CN112972438A

CN112972438A - Lignan compounds derived from radix paeoniae rubra as well as preparation method and application thereof

Info

Publication number: CN112972438A
Application number: CN201911275777.8A
Authority: CN
Inventors: 靳洪涛; 林生; 李恩灿; 钟万超; 李万芳; 夏桂阳; 郝瑞瑞; 夏欢
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2019-12-12
Filing date: 2019-12-12
Publication date: 2021-06-18
Anticipated expiration: 2039-12-12
Also published as: CN112972438B

Abstract

The invention belongs to the field of medicines, and relates to lignan compounds in red paeony root, a preparation method thereof and application of the lignan compounds in preventing and treating allergic diseases. Pharmacological experiments prove that the compound can effectively inhibit the release of beta-hexosaminidase (beta-HEX) and histamine (His) in sensitized RBL-2H3 cells, can obviously reduce the content of inflammatory mediators in the supernatant of RBL-2H3 cells, has good anti-allergic, anti-anaphylactoid and anti-inflammatory effects, and has an effective dose of 5 mu mol/L. Can be used for preparing medicines for preventing and treating allergic diseases.

Description

Lignan compounds derived from radix paeoniae rubra as well as preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a compound separated from a traditional Chinese medicine red paeony root and application thereof in preventing and treating allergic diseases.

Background

Allergic diseases, as a chronic disease, seriously affect the working efficiency of patients, reduce the production capacity thereof, and thus lead to an increase in social costs. Therefore, effective and safe treatment of allergic diseases is one of the major challenges facing public health. Allergic diseases include anaphylactic reactions (allergic reactions) and anaphylactoid reactions (anaphylactic reactions). The anaphylaxis is the most urgent and potentially severe manifestation of allergic diseases, and refers to a physiological change mainly caused by physiological dysfunction or tissue cell injury of an organism when the organism receives the stimulation of the same antigen again after the organism initially responds to the antigen substance. According to the report of the world allergy organization, people accompanied with allergic symptoms globally account for 30 to 40 percent of the total population; the number of patients is large, and the data are often underestimated because the symptoms seriously damaging the life health do not appear in the early and middle stages of allergic reaction. Most of the antiallergic drugs sold at present have serious adverse reactions, such as arrhythmia, mental dysfunction, gastrointestinal disorder, infection and the like. Therefore, the search for new effective treatment schemes is imminent.

The clinical symptoms of type I hypersensitivity and anaphylactoid reaction are similar, but the difference between the two action mechanisms is not completely understood at present. However, a large number of studies show that both anaphylactoid reaction and anaphylactic reaction can cause the body to release beta-hexosaminidase and histamine, cause smooth muscle contraction, increase vascular permeability and the like, and further cause a series of anaphylactic symptoms. The cytoplasm of RBL-2H3 cell contains abundant basophilic granules, and can cause degranulation phenomenon when cell has type I anaphylactic reaction or anaphylactoid reaction, i.e. the alkaline granules wrapping histamine and beta-hexosaminidase gradually approach to cell membrane, the vesicle membrane fuses with the cell membrane, and the content is released outside the cell to induce the generation of corresponding anaphylactic symptom. In view of this, many scholars consider: inhibiting the release of allergic mediators can become a feasible direction for the development of new antiallergic and antiallergic drugs.

Type I hypersensitivity and anaphylactoid hypersensitivity are difficult to distinguish by symptoms in clinical diagnosis, and the main pathological changes of the type I hypersensitivity and anaphylactoid hypersensitivity are smooth muscle contraction, capillary vessel dilatation, vascular permeability increase and gland secretion increase, so that the elimination and alleviation of the symptoms become necessary links for resisting anaphylactoid diseases. The series of symptoms are caused by a series of inflammatory mediators released by the body, wherein the main mediators comprise His and beta-HEX, so that the inhibition of the release of the two mediators becomes the key for treating the allergic diseases. The natural product has the advantages of small toxic and side effect, lasting curative effect, good overall regulation and synergistic effect and the like, and becomes a research hotspot of new medicines for treating allergic diseases in recent years. Although the curative effect evaluation and mechanism discussion of the natural products are still based on the laboratory research stage, and the clinical research does not provide a definitive conclusion, a large amount of data lays a certain theoretical basis, and a basis is laid for further preferably tamping the anti-allergic disease new drugs.

Radix Paeoniae Rubra (Paeoniaceae Rubra Radix) is dried root of Paeonia lactiflora Pall of Ranunculaceae. It enters liver meridian. The traditional Chinese medicine is often combined clinically to treat warm-toxicity macula, conjunctival congestion, swelling and pain, liver depression, hypochondriac pain, amenorrhea, dysmenorrhea, traumatic injury, carbuncle swelling, sore and ulcer and other symptoms. Modern pharmacology finds that the red paeony root has the drug effect of resisting bacteria, inflammation and pain. The red peony root has complex chemical components, mainly monoterpenes, fatty acids, phenols, lignans and other compounds.

The applicant researches and discovers that lignan compounds I and II obtained by separating from red paeony root have the following chemical structures, and pharmacological experiments prove that the lignan compounds can effectively reduce the release amount of allergen-induced RBL-2H3 cell histamine and beta-hexosaminidase, have good effect of inhibiting cell degranulation, show good potential for resisting allergic diseases and have the effective dose of 5 mu mol/L. At present, no research report on the application of the red paeony root extract in treating allergic diseases exists, and no patent document on a preparation method of the red paeony root compound monomer for inhibiting RBL-2H3 degranulation exists.

Disclosure of Invention

The invention aims to solve the technical problem of providing a novel medicament for resisting allergic diseases.

