Application of daphnetin in preparing medicine for treating lymphatic reflux disorder

Abstract

The present invention relates to the use of daphnetin in the preparation of a drug for treating lymphatic reflux disorder. When daphnetin or a pharmaceutically acceptable salt or solvate thereof is used for treating lymphatic reflux disorder, it has a good therapeutic effect. The present invention has found that daphnetin can improve the substantial symptoms of the lymphatic reflux system caused by lymphatic vessel dilation and distortion, lymphatic vessel valve insufficiency, etc. caused by venous reflux disorder; and, because it has an anti-inflammatory effect, it has a good therapeutic effect on lymphatic vessel obstruction caused by bacterial infection.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of daphnetin in preparing medicines for treating lymphatic reflux disorders. Background Art

[0002] Daphnetin is an active ingredient extracted from Daphne Korean Nakai, a plant of the genus Daphne. It is also known as Daphnetin. It is the first new drug obtained in my country through chemical synthesis. Its chemical name is 7,8-dihydroxycoumarin.

[0003] Daphne has the effects of dilating coronary blood vessels, increasing coronary blood flow, reducing myocardial oxygen consumption, improving myocardial metabolism and promoting cardiac function recovery, dilating peripheral blood vessels, resisting arterial thrombosis and inhibiting platelet aggregation; it also has the anti-inflammatory effect of stimulating the pituitary-adrenal cortex system.

[0004] After absorption, daphneline is widely distributed in the body, with the highest distribution in the kidney, followed by the lung, spleen, and plasma. The main excretion route is the kidney. Clinically, it is mainly used as an adjuvant treatment for thromboangiitis obliterans and other occlusive vascular diseases and coronary heart disease. Summary of the invention

[0005] The purpose of the present invention is to provide the use of daphnetin in the preparation of drugs for treating lymphatic reflux disorders. Daphnetin can improve substantial symptoms of the lymphatic reflux system caused by lymphatic vessel dilation and distortion, lymphatic valve insufficiency, etc. caused by venous reflux disorders; and, due to its anti-inflammatory effect, it has a good therapeutic effect on lymphatic vessel obstruction caused by bacterial infection.

[0006] To this end, in a first aspect, the present invention provides the use of daphnetin or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for treating lymphatic reflux disorder.

[0007] In some embodiments, the medicament uses daphnetin or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.

[0008] In some embodiments, the daphnetin or a pharmaceutically acceptable salt or solvate thereof is present in the medicament in an amount effective for treating the lymphatic drainage disorder.

[0009] In some embodiments, in the medicament, the content of daphnetin or a pharmaceutically acceptable salt or solvate thereof as an active ingredient is 0.1-99% (w / w).

[0010] In some embodiments, the lymphatic drainage disorder is specifically upper limb lymphatic drainage disorder or lower limb lymphatic drainage disorder.

[0011] In some embodiments, the lymphatic reflux disorder includes lymphatic vessel obstruction, lymphatic vessel dilation and distortion, lymphatic vessel valve insufficiency, etc. In some embodiments, daphnetin can be used to prepare any of the following drugs: drugs for treating lymphatic vessel obstruction, drugs for treating lymphatic vessel dilation and distortion, drugs for treating lymphatic vessel valve insufficiency, etc.

[0012] In some embodiments, the drug is administered orally, by injection, implant, spray or inhalation.

[0013] In some embodiments, the dosage form of the drug includes but is not limited to injection, tablet, granule, capsule, pill, sustained release agent, oral liquid preparation, powder or gel.

[0014] In some embodiments, the drug further comprises an effective ingredient that acts synergistically with daphnetin or a pharmaceutically acceptable salt or solvate thereof.

[0015] In some embodiments, the drug further comprises a drug carrier or a pharmaceutically acceptable excipient.

[0016] The second aspect of the present invention provides a pharmaceutical composition comprising an effective amount of daphnetoside or a pharmaceutically acceptable salt or solvate thereof for treating lymphatic reflux disorder.

