CN114377029B - Cage-like monoterpene glycoside compounds derived from red paeony root, and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medicines, relates to application of caged monoterpene glycoside compounds in resisting allergic diseases, and in particular relates to application of caged monoterpene glycoside compounds in red paeony root in preparing medicines for preventing and treating allergic diseases. Pharmacological experiments prove that the compound can effectively inhibit the release of hexosaminidase (beta-HEX) and Histamine (HIS) in sensitized RBL-2H3 cells, and the antiallergic effect of the compounds I-VI is stronger than that of paeoniflorin. In addition, representative compound II has been shown to reduce HIS release by binding to HDC and inhibit inflammatory factor release by blocking ERK1/2, JNK and p38 in MAPK signaling pathway, and is also important for repairing cytoskeletal and mitochondrial membrane damage caused by allergic reactions. In addition, in vivo experiments also prove the protective effect of the compound II on an allergic disease animal model. Therefore, caged monoterpenes from red peony can be considered as a promising dual HDC and MAPK signaling pathway inhibitor of natural origin.

Description

Cage-like monoterpene glycoside compounds derived from red paeony root, and preparation method and application thereof

Technical Field

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

Background

Allergic diseases are chronic diseases, which seriously affect the working efficiency of patients, and reduce the productivity of patients, thereby leading to the improvement of social cost. Thus, effective and safe treatment of allergic diseases is one of the major challenges facing public health. Allergic reactions are the most urgent and potentially serious manifestations of allergic diseases, and refer to a physiological change mainly caused by physiological dysfunction or tissue cell injury of an organism when the organism receives the same antigen stimulus again after the organism responds to an antigen substance for the first time. According to the report of the world allergic reaction organization, people with allergic symptoms worldwide account for 30% -40% of the general population; the number of patients is large, and the data are often underestimated because symptoms seriously damaging life health do not appear in early and middle stages of anaphylaxis. Most of the antiallergic drugs currently on the market have serious adverse effects such as arrhythmia, mental dysfunction, gastrointestinal disorder and infection. It is therefore imperative to search for new and effective therapeutic regimens.

A great deal of research shows that anaphylactic reaction can cause the organism to release beta-hexosaminidase and histamine, cause smooth muscle contraction, increase vascular permeability and the like, thereby inducing the organism to generate a series of anaphylactic symptoms. RBL-2H 3 cells contain abundant basophilic particles in cytoplasm, and can cause degranulation phenomenon when cells are subjected to anaphylaxis, namely, the alkaline particles coated with histamine and beta-hexosaminidase gradually approach to cell membranes, vesicle membranes are fused with the cell membranes, and the contents are released outside the cells to induce the generation of corresponding anaphylactic symptoms. In view of this, many scholars consider: inhibiting the release of allergic mediators can be a viable direction for the development of new antiallergic agents.

The major pathological changes of anaphylaxis are smooth muscle contraction, telangiectasia, increased vascular permeability and increased gland secretion, so that elimination and alleviation of the symptoms become necessary links for antiallergic. The above-mentioned series of symptoms are caused by a series of inflammatory mediators released by the body, wherein the mediators mainly play a role include HIS and beta-HEX, and thus, inhibition of the release of the two mediators becomes a key for the treatment of allergic diseases. The natural product has the advantages of small toxic and side effects, lasting curative effect, good overall regulation and synergistic effect and the like, and becomes a research hot spot of new medicines for treating allergic diseases in recent years. Although the efficacy evaluation and mechanism discussion of the natural products are still based on laboratory research stages, clinical research has not provided definitive conclusions, a great amount of data has laid a certain theoretical foundation, and a foundation is provided for further optimizing new anti-allergic disease drugs.

Radix Paeoniae Rubra (Paeoniae Rubra Radix) is dried root of radix Paeoniae Rubra (Paeonia lactiflora Pall) of Ranunculaceae. Enter liver meridian. The Chinese medicine is usually combined for treating symptoms such as toxic heat, spot, conjunctival congestion, swelling and pain, liver Yu Xie pain, amenorrhea, dysmenorrhea, traumatic injury, carbuncle, swelling and sore. Modern pharmacology finds that the radix paeoniae rubra has the effects of resisting bacteria, resisting inflammation and relieving pain. The red paeony root has complex chemical components, mainly monoterpenes, fatty acids, phenols, lignans and other compounds.

The applicant researches find that a cage-shaped monoterpene glycoside compound I, II, III, IV, V, VI is separated from red paeony root, has the chemical structure shown as follows, pharmacological experiments prove that the compound can effectively reduce the release amount of allergen-induced RBL-2H3 cell histamine and beta-aminohexosidase, has good effect of inhibiting cell degranulation, thus showing good antiallergic potential, the effective dose can reach 5 mu mol/L, and the toxicity evaluation proves that the safety of the compound I, II, III, IV, V, VI is superior to that of paeoniflorin, and the pharmacological activity is stronger than that of paeoniflorin, especially the compound II. At present, research reports on the application of the radix paeoniae rubra extract in treating allergic diseases are not seen, and patent documents of a preparation method of the radix paeoniae rubra compound monomer for inhibiting RBL-2H3 degranulation are not seen.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of cage-shaped monoterpene glycoside compounds and pharmaceutically acceptable salts thereof in preparing medicines for preventing, relieving and/or treating allergic diseases,

in order to solve the technical problems, the invention provides the following technical scheme:

the first aspect of the technical scheme of the invention provides application of a compound I, II, III, IV, V, VI derived from red paeony root and pharmaceutically acceptable salts thereof in preparing medicaments for preventing, relieving and/or treating allergic diseases, and the structure of the compound I, II, III, IV, V, VI is as follows:

the pharmaceutically acceptable salts of the compounds are selected from salts of the compounds with inorganic or organic acids.