In a first aspect, the invention provides a class of compounds derived from red peony root;

in a second aspect, the invention provides a process for the preparation of such compounds;

in a third aspect, the invention provides pharmaceutical compositions comprising such compounds;

in a fourth aspect, the invention provides the use of such compounds in the treatment of allergic diseases.

In order to solve the technical problems, the following technical scheme is adopted:

the invention provides compounds I, II, III and IV from red paeony root in a first aspect, and the structures of the compounds are as follows:

the second aspect of the present invention provides a method for preparing the above red peony root compound, which is characterized by comprising the following steps: soaking radix Paeoniae Rubra in distilled water, ultrasonic extracting, concentrating, separating and purifying the extract with organic solvent extraction, macroporous adsorbent resin chromatography, gel column chromatography, reversed phase silica gel column chromatography and preparative HPLC to obtain the compounds I, II, III, and IV, and analyzing and identifying their structures by UV, IR, NMR, MS, CD and other spectroscopic means to obtain lignan compounds.

In a third aspect, the invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of a compound and a pharmaceutically acceptable carrier. Typically, the pharmaceutical compositions of the present invention contain from 0.1% to 96% by weight of a compound of the present invention. The compound of the invention is generally present in an amount of 0.1 to 100mg in a unit dosage form, preferably 5 to 60mg in a unit dosage form.

Pharmaceutical compositions of the compounds of the invention may be prepared according to methods well known in the art. For this purpose, the compounds of the invention can, if desired, be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants and brought into a suitable administration form or dosage form for use as human or veterinary medicine.

The compounds of the present invention or pharmaceutical compositions containing them may be administered in unit dosage form by enteral or parenteral routes, such as oral, intramuscular, subcutaneous, nasal, oromucosal, dermal, ocular, peritoneal or rectal administration and the like.

The route of administration of the compounds of the invention or the pharmaceutical compositions containing them may be by injection. The injection includes intravenous injection, subcutaneous injection, intradermal injection, acupoint injection, etc.

The administration dosage form can be liquid dosage form or solid dosage form. For example, the liquid dosage form can be true solution, colloid, microparticle, emulsion, or suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, eye drop, lyophilized powder for injection, etc.

The compound can be prepared into common preparations, sustained release preparations, controlled release preparations, targeting preparations and various microparticle drug delivery systems.

For example, in order to form a unit dosage form into a tablet, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecylsulfonate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate, and the like; lubricants, for example, talc, silica, corn starch, stearate, perlitic acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets.

For example, to form the administration units into pills, various carriers well known in the art are widely used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, glycerin monostearate, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfonate, methylcellulose, ethylcellulose, etc.

For example, to encapsulate the administration unit, the active ingredient of the compounds of the present invention is mixed with the various carriers described above, and the mixture thus obtained is placed in hard gelatin capsules or soft gelatin capsules. The effective component of the compound can also be prepared into microcapsules, and the microcapsules can be suspended in an aqueous medium to form a suspension, and can also be filled into hard capsules or prepared into injections for application.

For example, the compounds of the present invention may be formulated as injectable preparations, such as solutions, suspensions, emulsions, lyophilized powders, which may be aqueous or non-aqueous, and may contain one or more pharmaceutically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants or dispersants. For example, the diluent is selected from water, ethanol, polyethylene glycol, 1, 3-propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitol fatty acid ester. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added. These adjuvants are commonly used in the art.

In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired.

For the purpose of administration and enhancing the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known administration method.

The dosage of the pharmaceutical composition of the compound of the present invention to be administered depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, body weight, character and individual response of the patient or animal, the administration route, the number of administrations and the therapeutic purpose, and thus the therapeutic dosage of the present invention can be widely varied. Generally, the dosage of the pharmaceutical ingredients of the present invention used is well known to those skilled in the art. The prophylactic or therapeutic objectives of the present invention can be accomplished by appropriate adjustment of the actual amount of drug contained in the final formulation of the compound composition of the present invention to achieve the desired therapeutically effective amount. Suitable daily dosage ranges for the compounds of the invention: the amount of the compound of the present invention is 0.001 to 100mg/Kg body weight, preferably 0.01 to 75mg/Kg body weight, more preferably 0.05 to 50mg/Kg body weight, and most preferably 0.06 to 10mg/Kg body weight. The compound of the invention is taken by an adult patient at 1-300 mg, preferably 4-150 mg, once or 2-3 times; the dosage of the composition for children is 0.01-15 mg/kg body weight, preferably 0.06-5 mg/kg body weight. The above-mentioned dosage may be administered in a single dosage form or divided into several, e.g., two, three or four dosage forms, which is limited by the clinical experience of the administering physician and the dosage regimen of the therapeutic means. The compounds or compositions of the present invention may be administered alone or in combination with other therapeutic or symptomatic agents.

The fourth aspect of the invention relates to the application of the red paeony root compound in resisting allergic diseases. In-vitro antiallergic and antiallergic pharmacological test results show that the compound can effectively inhibit the release of histamine and beta-hexosaminidase from RBL-2H3 cells, and the survival rates of the RBL-2H3 cells are 87.31%, 99.66%, 80.33% and 85.45% respectively when the administration concentration reaches 50 mu M. For allergic reactions, the compounds can change the histamine release rate from 55.51% to 30.88%, 35.64%, 37.41%, 35.64%; the compound can change the release rate of beta-hexosaminidase from 43.73% to 26.42%, 33.81%, 33.64% and 30.56%. For anaphylactoid reaction, the compound can reduce histamine release rate from 49.23% to 25.49%, 30.92%, 24.21% and 32.39%, respectively; the compound can reduce the release rate of beta-hexosaminidase from 12.73% to 6.38%, 8.32%, 6.34% and 8.25%. Therefore, the medicament achieves the purpose of inhibiting type I hypersensitivity and anaphylactoid reaction through selectively inhibiting histamine and the release of beta-hexosaminidase.