[0017] In some embodiments, the pharmaceutical composition further comprises an effective ingredient that acts synergistically with daphnetin or a pharmaceutically acceptable salt or solvate thereof.

[0018] In some embodiments, the drug further comprises a drug carrier or a pharmaceutically acceptable excipient.

[0019] The third aspect of the present invention provides a therapeutic method for treating lymphatic reflux disorder, which comprises administering to a subject an effective amount of a drug comprising daphnetin or a pharmaceutically acceptable salt or solvate thereof.

[0020] In some embodiments, the subject includes a mammal; for example, a human, a rabbit, a pig, a sheep, a cow, a rat, a mouse, a monkey, etc.

[0021] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0022] (1) The present invention provides the use of daphnetin in the preparation of a drug for treating lymphatic reflux disorders, which not only expands the application scope of daphnetin, but also provides a new option for the treatment of lymphatic reflux disorders.

[0023] (2) According to the technical solution of the present invention, daphneline has excellent effects in treating lymphatic reflux disorders, and because the safety of daphneline has been clinically verified, using daphneline to treat lymphatic reflux disorders, or developing drugs for treating lymphatic reflux disorders with daphneline as an active ingredient has good clinical application prospects.

[0024] (3) Animal experiments have shown that different doses of daphnetin have no obvious toxic effects on mice, and have excellent therapeutic effects on the lymphatic reflux disorder mouse model, with an effect close to that of the positive control. In addition, the therapeutic effect of daphnetin on the lymphatic reflux disorder mouse model showed a certain dose dependence. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only used for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. In the accompanying drawings:

[0026] Figure 1 : For the mouse model of lower limb lymphatic drainage disorder, the results of comparison of the swelling degree of the lower limbs of mice after treatment with different doses of daphneline;

[0027] Figure 2 : For the mouse model of lower limb lymphatic drainage disorder, the weight changes of mice after treatment with different doses of daphneline;

[0028] Figure 3 : For the mouse model of lower limb lymphatic drainage disorder, the changes in the circumference of the mouse lower limbs after treatment with different doses of daphneline;

[0029] Figure 4 : For the mouse model of lower limb lymphatic drainage disorder, the volume changes of the lower limbs of mice after treatment with different doses of daphneline;

[0030] Figure 5 : HE staining imaging results of the mouse model of lower limb lymphatic drainage disorder after treatment with different doses of daphneline, the scale bar is 100μm;

[0031] Figure 6 :For the mouse model of lower limb lymphatic drainage disorder, the results of LYVE-1 immunostaining imaging of the lesion site of mice after treatment with different doses of daphneline, the scale bar is 100μm;

[0032] Figure 7 : For the mouse model of lower limb (tail) lymphatic drainage disorder, the results of comparison of the degree of tail swelling of mice after treatment with different doses of daphneline;

[0033] Figure 8 :For the mouse model of lower limb (tail) lymphatic drainage disorder, the weight changes of mice after treatment with different doses of daphneline;

[0034] Fig. 9 :For the mouse model of lower limb (tail) lymphatic drainage disorder, the changes in the swelling volume of the mouse tail after treatment with different doses of daphneline;

[0035] Fig.10 :For the mouse model of lower limb (tail) lymphatic drainage disorder, HE staining imaging results of the mouse lesion site after treatment with different doses of daphneline, the scale bar is 100μm;

[0036] Fig.11 :For the mouse model of lower limb (tail) lymphatic drainage disorder, the results of LYVE-1 immunostaining imaging of the lesion site of mice after treatment with different doses of daphneline, the scale bar is 520μm;

[0037] In the above figures, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001. DETAILED DESCRIPTION

[0038] The exemplary embodiments of the present disclosure will be described in detail below. It should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0039] The "range" disclosed in the present invention is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of a particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 60 to 120 and 80 to 110 is listed for a particular parameter, it is understood that a range of 60 to 110 and 80 to 120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and the maximum range values ​​3, 4 and 5 are listed, the following ranges can all be expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 and 2 to 5. In this application, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0040] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions.

[0041] Unless otherwise stated, all technical features and optional technical features of the present invention can be combined with each other to form a new technical solution.

