The mode of administration of the active ingredients or compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical (edema site).

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active ingredient is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, e.g., glycerin; (d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent, such as paraffin; (f) an absorption accelerator, e.g., a quaternary amine compound; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) an adsorbent, for example, kaolin; and (i) a lubricant, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active ingredient in such a composition may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active ingredient may also be in the form of microcapsules with one or more of the above excipients, if desired.

Liquid dosage forms for oral administration include pharmaceutically or nutraceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.

In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active ingredient, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-agar or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms of the active ingredients of the present invention for topical application include gels (e.g., injectable hydrogels), patches, ointments, powders, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The active ingredients of the present invention may be administered alone or in combination with other therapeutic agents. In another preferred embodiment, the composition further comprises one or more additional therapeutic agents. Preferably, the therapeutic agent is selected from the group consisting of: diosmin, furosemide, busulfan, spironolactone, su Zia ply, or combinations thereof.

In certain embodiments, the active ingredients of the present invention are used simultaneously with, or sequentially with, other agents that are part of a combination therapeutic regimen in the same or separate formulations.

The general range of therapeutically effective doses for the compositions of the active ingredients of the present invention will be: about 1 to 2000 mg/day, about 10 to about 1000 mg/day, about 10 to about 500 mg/day, about 10 to about 250 mg/day, about 10 to about 100 mg/day, or about 10 to about 50 mg/day. A therapeutically effective dose will be administered in one or more doses. However, it will be appreciated that the particular dosage of a compound of the invention for any particular patient will depend on a variety of factors, such as the age, sex, weight, general health, diet, individual response of the patient to be treated, the time of administration, the severity of the disease to be treated, the activity of the particular compound administered, the dosage form, the mode of application and concomitant medication. The therapeutically effective amount for a given situation can be determined by routine experimentation and is within the ability and judgment of a clinician or physician. In any event, the active ingredient or composition will be administered in multiple doses based on the individual circumstances of the subject to be administered and in a manner that allows for the delivery of a therapeutically effective amount.

Examples of subjects to which the active ingredients or compositions of the present invention are administered include mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cattle, sheep, monkeys, etc.).

The main advantages of the invention include:

a) Ginsenoside Re can treat lymphedema of patients with low TNF-alpha content, so that accurate medication of the medicines is realized;

b) Ginsenoside Re can stimulate VEGF-C mRNA expression of lymphatic endothelial cells or the expression level of protein thereof, promote lymphatic endothelial cell migration, and improve lymphedema.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

In the following experimental examples, ginsenoside Re (with a structural formula shown in FIG 1) is extracted from dried roots and rhizomes of pseudo-ginseng by using a commercially available product or according to a common extraction method in the field of traditional Chinese medicines.

In the following examples of the present invention, M is the molar concentration, i.e., mol/L; mu M is micromole per liter; nM is nanomole per liter.

EXAMPLE 1 detection of ginsenoside Re expression of VEGF-C protein in lymphatic endothelial cells

Ginsenoside Re (0, 2.5, 5, 10, 25, 50. Mu.M) pre-treated lymphatic endothelial cells for 24h. Extracting cell total protein, and detecting the VEGF-C protein expression condition of the lymphatic endothelial cells by using a Western bolt technology. The BCA method is used for measuring the protein concentration, and the activation index level is detected through electrophoresis, membrane transfer, antigen-antibody reaction and exposure and development after denaturation. The results are shown in FIG. 2.

EXAMPLE 2 detection of ginsenoside Re expression of VEGF-C mRNA in lymphatic endothelial cells

Ginsenoside Re (0, 2.5, 5, 10, 25, 50. Mu.M) pre-treated lymphatic endothelial cells for 24h. Cell mRNA is extracted, and the expression of VEGF-C mRNA of lymphatic endothelial cells is detected by RT-qPCR technology. Reverse transcription of mRNA, cDNA amplification, fluorescent real-time quantification technique, detection of VEGF-C mRNA expression. The results are shown in fig. 3 (×p <0.05, ×p < 0.01).