The beneficial technical effects are as follows:

1. the red paeony root compound has obvious antiallergic and antiallergic effects, and can obviously inhibit the release of histamine, and the drug effect can reach 5 mol/L. Secondly, the release of hexosaminidase can be obviously inhibited, and the drug effect also reaches 5 mol/L.

2. The red paeony root compound has the potential of being further developed into medicines for preventing and treating allergy and anaphylactoid diseases.

Drawings

FIG. 1, the preparation process of the red peony parts CS-1, CS-2, CS-3 and CS-4.

Detailed Description

The following examples and pharmacological activity experiments are intended to further illustrate the invention, but are not meant to be limiting in any way.

1. Preparation method of red peony root monomeric compounds I, II, III and IV

Example 1 preparation of Compounds I, II, III, IV

Soaking 50kg of red peony root decoction pieces in distilled water, and ultrasonically extracting for 3 times, 1 hour each time. Separating the water extractive solution with macroporous adsorbent resin, eluting with 50% ethanol, and concentrating the eluate under reduced pressure. And continuously separating the 50% ethanol elution part by using an MCI column, sequentially eluting by using 30% ethanol and 50% ethanol, and concentrating the eluent under reduced pressure, wherein the 30% ethanol elution sample is CS-3(2000g), and the 50% ethanol elution sample is CS-4(316 g). Concentrating the residue with 95% ethanol extractive solution until there is no ethanol, extracting with ethyl acetate of equal volume for 5 times, and concentrating the extractive solution under reduced pressure to obtain ethyl acetate part CS-1(580g) and water phase part CS-2(270g), as shown in figure 1.

Previous activity screening finds that only CS-4 in the red peony root part has an antiallergic effect. Therefore, the red peony root part CS-4 is further extracted: further extracting the red peony root after dissolving the red peony root part in CS-4 methanol: after dissolving the CS-4 methanol in the red peony root part, separating the dissolved CS-4 methanol by a SephadexLH-20 column, and respectively eluting the 50 percent methanol and the methanol to obtain components CS-4-1 to CS-4-8. CS-4-4(12.9g) was isolated by reverse phase medium pressure chromatography under the conditions: 20% methanol-water (40min), 20% -100% methanol-water (230min), 100% methanol (60min) to obtain components A1, A2, wherein component A2(8.3g) is separated by silica gel column chromatography (dichloromethane-methanol 50:1-10:1) to obtain components B1-B9. Separating B3 by silica gel column chromatography (petroleum ether-acetone 10:1-2:1) to obtain D1-D11, separating D7 by preparing thin layer chromatography plate (dichloromethane-acetone-methanol 15:1:1) to obtain D7-1-D7-4, separating D7-3 by preparing liquid chromatography (RpC18, 31% acetonitrile-water, 230nm) to obtain D7-3-1-D7-3-3; d7-3-2 was separated by preparative liquid chromatography (RpC18, 20% acetonitrile-water, 230nm) to give compound III (8 mg); d7-3-3 was then separated by preparative liquid chromatography (RpC18, 22% acetonitrile-water, 230nm) to give compound I (11 mg). Separating A1 with SephadexLH-20 (dichloromethane-methanol 1:1) to obtain A1-1 and A1-2; separating the A1-1 by silica gel column chromatography (dichloromethane-methanol 100:1-1:1) to obtain H1-H7; h5 is separated by a preparative thin layer chromatography plate (dichloromethane-methanol 8:1) to obtain H5-1-H5-4, and H5-1 is separated by preparative liquid chromatography (RpC18, 14% acetonitrile-water, 230nm) to obtain a compound II (9 mg); h6 was separated by preparative TLC plate (dichloromethane-methanol 15:1) to give H6-1-H6-4, and H6-2 was separated by preparative liquid chromatography (RpC18, 20% acetonitrile-water, 230nm) to give compound IV (5 mg). The structure of the lignan compound is analyzed and identified by means of spectroscopy such as UV, IR, NMR, MS and the like, and the lignan compound is obtained.

The spectral information of the above compounds is as follows:

i: a white powder; ESI-MS M/z 385[ M + Na ]]⁺,401[M+K]⁺,361[M-H]^-,397[M+Cl]^-；¹H NMR(CD3OD,600MHz)δ:6.60(2H,d,J＝1.9Hz,H-2,2′),6.67(2H,d,J＝7.9Hz,H-5,5′),6.56(2H,dd,J＝7.9,1.9Hz,H-6,6′),2.57(2H,dd,J＝13.8,7.8Hz,H-7a,7′a),2.67(2H,dd,J＝13.8,6.9Hz,H-7b,7′b),1.92(2H,m,H-8,8′),3.60(4H,qd,J＝11.1,5.0Hz,H-9,9′),3.75(6H,s,3,3′-OMe)；¹³C NMR(CD₃OD,150MHz)δ:134.0(C-1,1′),113.5(C-2,2′),149.0(C-3,3′),145.6(C-4,4′),115.9(C-5,5′),122.9(C-6,6′),36.2(C-7,7′),44.3(C-8,8′),62.3(C-9,9′),56.3(3,3′-OMe)。