[0042] Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, mentioning that the method may also include step (c) means that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0043] Unless otherwise specified, the "include" and "comprising" mentioned in the present invention represent open-ended or closed-ended expressions. For example, the "include" and "comprising" may represent that other components not listed may also be included or comprised, or may only include or comprise the listed components.

[0044] Unless otherwise indicated, the term "effective amount" of the present invention specifies the amount of the compound sufficient to produce the desired response, such as improvement or complete cure of lymphatic reflux disorders. For therapeutic purposes, the effective amount is also the amount in which any harmful effects of the compound are offset by the beneficial effects of the treatment. The specific effective amount or sufficient amount will vary with factors such as the specific condition being treated, the patient's physical condition (e.g., the patient's weight, age or gender, the type of object being treated, the duration of treatment). The effective amount can be, for example, expressed in grams, milligrams or micrograms or in milligrams per kilogram of body weight (mg / kg); in some embodiments, the effective amount is 25 mg / kg, 50 mg / kg, 100 mg / kg, etc. of daphneline. Alternatively, the effective amount can be expressed in terms of the concentration of the active component (e.g., daphneline of the present disclosure), such as molar concentration, mass concentration, volume concentration, molar concentration, molar fraction, mass fraction and mixing ratio. In addition, those skilled in the art can calculate the human equivalent dose of a drug (e.g., a drug containing daphneline of the present invention) based on the dose determined from an animal model.

[0045] Unless otherwise indicated, the term "treatment" refers to the effect of administering an effective amount of daphnetoside to a subject or patient, so that a person suffering from lymphatic reflux disorder shows improvement in the symptoms of lymphatic reflux disorder. The improvement is any observable or measurable improvement, and therefore, those skilled in the art will understand that treatment can improve the condition of the subject or patient, but may not be a complete cure of the disease.

[0046] Unless otherwise indicated, the terms "subject" or "patient" are used interchangeably herein to refer to a mammal that can be treated by the medicament or pharmaceutical composition provided by the present invention. The term "mammal" refers to all members of the mammal class, including humans, primates, livestock and domestic animals, such as rabbits, pigs, sheep, cattle, etc. "Subject" or "patient" refers to both male (male) and female (female) genders unless a gender is specifically indicated. In a preferred embodiment, the subject is a human.

[0047] Unless otherwise specified, the term "drug carrier" refers to a system that can change the way a drug enters the human body and its distribution in the body, control the release rate of the drug, and deliver the drug to the target organ. The present invention does not limit the specific types of drug carriers. In some embodiments, the drug carriers that can be used in the drugs or pharmaceutical compositions provided by the present invention include but are not limited to microcapsules, microspheres, nanoparticles, and liposomes.

[0048] Unless otherwise specified, the term "pharmaceutically acceptable excipient" refers to an excipient that, in addition to the active ingredient, has no significant irritation to the organism and does not damage the biological activity and performance of the active ingredient. The use of pharmaceutically acceptable excipients to prepare pharmaceutical preparations is well known to those of ordinary skill in the art. In some embodiments, the pharmaceutically acceptable excipients that can be used in the medicament or pharmaceutical composition provided by the present invention include, but are not limited to: propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, and release retardants.

[0049] In the prior art, daphnetin, as a safe clinical drug, has been widely reported, but its application in lymphatic reflux disorder has not been found. The present invention provides the use of daphnetin or its pharmaceutically acceptable salt or solvate in the preparation of a drug for treating lymphatic reflux disorder through research.

[0050] Daphnetin, also known as daphnetolide, ancestral dapoxetine, and daphnetin, is the active ingredient of Daphne Korean Nakai. It is the first new drug obtained by chemical synthesis in my country. Its chemical name is 7,8-dihydroxycoumarin, and its molecular formula is C9H6O4. Its chemical structure is shown in formula (I).

[0051]

[0052] The first aspect of the present invention provides the use of daphnetin or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for treating lymphatic reflux disorder.