As shown in fig. 2 and 3, the expression level of VEGF-C protein and mRNA of lymphatic endothelial cells is remarkably increased after the intervention of ginsenoside Re, and a certain dose-dependent effect is shown along with the increase of the concentration of ginsenoside Re. When 50. Mu.M ginsenoside Re was added, the mRNA level reached 2 times the level of ginsenoside Re not added.

Example 3 detection of the function of ginsenoside Re in the migration of lymphatic endothelial cells

Lymphatic endothelial cells were seeded in 6-well plates and scored in the middle with a pipette tip after cell confluence. The original medium was replaced with a medium containing different concentrations of ginsenoside Re (0, 2.5, 5, 10, 25, 50. Mu.M). After 24 hours, scratch was observed again and representative micrographs were taken. The results are shown in FIG. 4.

As shown in fig. 4, the intervention of ginsenoside Re can promote lymphatic endothelial cell migration, and exhibits a certain dose-dependent effect with the increase of the concentration of ginsenoside Re. When 50 mu M of ginsenoside Re is added, the endothelial cells of the lymphatic vessels migrate in a large quantity within 24 hours, the scratch size is obviously reduced, and the lymphedema can be improved.

The ginsenoside Re is prompted to increase VEGF-C expression, and the generation of lymphatic vessels is promoted, so that the lymphatic vessel reflux function is promoted, and lymphedema is improved.

Comparative example 1 ginsenoside Rg1 promotes expression of VEGF-C mRNA in lymphatic endothelial cells under TNF-alpha stimulation

After treating lymphatic endothelial cells with ginsenoside Rg1 at different concentrations (0, 10, 50, 100 nM) for 24h with or without TNF-alpha (1 ng/mL) stimulation, cellular mRNA was extracted and assayed for VEGF-C mRNA expression by RT-qPCR technique. Reverse transcription of mRNA, cDNA amplification, fluorescent real-time quantification technique, detection of VEGF-C mRNA expression, use 2 ^﹣ΔΔCt The data were analyzed by the method.

The relative expression of VEGF-C mRNA in the absence and presence of TNF-alpha stimulation by Rg1 is shown in Table 1 and FIG. 5.

TABLE 1 relative mRNA expression of VEGF-C and beta-actin in the absence and presence of TNF-alpha stimulation by Rg1

Comparative example 2 ginsenoside Rg1 promotes expression of VEGF-C protein in lymphatic endothelial cells under TNF-alpha stimulation

Protein sample extraction was performed after treatment of lymphatic endothelial cells with varying concentrations (0, 10, 50, 100 nM) of ginsenoside Rg1, or VEGF-c protein (0.34 nM) for 24h with or without TNF- α (1 ng/mL) stimulation. And then detecting the VEGF-C protein expression condition of the lymphatic endothelial cells by using a Western bolt technology. The BCA method is used for measuring the protein concentration, and the activation index level is detected through electrophoresis, membrane transfer, antigen-antibody reaction and exposure and development after denaturation.

The relative beta-actin expression of VEGF-C protein by Rg1 in the absence of TNF-alpha stimulation (FIG. 6) and in the presence of TNF-alpha stimulation (FIG. 7) is shown in tables 2 and 3 below.

TABLE 2 relative protein expression of VEGF-C and beta-actin in absence of TNF-alpha stimulation by Rg1

	Blank space	Rg1 5nM	Rg1 10nM	Rg1 50nM	Rg1 100nM
						1	0.81	0.85	0.97	0.94	1.14
2	0.89	0.80	1.06	0.98	1.10
						3	0.82	0.82	1.03	0.94	1.13
Average of	0.84	0.82	1.02	0.95	1.12

TABLE 3 relative protein expression of VEGF-C and beta-actin in the presence of TNF-alpha stimulation by Rg1

Experiments show that under the condition of not adding TNF-alpha, compared with a control group, the expression conditions of VEGF-C protein and mRNA have no statistical difference when ginsenoside Rg1 is added; under the stimulation of TNF-alpha, compared with a treatment group with only TNF-alpha, the treatment group with ginsenoside Rg1+TNF-alpha shows a remarkably improved effect of promoting the mRNA and protein expression of VEGF-C, and a certain dose-dependent effect is shown along with the increase of the concentration of ginsenoside Rg 1. It is explained that ginsenoside Rg1 must exhibit the effect of promoting VEGF-C expression in the presence of TNF-alpha, and that ginsenoside Rg1 does not have a significant promoting effect in the absence of TNF-alpha. Furthermore, the effect of adding TNF- α and 100nM ginsenoside Rg1 was still less than that of adding 0.34nM VEGF-C.