II: a light yellow powder; [ alpha ] to]²⁰ _D+44.7(c 0.15,MeOH)；ESI-MS m/z 399[M+Na]⁺,411[M+Cl]^-；¹H NMR(CD₃OD,500MHz)δ:7.00(2H,d,J＝1.9Hz,H-2,2′),6.75(2H,d,J＝8.1Hz,H-5,5′),6.84(2H,dd,J＝8.1,1.9Hz,H-6,6′),4.90(2H,d,J＝8.5Hz,H-7,7′),2.28(2H,ddd,J＝8.5,5.0,3.1Hz,H-8,8′),3.66(2H,dd,J＝11.4,3.1Hz,H-9a,9′a),3.57(2H,dd,J＝11.4,5.0Hz,H-9b,9′b),3.85(6H,s,3,3′-OMe)；¹³C NMR(CD₃OD,125MHz)δ:134.9(C-1,1′),111.2(C-2,2′),149.1(C-3,3′),147.3(C-4,4′),116.0(C-5,5′),120.5(C-6,6′),84.4(C-7,7′),55.3(C-8,8′),61.7(C-9,9′),56.4(3,3′-OMe)。

III: a light brown oily liquid; ESI-MS M/z 383[ M + Na ]]⁺,399[M+K]⁺,395[M+Cl]^-；¹H NMR(CD3OD,500MHz)δ:6.91(1H,d,J＝1.9Hz,H-2),6.73(1H,d,J＝8.1Hz,H-5),6.79(1H,dd,J＝8.1,1.9Hz,H-6),5.46(1H,d,J＝6.2Hz,H-7),3.43(1H,m,H-8),3.72(1H,dd,J＝11.0,7.2Hz,H-9a),3.80(1H,dd,J＝11.0,5.5Hz,H-9b),6.69(1H,s,H-2′),6.69(1H,s,H-6′),2.59(2H,t,J＝6.6Hz,H-7′),1.78(2H,dt,J＝6.6,6.6Hz,H-8′),3.53(2H,t,J＝6.6Hz,H-9′),3.78(3H,s,3-OMe),3.82(3H,s,3′-OMe)；¹³C NMR(CD₃OD,125MHz)δ:136.9(C-1),114.0(C-2),147.5(C-3),147.4(C-4),116.1(C-5),117.9(C-6),89.0(C-7),55.5(C-8),64.9(C-9),129.8(C-1′),110.4(C-2′),145.2(C-3′),149.0(C-4′),134.8(C-5′),119.7(C-6′),35.8(C-7′),32.9(C-8′),62.2(C-9′),56.3(3-OMe),56.6(3′-OMe)。

IV: a white powder; ESI-MS M/z 401[ M + Na ]]⁺,417[M+K]⁺,413[M+Cl]^-；¹H NMR(CD₃OD,500MHz)δ:6.99(1H,d,J＝1.7Hz,H-2),6.94(1H,d,J＝8.2Hz,H-5),6.82(1H,dd,J＝8.2,1.7Hz,H-6),4.84(1H,d,J＝6.0Hz,H-7),4.16(1H,ddd,J＝6.0,5.2,4.0Hz,H-8),3.41(1H,dd,J＝11.9,5.2Hz,H-9a),3.67(1H,dd,J＝11.9,4.0Hz,H-9b),6.82(1H,d,J＝2.0Hz,H-2′),6.72(1H,d,J＝8.1Hz,H-5′),6.68(1H,dd,J＝8.1,2.0Hz,H-6′),2.59(2H,t,J＝7.5Hz,H-7′),1.78(2H,m,H-8′),3.52(2H,t,J＝6.5Hz,H-9′),3.79(3H,s,3-OMe),3.82(3H,s,3′-OMe)；¹³C NMR(CD₃OD,125MHz)δ:138.3(C-1),111.8(C-2),149.0(C-3),147.3(C-4),116.0(C-5),120.9(C-6),74.3(C-7),87.9(C-8),62.0(C-9),133.9(C-1′),114.0(C-2′),151.8(C-3′),147.7(C-4′),119.7(C-5′),122.2(C-6′),32.9(C-7′),35.7(C-8′),62.3(C-9′),56.4(3-OMe),56.6(3′-OMe)。

Pharmacological activity experiment of radix Paeoniae Rubra

Experimental example 1

Exploration of cytotoxicity of red peony root site

RBL-2H3 cells in logarithmic growth phase were removed from the incubator and digested to prepare a cell suspension. Cell density was calculated using a hand-held cell counter (model: scepter). Cell number was adjusted to 1X 10 with fresh complete medium⁵One per ml, inoculated in 96-well plates at 200. mu.L per well,uniformly mixing cell suspension once every three inoculated holes, culturing for 24h, discarding supernatant, adding the to-be-screened drugs with different concentrations (0.2 mu g/ml, T2 2 mu g/ml and T3 20 mu g/ml) prepared by fresh culture medium respectively, arranging 3 duplicate holes in each group, arranging a normal group (cell blank hole without drugs) and a zero-adjusting hole (cell blank hole without inoculation), culturing for 24h, discarding supernatant, adding 200 mu L of MTT solution prepared without serum (serum-free culture medium: 5 mg/ml: MTT 1:10), incubating for 4h, centrifuging for 400g/5min, discarding supernatant, adding 150 mu L DMSO, fully shaking to dissolve crystals, and determining the OD value of each hole at 570 nm.

Cell viability (%) - (drug group OD value-zero group OD value)/(normal group OD value-zero group OD value) × 100%

The results are shown in Table 1. As shown in Table 1, after each drug acts on RBL-2H3 cells, the cell survival rate is higher than 95%, so that the cytotoxicity of the red peony root part is low, and the red peony root part is not cytotoxic when the administration dose is 20 mu g/ml. The results show that the red peony root has low cytotoxicity and high safety. 0.2-20 mug/ml can be selected as the dosage selection range for administration, and the influence of the part on the HIS and beta-HEX release rate of antigen-induced RBL-2H3 cell activation degranulation is further discussed.