[0053] The present invention has found through research that daphnetin can improve substantial symptoms of the lymphatic return system caused by lymphatic vessel dilation and distortion, lymphatic valve insufficiency, etc. caused by venous reflux obstruction; and, due to its anti-inflammatory effect, it has a good therapeutic effect on lymphatic vessel obstruction caused by bacterial infection.

[0054] In some embodiments, the drug uses daphnetin or a pharmaceutically acceptable salt or solvate thereof as an active ingredient. For example, daphnetin or a pharmaceutically acceptable salt or solvate thereof can be the only active ingredient of the drug, or one of the active ingredients.

[0055] In some embodiments, the lymphatic drainage disorder is specifically upper limb lymphatic drainage disorder or lower limb lymphatic drainage disorder.

[0056] In some embodiments, the lymphatic reflux disorder includes lymphatic vessel obstruction, lymphatic vessel dilation and distortion, lymphatic vessel valve insufficiency, etc. In some embodiments, daphnetin can be used to prepare any of the following drugs: drugs for treating lymphatic vessel obstruction, drugs for treating lymphatic vessel dilation and distortion, drugs for treating lymphatic vessel valve insufficiency, etc.

[0057] In some embodiments, the daphnetin or a pharmaceutically acceptable salt or solvate thereof is present in the medicament in an amount effective for treating lymphatic reflux disorder.

[0058] In some embodiments, the daphnetoside or a pharmaceutically acceptable salt or solvate thereof is present in the drug in an effective amount capable of treating lymphatic reflux disorders, specifically referring to: based on the body weight of the drug subject in kg, 25 to 100 mg / kg of daphnetoside or a pharmaceutically acceptable salt or solvate thereof can be provided to the drug subject.

[0059] In some embodiments, in the drug, the mass percentage of daphnetin or its pharmaceutically acceptable salt or solvate as the active ingredient is 0.1-99%; for example, it can be about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, %, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% etc.

[0060] In some embodiments, the drug is administered orally, by injection, implant, spray or inhalation.

[0061] In some embodiments, the dosage form of the drug includes but is not limited to injection, tablet, granule, capsule, pill, sustained-release agent, oral liquid preparation, powder or gel.

[0062] In some embodiments, the drug further comprises a drug carrier or a pharmaceutically acceptable excipient.

[0063] In some embodiments, the dosage form of the drug is a capsule, and the raw materials thereof include, by mass percentage: 80-90% daphnetin, 10-20% filler, 1-5% binder, and 1-5% lubricant.

[0064] In some embodiments, the method for preparing the drug comprises:

[0065] S1. Weighing: Weigh the raw materials according to the following mass percentages: 80-90% daphne, 10-20% filler, 1-5% binder, 1-5% lubricant;

[0066] S2. Prepare the adhesive: dissolve the adhesive in water;

[0067] S3 wet granulation: The daphne is added to the wet granulator, the binder prepared in step S2 is added to the wet granulator, stirred evenly, and the wet granules are formed by a wet granulator;

[0068] S4. Wet granulation: adding the wet granules to a square vibrating screening machine, passing through a sieve of a first mesh number (eg, 20 meshes), to obtain first granules;

[0069] S5. Drying: putting the first particles into a hot air circulation oven to obtain second particles after drying;

[0070] S6. Particle selection: adding the second particles to an oscillating sieve, passing through a second mesh number (eg, 30 mesh) sieve to obtain third particles;

[0071] S7. Granulation: The particles that have not passed the second mesh sieve are added to a multifunctional granulator, and passed through a third mesh sieve (eg, Φ1 sieve) to obtain fourth particles;

[0072] S8 mixing: the steps S6, S7 obtained particles and lubricants, fillers into a three-dimensional mixer and mixed evenly to obtain daphne granules;

[0073] S9. Filling the mixed particles to obtain the drug.

[0074] Example 1

[0075] The experimental animals, 6-8 weeks old male BALb / c mice, were used as the research objects to explore the effects of different doses of daphneline on the mouse model of lower limb lymphatic drainage disorder. The lower limbs (including appearance and pathological sections) and the expression of related markers of mice after treatment with different doses of drug administration were observed, and the therapeutic effect of daphneline on mice with lower limb lymphatic drainage disorder was analyzed.