Comparative example 3 ginsenoside Rg1 promotes tube formation and migration of lymphatic endothelial cells under TNF- α stimulation

Matrigel (Gibco Life Technologies Corporation, grand Island, NY, USA, orderAccession number A14132-02, 50. Mu.L) was pipetted into a 96-well plate and polymerized at 37℃for 30min. Lymphatic endothelial cells were grown at 3X 10 ⁴ The density of individual cells/wells was seeded onto the matrigel layer, then TNF-. Alpha.was added (1 ng/mL) and Rg1 (0, 10, 50, 100 nM) or VEGF-C (0.34 nM) at various concentrations, after 6 hours, tube formation was analyzed by microscopy (Leica TCS-SP5, germany) (FIG. 8A).

LECs were cultured in 12-well plates (2X 10) ⁵ Individual cells/well) and after 24 hours the fused monolayer was scored intermediately with a pipette. And the original medium in the wells was replaced with medium containing TNF- α (1 ng/mL) and varying concentrations of Rg1 (0, 10, 50, 100 nM) or VEGF-C (0.34 nM). Wound closure was monitored 24 hours after Rg1 or VEGF-C treatment and representative micrographs were taken (B in FIG. 8).

As shown in FIG. 8, ginsenoside Rg1 showed an effect of promoting the generation and migration of lymphatic endothelial cells under TNF- α stimulation.

Discussion of the invention

Unlike ginsenoside Rg1 which can promote the expression of VEGF-C mRNA of lymphatic endothelial cells only by the stimulation of TNF-alpha, ginsenoside Re can effectively promote the expression of VEGF-C mRNA without the stimulation of TNF-alpha, increase the protein expression quantity, promote the migration of lymphatic endothelial cells and further improve lymphedema, so that the accurate application of the medicines can be realized.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. Use of ginsenoside Re, or a pharmaceutically acceptable salt or an extract thereof, for the preparation of a composition for preventing and/or treating lymphedema;

2. the use of claim 1, wherein the lymphedema is suitable for subjects with a low level of TNF- α in the body, wherein a low level of TNF- α means a TNF- α level of less than 1.5ng/mL.

3. The use of claim 1, wherein the lymphedema is selected from the group consisting of: primary lymphedema and secondary lymphedema.

4. The use according to claim 1, wherein the secondary lymphedema is selected from the group consisting of: infectious lymphedema, damaging lymphedema, malignant lymphedema, or a combination thereof.

5. The use according to claim 1, wherein the prevention and/or treatment of lymphedema comprises one or more features selected from the group consisting of:

(i) Promoting VEGF-C expression of lymphatic endothelial cells;

(ii) Stimulating secretion of VEGF-C by lymphatic endothelial cells;

(iii) Stimulating lymphangiogenesis;

(iv) Promoting lymphatic endothelial cell migration;

(v) Preventing the regeneration of lymphatic effusion;

(vi) Improving lymphatic return function.

6. The use according to claim 1, wherein the composition comprises ginsenoside Re, or a pharmaceutically acceptable salt thereof, or an extract thereof, at a concentration of 1 μm or more.

7. The use according to claim 1, wherein the content of ginsenoside Re in the extract is greater than or equal to 7wt%, wherein the content C1 is based on dry weight of the extract.

8. A pharmaceutical composition for treating lymphedema, the pharmaceutical composition comprising:

(b) A pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of: gel, patch, tablet, capsule, powder, ointment, powder, injection, aqua, enteric sustained-release preparation or injection.

10. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of: oral dosage forms, transdermal injection dosage forms, intravenous injection dosage forms, intramuscular injection dosage forms.