TABLE 1 cytotoxic effect of radix Paeoniae Rubra fraction on RBL-2H3 cells (mean. + -. standard deviation, n ═ 6)

Experimental example 2

Research on influence of red peony root on RBL-2H3 cell degranulation caused by anaphylactic reaction

Digesting the cells in logarithmic growth phase, and adjusting cell density to 1 × 10⁵One per ml. 200 u L/hole into 96 hole plate, set zero hole, blank control hole, total enzyme hole and each dosing hole. The administration wells were divided into model control group, T1, T2 and T3 groups, wherein the final concentration of T1 was 0.2. mu.g/ml, T2 was 2. mu.g/ml and T3 was 20. mu.g/ml. Incubating overnight, adding complete culture medium into zero, normal and total enzyme wells, culturing, and adding final concentration into model group200 mu L of anti-DNP-IgE prepared by a complete culture medium with the concentration of 750ng/mL, 200 mu L of each concentration of medicine and 200 mu L of anti-DNP-IgE prepared by a final concentration of 750ng/mL are respectively added into dosing holes, incubation is carried out for 24h, centrifugation is carried out, improved desktop liquid is added to wash the mixture until no residual culture medium exists, 200 mu L of blank improved desktop liquid is added into zero-adjusting holes and blank control holes, 200 mu L of TritonX-100 lysate is added into total enzyme holes, DNP-BSA prepared by the improved desktop liquid is added into the dosing group and the model control group, the supernatant is obtained by centrifugation at 3000r/5min after the mixture is cultured for 2h, the release amount of histamine and beta-aminoglycoside is measured, and the cell morphology is observed by a microscope. The histamine release amount determination method is as follows: taking 100 mu L of cell supernatant, adding 20 mu L of histamine substrate, adding 20 mu L of NaOH, incubating for 15min at 37 ℃, adding 3% HCL solution of stop solution to stop the reaction, stabilizing for 15min, and measuring fluorescence values of each group at an excitation wavelength of 355nm and an emission wavelength of 460 nm. Based on the fluorescence values measured for each group, the histamine release rate was calculated according to the following formula:

histamine release rate (%) - (sample supernatant fluorescence value-fluorescence value of supernatant in zero-adjusted group)/(total enzyme well fluorescence value-fluorescence value of supernatant in zero-adjusted group) × 100%

The method for measuring the release amount of the beta-aminoglycoside comprises the following steps: taking 50 mu L of cell supernatant, adding beta-aminoglycoside substrate, incubating at 37 ℃ for 45min, adding NaHCO3/Na2CO 3200 mu L of termination solution to terminate the reaction, and measuring the absorbance of each well at 405 nm. According to the OD values measured in each group, the release rate of beta-aminoglycoside is calculated by the following formula

Beta-aminoglycoside release rate (%) - (sample supernatant value-zeroed value)/(total enzyme pore value-zeroed value) × 100%

As shown in Table 2, the results of measuring the release rate of histamine and beta-aminoglycoside show that the content of histamine and beta-aminoglycoside in the cell culture supernatant can be remarkably improved by 750ng/mL anti-DNP-IgE stimulation and 1 mu g/mL DNP-BAS stimulation (P is less than 0.001, and P is less than 0.001), while the red peony root compound can inhibit the release of histamine and beta-aminoglycoside at 20 mu g/mL (P is less than 0.001, and P is less than 0.001).

TABLE 2 Effect of red peony site on cell degranulation by allergic reaction (0.2. mu.g/mL, 2. mu.g/mL, 20. mu.g/mL, mean. + -. standard deviation, n ═ 6)

^###P<0.001vs blank control group, P<0.05，**P<0.01，***P<0.001vs model control group

Experimental example 3

Effect of red peony site on cell degranulation by anaphylactoid reaction (0.2. mu.g/mL, 2. mu.g/mL, 20. mu.g/mL, mean. + -. standard deviation, n ═ 6)

A zero-setting hole, a blank control hole, a total enzyme hole and each administration hole are arranged in a 96-well plate. The administration wells were divided into model control group, T₁、T₂And T₃Group of which T₁The final concentration of the drug is 0.2 mu g/mL, T₂Is 2. mu.g/mL, T₃20. mu.g/mL. Model control group C48/80 solution prepared from modified benchtop solution was added to a final concentration of 15. mu.g/ml. Adding medicines with various concentrations prepared by the improved desktop liquid and 200 microliter of C48/80 with the final concentration of 15 microgram/ml into the administration holes respectively, adding 200 microliter of blank improved desktop liquid into the zero-setting holes and the blank control holes, adding 200 microliter of 1% TritonX-100 lysate into the total enzyme holes, incubating for 1h, centrifuging at 3000r/5min, taking the supernatant, and determining the release amount of histamine and beta-glucosaminidase. The measurement method is the same as above.

As shown in Table 3, the results of measuring the release rates of histamine and beta-aminoglycoside show that C48/80 with the concentration of 15 μ g/mL can significantly increase the content of histamine and beta-aminoglycoside in cell culture supernatant (P <0.001), and the red peony root compound can selectively inhibit the release of histamine or beta-aminoglycoside within the dosage range of 2-20 μ g/mL. Wherein the ratio of histamine: at an administration dose of 20 μ g/mL, the release amount may be significantly reduced (P < 0.001). For beta-aminoglycosides: the release amount can be obviously reduced under the administration dosage of 2 mu g/mL (P < 0.001).