[0076] (I) Establishment of a mouse model of lower limb lymphatic drainage disorder

[0077] The mouse model of lower limb lymphatic dysfunction was established using the following method:

[0078] After weighing and anesthetizing 6-8 week old male mice, 0.1 ml of patented blue V solution (25 mg / ml, purchased from MCE) prepared with normal saline was injected into the web of the mouse's foot (the thin membrane between the toes) via intraperitoneal injection, and the injection site was fully rubbed and squeezed for about 1 min.

[0079] Place the mouse in prone position and make a longitudinal incision about 0.5 cm long about 1.5 cm above the popliteal fossa. Carefully separate the subcutaneous tissue to the fascia layer, and you can see the blue-stained lymphatic vessels and the round-shaped popliteal lymph nodes. Carefully ligate the lymphatic vessels with 10-0 sutures, separate and remove the popliteal lymph nodes, and suture the incision layer by layer.

[0080] The mouse was placed in supine position and an oblique incision of about 1 cm long was made about 1 cm below the groin of the mouse. The subcutaneous tissue was carefully separated to the fascia layer, and the blue-stained lymphatic vessels were seen. The blue-stained lymphatic vessels were carefully ligated with 10-0 thread. Clusters of fat pad tissue were seen about 1 cm above the incision, and the inguinal lymph nodes were wrapped in the fat pad tissue. All the fat pad tissue was carefully separated and removed, and the incision was sutured layer by layer.

[0081] (II) Treatment with different doses of daphneline

[0082] All animals were healthy male BALb / c mice purchased from Beijing Weitonglihua, SPF grade, and housed and tested in the Animal Experiment Center of Jilin University School of Basic Medical Sciences. 35 BALb / c mice weighing between 17-20 g (4-6 weeks old) were selected.

[0083] Mice were randomly divided into a blank group, a sham operation group, a model group, a low-dose group (25 mg / kg / d of daphnetin), a medium-dose group (50 mg / kg / d of daphnetin), a high-dose group (100 mg / kg / d of daphnetin) and a positive control group (melilotus oleraceus fluid extract tablets). The model group and the drug group both adopted the surgical method of constructing the lower limb model in (I), in which the model group was injected with the same dose of normal saline through the tail vein; the blank group mice were not treated in any way, and the same dose of normal saline was injected through the tail vein; the sham operation group mice were only made the same incision at the corresponding part, and then the incision was sutured, and the same dose of normal saline was injected through the tail vein; on the second day after the model was successfully established, except for the blank group, sham operation group and model group, the mice in the other groups were gavaged once a day for 7 days. Food and water were provided normally during the administration.

[0084] 3. Evaluation of treatment effect

[0085] 1. Comparison of swelling degree of lower limbs of mice

[0086] After 7 days of drug treatment, the swelling degree of the lower limbs of mice in each group was compared. Figure 1 As shown. Figure 1 Compared with the control group, there was no significant change in the sham operation group; compared with the model group, the swelling in the low-dose group was slightly reduced, and the swelling in the medium-dose, high-dose and positive control groups was significantly reduced.

[0087] 2. Changes in mouse weight

[0088] During the treatment, the weight changes of mice were as follows Figure 2 As shown. It can be seen that compared with the control group, the body weight of the sham operation group, low-dose group, medium-dose group, high-dose group and positive control group did not change significantly, and increased with the increase of treatment time; while the model group began to lose weight on the third day of treatment.

[0089] 3. Changes in the circumference of mouse lower limbs

[0090] During the treatment, the circumference of the lower limbs of mice changed as follows Figure 3 As shown. Compared with the control group, the sham operation group had no significant difference in the circumference of the lower limbs; the model group had significant differences during the experiment (P<0.0001), and showed an increasing trend with the increase of time; although there were significant differences in the low, medium and high dose groups during the experiment, the circumference of the lower limbs gradually decreased with the increase of treatment time.