TABLE 3 Effect of red peony site on cell degranulation by anaphylactoid reaction (0.2. mu.g/mL, 2. mu.g/mL, 20. mu.g/mL, mean. + -. standard deviation, n ═ 6)

^###P<0.001vs blank control group, P<0.05，**P<0.01，***P<0.001vs model control group from the results of Experimental example 2 and Experimental example 3, it was found that CS-1, CS-2, and CS-3 had no antiallergic or antiallergic effect at each dose. Under the administration dosage of 20 mu g/mL, only the CS-4 at the red paeony root part can remarkably inhibit the release of histamine and beta-aminoglycoside (P) in cell degranulation caused by anaphylactoid reaction and anaphylactoid reaction<0.001). Thus, the CS-4 site was further extracted and isolated.

Pharmacological activity experiment of red peony root monomer compound

The research at home and abroad finds that the common allergens in the nature, such as pollen, dust, catkin, animal fur, oil smoke, food, medicines and the like, appear in each link of life, and the allergens can cause the organism to generate allergy or anaphylactoid reaction. Pharmacological experiments show that the compounds I, II, III and IV have obvious effects of inhibiting the release of histamine and beta-hexosaminidase and can be used for preparing medicaments for preventing or treating allergic diseases.

Experimental example 4

Exploration of cytotoxicity of red peony compounds

RBL-2H3 cells in logarithmic growth phase were removed from the incubator and digested to prepare a cell suspension. Cell density was calculated using a hand-held cell counter (model: scepter). Cell number was adjusted to 1X 10 with fresh complete medium⁵And inoculating the mixed solution into a 96-well plate, inoculating 200 mu L of the mixed solution into each well, uniformly mixing cell suspension once per three inoculated wells, culturing for 24h, removing supernatant, adding medicaments to be screened with different concentrations (0.08, 0.2, 4, 10 and 50 mu M) prepared by a fresh culture medium into each group of 3 duplicate wells, additionally arranging a normal group (cell blank wells without medicaments) and a zero-adjusting well (cell blank wells without inoculation), culturing for 24h, removing supernatant, adding 200 mu L of serum-free MTT solution (serum-free culture medium: 5 mg/ml: MTT 1:10), incubating for 4h, centrifuging for 400g/5min, removing supernatant, adding 150 mu L DMSO, fully shaking to dissolve crystals, and determining the OD value of each well at 570 nm.

The results are shown in Table 1. As shown in Table 1, after the medicines act on RBL-2H3 cells, the cell survival rate is higher than 80%, so that the red paeony root compound has low cytotoxicity and high safety. 0.08-50 mu M can be selected as the dosage selection range of administration, and the influence of each medicament on the release rate of the antigen-induced RBL-2H3 cell activated degranulation His and beta-HEX is further discussed.

Table 4 cytotoxic effect of red peony compounds on RBL-2H3 cells (mean ± sd, n ═ 6)

Experimental example 5

The influence of the red paeony root compound on RBL-2H3 cell degranulation caused by anaphylactic reaction is explored

Digesting the cells in logarithmic growth phase, and adjusting cell density to 1 × 10⁵One per ml. 200 u L/hole into 96 hole plate, set zero hole, blank control hole, total enzyme hole and each dosing hole. The administration wells were divided into model control group, T₁、T₂And T₃Group of which T₁The final concentration of administration was 50. mu.M, T₂Is 25 μ M, T₃At 5. mu.M. Incubating overnight, adding complete culture medium into zero-adjustment holes, normal holes and total enzyme holes for normal culture, adding 200 muL of anti-DNP-IgE prepared by the complete culture medium with the final concentration of 750ng/mL into a model group, adding 200 muL of medicaments with each concentration and the anti-DNP-IgE prepared by the complete culture medium with the final concentration of 750ng/mL into an administration hole respectively, incubating for 24h, centrifuging, adding improved bench type liquid for cleaning until no residual culture medium exists, adding 200 muL of blank improved bench type liquid into the zero-adjustment holes and blank control holes, adding 200 muL of 1% TritonX-100 lysate into the total enzyme holes, adding DNP-BSA prepared by the improved bench type liquid into the administration group and the model control group, culturing for 2h, centrifuging at 3000r/5min to obtain supernatant, measuring the release amount of histamine and beta-hexosaminidase, and observing cell morphology under microscope. The histamine release amount determination method is as follows: taking 100 mu L of cell supernatant, addingAdding histamine substrate 20 μ L, adding NaOH 20 μ L, incubating at 37 deg.C for 15min, adding 3% HCl solution as stop solution to stop reaction, stabilizing for 15min, and measuring fluorescence value of each group at excitation wavelength of 355nm and emission wavelength of 460 nm. Based on the fluorescence values measured for each group, the histamine release rate was calculated according to the following formula:

The method for measuring the release amount of the beta-hexosaminidase comprises the following steps: collecting cell supernatant 50 μ L, adding beta-hexosaminidase substrate, incubating at 37 deg.C for 45min, and adding termination solution NaHCO₃/Na₂CO₃The reaction was stopped at 200. mu.L, and the absorbance at 405nm was measured for each well. According to the OD values measured in each group, the release rate of beta-hexosaminidase was calculated by the following formula

Beta-hexosaminidase release rate (%) (sample supernatant value-zero)/(total enzyme pore value-zero) x 100%

Microscopic observation results show that the RBL-2H3 cells in the normal group are in a long fusiform shape, complete in edge and compact in structure. The cell volume of the model group is increased, the edge is not integral, a large number of vacuoles or particle-like structures appear, most cell membranes are broken, and particle-like substances are exuded. The cell state of the red paeony root compound is obviously improved, and the vacuole-like structure is obviously reduced, so that the red paeony root compound can effectively protect the intact cell form and inhibit the exudation of the granular-like substances.

As shown in Table 5, the results of measuring the release rate of histamine and beta-hexosaminidase show that the stimulation of anti-DNP-IgE by 750ng/ml and the excitation of DNP-BAS by 1 mu g/ml can significantly improve the content of histamine and beta-hexosaminidase in cell culture supernatant (P <0.001 and P <0.001), while the red peony root compound can selectively inhibit the release of histamine or beta-hexosaminidase within the dosage range of 5-50 mu M. Wherein the ratio of histamine: the release amount can be obviously reduced under the administration dosage of 25 mu M (P <0.001, P <0.01, P <0.001, P < 0.01). For beta-hexosaminidase: the release amount can be remarkably reduced under the administration dosage of 25 mu M (P <0.001, P <0.05, P <0.05, P < 0.01). The compound I can still remarkably reduce the content of beta-hexosaminidase in cell supernatant under the dosage of 5 mu M (P < 0.05).

Table 5 effect of red peony compounds on cell degranulation by allergic reaction (5 μ M, 25 μ M, 50 μ M, mean ± standard deviation, n ═ 6)

^###P<0.001vs blank control group P<0.05，**P<0.01，***P<0.001vs model control group

Experimental example 6

Effect of red peony compounds on cell degranulation by anaphylactoid reaction (50 μ M, 25 μ M, 5 μ M, mean ± standard deviation, n ═ 6)

A zero-setting hole, a blank control hole, a total enzyme hole and each administration hole are arranged in a 96-well plate. The administration wells were divided into model control group, T₁、T₂And T₃Group of which T₁The final concentration of administration was 50. mu.M, T₂Is 25 μ M, T₃At 5. mu.M. Model control group C48/80 solution prepared from modified benchtop solution was added to a final concentration of 15. mu.g/ml. Adding medicines with various concentrations prepared by the improved desktop liquid and 200 mul of C48/80 with the final concentration of 15 mug/ml into the administration holes respectively, adding 200 mul of blank improved desktop liquid into the zero adjustment holes and the blank control holes, adding 200 mul of 1% TritonX-100 lysate into the total enzyme holes, incubating for 1h, centrifuging at 3000r/5min, taking the supernatant, and determining the release amount of histamine and beta-hexosaminidase. The measurement method is the same as above.

As shown in Table 6, the results of measuring the release rates of histamine and beta-hexosaminidase show that C48/80 with a concentration of 15 μ g/ml can significantly increase the content of histamine and beta-hexosaminidase in the cell culture supernatant (P <0.001), and the red peony root compound can selectively inhibit the release of histamine or beta-hexosaminidase within the dosage range of 5-50 μ M. Wherein the ratio of histamine: the release amount can be remarkably reduced under the administration dosage of 50 mu M (P < 0.001). Wherein the compounds I and III can still significantly reduce the histamine content in cell supernatant (P <0.001, P <0.01)) under the dosage of 5 mu M. For beta-hexosaminidase: at 5 μ M dose, compounds I and III still significantly reduced their release (P < 0.05).

Table 6 effect of red peony compounds on cell degranulation by anaphylactoid reaction (5 μ M, 25 μ M, 50 μ M, mean ± standard deviation, n ═ 6)

From the results of experimental examples 2 and 3, it is known that the compounds I, II, III and IV can significantly inhibit the release of histamine and beta-hexosaminidase in the cell degranulation caused by anaphylactoid reaction and anaphylactoid reaction (P <0.001) at the administration dosage of 50 μ M. At the administration dose of 25 mu M, the compounds I and II still show strong antiallergic effect. At 5 μ M dosing, compounds I and III still selectively inhibited the release of histamine and β -hexosaminidase.

Experimental example 7

Effect of Red peony root Compounds on TNF-alpha and IL-4 in cell supernatant caused by anaphylaxis

Digesting the cells in logarithmic growth phase, and adjusting cell density to 1 × 10⁵One per ml. 500 μ L/well was added to a 24-well plate, and a zero well, a blank control well, and each administration well were set. The administration wells were divided into model control group, T₁、T₂And T₃Group of which T₁The final concentration of administration was 50. mu.M, T₂Is 25 μ M, T₃At 5. mu.M. Incubating overnight, adding complete culture medium into zero, normal and total enzyme wells, culturing normally, adding 200 μ L of anti-DNP-IgE prepared from complete culture medium with final concentration of 750ng/ml into model group, adding 200 μ L of drugs with different concentrations and anti-DNP-IgE with final concentration of 750ng/ml into administration wells, incubating for 24h, centrifuging, adding modified enzymeCleaning the Liangtai liquid until no residual culture medium exists, adding 200 mu L of blank improved desktop liquid into zero adjusting holes and blank control holes, adding DNP-BSA (deoxyribose nucleic acid) -with the final concentration of 1 mu g/mL prepared by the improved desktop liquid into an administration group and a model control group, culturing for 2h, centrifuging at 3000r/5min, taking supernatant, and detecting the contents of inflammatory factors TNF-alpha and IL-4 in the supernatant according to the method of a kit specification of rats TNF-alpha and IL-4 of Jiangsu Jingmei biological science and technology Limited.