[0091] 4. Changes in the volume of mouse lower limbs

[0092] During the treatment, the volume changes of the lower limbs of mice were as follows Figure 4 As shown. Compared with the control group, the sham operation group had no significant difference in lower limb volume; the model group had significant differences during the experiment (P<0.0001); the low-dose group had significant differences during the experiment, but as the treatment time increased, the lower limb volume gradually decreased; the medium-dose group and the high-dose group had significant differences compared with the control group in the early stage of treatment, but as the treatment time increased, the lower limb volume gradually decreased, and there was no significant difference in the late stage of treatment.

[0093] 5. Mouse subcutaneous tissue pathology

[0094] After 7 days of drug treatment, the subcutaneous tissue of the lesion site of the mouse was taken for HE staining observation. The imaging results are as follows: Figure 5 As shown in the figure, according to the results of HE staining, compared with the control group and the sham operation group, the model group, positive control group, low-dose group, medium-dose group and high-dose group all had a certain degree of inflammatory cell infiltration. Compared with the model group, the inflammatory cell infiltration gradually decreased with the increase of the treatment dose of daphnetoclax.

[0095] 6. Mouse Lymphatic Vessel Endothelial Receptor-1 (LYVE-1) Immunostaining Analysis

[0096] Lymphatic Vessel Endothelial Receptor-1 (LYVE-1) is a close relative of the leukocyte receptor CD44 and is the main receptor for hyaluronic acid (HA) in lymphatic endothelial cells. It is a commonly used marker to distinguish blood vessels from lymphatic vessels. Seven days after administration, tissues from the lesion were taken for immunohistochemical staining of LYVE-1. The results are as follows: Figure 6 As shown. It can be seen that compared with the control group and the sham operation group, the lymphatic vessels in the model group, positive control group, low-dose group, medium-dose group and high-dose group showed thin walls, enlarged lumens and curling. Compared with the model group, the degree of lymphatic expansion gradually decreased with the increase of the treatment dose of daphnetoside.

[0097] Example 2

[0098] The experimental animals, 6-8 weeks old male BALb / c mice, were used as the research objects to explore the effects of different doses of daphnetin on the mouse model of tail lymphatic reflux disorder, observe the expression of the tail and related markers of mice after treatment with different doses of drug administration, and analyze the therapeutic effect of daphnetin on mice with tail lymphatic reflux disorder.

[0099] (I) Establishment of a mouse model of tail lymphatic reflux disorder

[0100] The tail lymphatic reflux disorder mouse model was established using the following method:

[0101] After weighing and anesthetizing with intraperitoneal injection, 6-8 week old male mice were sterilized with 75% ethanol at the tail, and the diameter of the tail was measured 2 cm from the base of the tail. A circular incision was made 1 cm from the base of the tail, and the skin of the tail was removed about 2 mm deep, and the deep fascia was removed at the same time. 5 μL of patented blue V solution (25 mg / ml, purchased from MCE) prepared with normal saline was injected intradermally at the tip of the tail, and the injection site was fully rubbed and squeezed for about 1 min, and the color of the lymphatic vessels accompanying the skin excision site and the blood vessels on both sides of the tail was observed. Under the assistance of a surgical microscope, the blue-stained exposed lymphatic vessels were excised, and the cut ends and the skin at the wound edge were cauterized to prevent the lymphatic vessels from recanalizing. Attention was paid to maintaining the integrity of the main subcutaneous blood vessels and tendons to avoid necrosis of the distal tail incision. The surgical wound was bandaged with vaseline gauze.

[0102] (II) Treatment with different doses of daphneline

[0103] All animals were healthy male BALb / c mice purchased from Beijing Weitonglihua, SPF grade, and housed and tested in the Animal Experiment Center of Jilin University School of Basic Medical Sciences. 35 BALb / c mice weighing between 17-20 g (4-6 weeks old) were selected.