As shown in Table 7, the test results of inflammatory mediators TNF-alpha and IL-4 release show that 750ng/ml anti-DNP-IgE stimulation and 1 mu g/ml DNP-BAS stimulation can obviously improve the content of TNF-alpha and IL-4 in cell culture supernatant (P <0.001 and P <0.001), and the red peony root compound can obviously inhibit the release of TNF-alpha and IL-4 (P <0.001) under the effect of 50 mu M dose. Wherein TNF- α: the content of the compound I in the supernatant can be still obviously inhibited under the action of 5 mu M dose (P < 0.05). IL-4: under the action of 25 mu M dosage, the compounds I, II, III and IV can obviously inhibit the content of IL-4 in supernatant (P <0.001, P <0.01, P <0.001 and P < 0.05). The compound I can still obviously inhibit the content of IL-4 in the supernatant under the action of 5 mu M dose (P < 0.05).

TABLE 7 Effect of red peony compounds on TNF-. alpha.and IL-4 in cell supernatants induced by allergic reactions (5. mu.M, 25. mu.M, 50. mu.M, mean. + -. standard deviation, n ═ 6)

Experimental example 8

Effect of Red peony root Compounds on TNF-alpha and IL-4 in cell supernatant resulting from anaphylactoid reaction

Digesting the cells in logarithmic growth phase, and adjusting cell density to 1 × 10⁵One per ml. 500 μ L/well plusPut into a 24-well plate, and set with a zero-setting well, a blank control well, and each administration well. The administration wells were divided into model control group, T₁、T₂And T₃Group of which T₁The final concentration of administration was 50. mu.M, T₂Is 25 μ M, T₃At 5. mu.M. Model control group C48/80 solution prepared from modified benchtop solution was added to a final concentration of 15. mu.g/ml. The medicines with various concentrations prepared by the improved desktop liquid and the C48/80 with the final concentration of 15 mug/ml are respectively added into the other administration holes for 200 mug, and the blank improved desktop liquid is added into the zero setting hole and the blank control hole for 200 mug. And centrifuging at 3000r/5min after incubation for 1h, taking the supernatant, and detecting the contents of inflammatory factors TNF-alpha and IL-4 in the supernatant according to the method of a kit specification of rats TNF-alpha and IL-4 of Jiangsu Jingmei Biotechnology limited company.

As shown in Table 8, the test results of the release of inflammatory mediators TNF-alpha and IL-4 show that C48/80 with the concentration of 15 mu g/ml can remarkably improve the content of TNF-alpha and IL-4 in cell culture supernatant (P <0.001), and the red paeony root compound can remarkably inhibit the release of TNF-alpha and IL-4 (P <0.001) under the effect of 50 mu M dose. Wherein TNF- α: the content of the compound I in the supernatant can be still obviously inhibited under the action of 5 mu M dose (P < 0.05). IL-4: under the action of 5 mu M dose, the compounds II and III can obviously inhibit the content of IL-4 in cell supernatant (P <0.01 and P < 0.05).

TABLE 8 Effect of red peony compounds on TNF-. alpha.and IL-4 in cell supernatants due to anaphylactoid reaction (5. mu.M, 25. mu.M, 50. mu.M, mean. + -. standard deviation, n ═ 6)

According to the experimental examples 7 and 8, the red paeony root compound can selectively inhibit the contents of TNF-alpha and IL-4 in RBL-2H3 cell supernatant, and has good anti-inflammatory effect. From the experimental examples 5 and 6, it is known that the red peony root compound can significantly inhibit RBL-2H3 cell degranulation caused by allergy and anaphylactoid reaction, reduce the release of cell histamine and beta-hexosaminidase, and has good antiallergic and antiallergic effects. As is clear from example 4 and Experimental example 1, the above-mentioned radix Paeoniae Rubra fraction and radix Paeoniae Rubra compound are highly safe. From the results of experimental examples 2 and 3, it is known that the red peony site can significantly inhibit the release of histamine and β -aminoglycoside in cell degranulation caused by allergic reaction and anaphylactoid reaction at the administration dose of 20 μ g/mL (P < 0.001). The concentration of the red peony monomeric compound which generally generates significant difference is 25 mu M/L, the relative molecular masses of the four red peony compounds are 362, 376, 360 and 378 respectively, the highest concentrations of the compound action after converting the unit are 9.05 mu g/mL, 9.40 mu g/mL, 9.00 mu g/mL and 9.45 mu g/mL respectively, and the concentrations are less than the dosage of the administration part of 20 mu g/mL, therefore, the antiallergic and anaphylactoid effects of the red peony monomeric compound are stronger than those of the red peony part (CS-4)

Therefore, the experimental results show that the lignin red paeony root compound has higher safety, good anti-allergic, anti-anaphylactoid and anti-inflammatory activities, and the red paeony root monomer has stronger activity with the part.

Note: the anaphylaxis is divided into two stages, namely a stimulation stage and an excitation stage, in the experiment, the anaphylaxis positive medicine is divided into an excitant and an excitation stage, wherein the excitant is anti-DNP-IgE, and the Chinese name is anti-dinitrophenol IgE monoclonal antibody. The exciting agent is DNP-BSA, and the name of the exciting agent is dinitrophenol-bovine serum albumin; the anaphylactoid reaction positive medicine is C48/80, and belongs to mast cell activator.

Claims

1. Application of lignin compounds and pharmaceutically acceptable salts thereof in preparing medicaments for preventing, relieving and/or treating allergic diseases is characterized in that the compounds are selected from the following groups,

2. the use of claim 1, wherein the pharmaceutically acceptable salt of the compound is selected from the group consisting of salts of the compound with inorganic or organic acids.

3. The application of a pharmaceutical composition in preparing a medicament for preventing, relieving and/or treating allergic diseases is characterized in that the pharmaceutical composition contains a therapeutically effective amount of the following compounds and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier,

4. use according to claim 3, characterized in that the pharmaceutical composition is in a dosage form selected from the group consisting of tablets, capsules, pills, granules, oral liquids or tear suspensions, eye drops, inhalants.