[0104] Mice were randomly divided into a blank group, a sham operation group, a model group, a low-dose group (25 mg / kg / d of daphnetin), a medium-dose group (50 mg / kg / d of daphnetin), a high-dose group (100 mg / kg / d of daphnetin) and a positive control group (melilotus odoratus fluid extract tablets). The model group and the drug group both adopted the surgical method of constructing the model in (I), in which the model group was gavaged with the same dose of normal saline; the blank group mice did not receive any treatment, and the same dose of normal saline was injected into the tail tip; the sham operation group mice only made the same incision at the corresponding site, and then the incision was sutured, and the same dose of normal saline was injected into the tail tip; on the second day after the model was successfully established, except for the blank group, sham operation group and model group, the mice in the other groups were gavaged once a day for 7 days. Food and water were provided normally during the administration.

[0105] 3. Evaluation of treatment effect

[0106] 1. Comparison of mouse tail swelling

[0107] After 7 days of drug treatment, the degree of tail swelling of mice in each group was compared. Figure 6 As shown. Figure 7 Compared with the control group, there was no significant change in the sham operation group; compared with the model group, the swelling in the low-dose group was slightly reduced, and the swelling in the medium-dose group, high-dose group, and positive control group was significantly reduced.

[0108] 2. Changes in mouse weight

[0109] During the treatment, the weight changes of mice were as follows Figure 8 As shown, compared with the control group, the body weight of the sham operation, low-dose, medium-dose, high-dose and positive control groups did not change significantly.

[0110] 3. Changes in the volume of mouse tail swelling

[0111] During the treatment, the volume changes of the mouse tail swelling were as follows Fig. 9 As shown. It can be seen that compared with the control group, there was no significant difference in the tail swelling volume in the sham operation group; there was a significant difference in the model group during the experimental period (P<0.0001), and it showed an increasing trend with the increase of time; although there were significant differences in the low, medium and high dose groups during the experimental period, the tail swelling volume gradually decreased with the increase of treatment time.

[0112] 4. Mouse tail tissue pathology

[0113] After 7 days of drug treatment, pathological sections of the mouse tail tissue were taken for HE staining. The imaging results are as follows: Fig.10 As shown, compared with the control group and the sham operation group, the edema degree in the model group, positive control group, low-dose group, medium-dose group and high-dose group increased, accompanied by a certain degree of lymphangiectasia and inflammatory cell infiltration; compared with the model group, the edema degree, lymphangiectasia and inflammatory cell infiltration were gradually reduced with the increase of the treatment dose of daphnetoclax.

[0114] 5. Mouse Lymphatic Vessel Endothelial Receptor-1 (LYVE-1) Immunostaining Analysis

[0115] Seven days after administration, tissues from the lesion site of the tail were collected for immunohistochemical staining of LYVE-1. The results were as follows: Fig.11 As shown. It can be seen that compared with the control group and the sham operation group, the lymphatic vessels in the model group, positive control group, low-dose group, medium-dose group and high-dose group showed thin walls, enlarged lumens and curling. Compared with the model group, the degree of lymphatic expansion gradually decreased with the increase of the treatment dose of daphnetoside.

[0116] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. Use of daphnetin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating lymphatic reflux disorder.

2. The use according to claim 1, characterized in that The medicine uses daphnetin or a pharmaceutically acceptable salt thereof as an active ingredient.

3. The use according to claim 1 or 2, characterized in that: The daphnetin or a pharmaceutically acceptable salt thereof is present in the medicine in an amount effective for treating the lymphatic reflux disorder.

4. The use according to any one of claims 1 to 3, characterized in that: In the medicine, the content of daphnetin or its pharmaceutically acceptable salt as an active ingredient is 0.1-99% (w / w).

5. The use according to claim 1, characterized in that The medicine is an oral administration agent, an injection administration agent, an implant administration agent, a spray administration agent or an inhalation administration agent.

6. The use according to claim 1, characterized in that The dosage form of the drug includes, but is not limited to, injection, tablet, granule, capsule, pill, sustained-release preparation, oral liquid preparation, powder or gel.

7. The use according to claim 1, characterized in that The medicine also includes a drug carrier or a pharmaceutically acceptable excipient.

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