The allergic diseases comprise allergic conjunctivitis, allergic rhinitis and allergic asthma.

The results of in vitro antiallergic pharmacological tests show that the compound can effectively inhibit the release of RBL-2H3 cell histamine and beta-aminohexosidase, and when the administration concentration reaches 50 mu M, the survival rate of RBL-2H3 cells is 99.95%, 92.33%, 81.69%, 89.48%, 95.88% and 91.25% respectively. For allergic reactions, compounds I-VI may change the histamine release rate from 55.51% to 26.70%, 14.85%, 20.21%, 30.53%, 28.09%, 29.45%; compounds I-VI can change the release rate of beta-hexosaminidase from 43.73% to 22.51%, 14.29%, 17.76%, 23.88%, 23.43%, 25.19%. Therefore, the medicine can achieve the aim of well inhibiting anaphylactic reaction by selectively inhibiting the release of histamine and beta-hexosaminidase.

According to a second aspect of the present invention, there is provided a method for preparing the above red peony root compound, comprising the steps of: soaking radix Paeoniae Rubra in distilled water, ultrasonic extracting, concentrating, extracting with organic solvent, macroporous adsorption resin chromatography, gel column chromatography, reversed phase silica gel column chromatography, and preparative HPLC to obtain paeoniflorin and I, II, III, IV, V, VI, and analyzing and identifying its structure by UV, IR, NMR, MS and CD spectroscopy.

In a third aspect of the present invention there is provided the use of a pharmaceutical composition comprising a pharmaceutically effective amount of a compound and a pharmaceutically acceptable carrier in the manufacture of a medicament for the prevention, alleviation and/or treatment of allergic diseases. The compound is a compound I, II, III, IV, V, VI from red paeony root and pharmaceutically acceptable salts thereof. Typically, the pharmaceutical compositions of the present invention contain 0.1% to 96% by weight of the compound of the present invention. The compounds of the invention are generally present in unit dosage forms in amounts of from 0.1 to 100mg, with preferred unit dosage forms containing from 5 to 60mg.

Pharmaceutical compositions of the compounds of the present invention may be prepared according to methods well known in the art. For this purpose, the compounds of the invention may, if desired, be combined with one or more solid or liquid pharmaceutical excipients and/or auxiliaries, in suitable administration forms or dosage forms which can be used as human or veterinary medicine.

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

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

The administration dosage form may be liquid dosage form or solid dosage form. For example, the liquid dosage form may 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 of the invention can be prepared into common preparations, sustained release preparations, controlled release preparations, targeted preparations and various microparticle administration systems.

For example, in order to prepare a unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers 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, etc.; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, dextrose solution, gum arabic slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, and the like; disintegrants such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oils and the like; absorption promoters such as quaternary ammonium salts, sodium lauryl sulfate, and the like; lubricants such as talc, silica, corn starch, stearate, stearic 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 bilayer and multilayer tablets.

For example, in order to make the administration unit into a pill, various carriers well known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, glycerol monostearate, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, and the like; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfonate, methylcellulose, ethylcellulose, etc.

For example, in order to make the administration unit into a capsule, the active ingredient of the compound of the present invention is mixed with the above-mentioned various carriers, and the thus-obtained mixture is placed in a hard gelatin capsule or a soft capsule. The active ingredient of the compound can be prepared into microcapsules, and the microcapsules can be suspended in an aqueous medium to form a suspension, or can be filled into hard capsules or prepared into injection for application.

For example, the compounds of the present invention may be formulated as injectable formulations, such as solutions, suspensions, emulsions, freeze-dried powder injection solutions, 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 may be selected from water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxy isostearyl alcohol, polyoxyethylene sorbitol fatty acid ester. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and further, a conventional cosolvent, a buffer, a pH adjuster, and the like may be added. These adjuvants are commonly used in the art.

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

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

The dosage of the pharmaceutical composition 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, weight, character and individual response of the patient or animal, the route of administration, the number of times of administration, the purpose of treatment, and thus the therapeutic dosage of the present invention may vary widely. Generally, the dosages of pharmaceutical ingredients used in the present invention are well known to those skilled in the art. The amount of the actual drug contained in the final formulation of the compound composition of the present invention may be appropriately adjusted to achieve the therapeutically effective amount thereof, thereby achieving the preventive or therapeutic object of the present invention. Daily suitable dosage range of 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, most preferably 0.06 to 10mg/Kg body weight. The compound of the invention is taken by adult patients at a daily dose of 1-300 mg, preferably 4-150 mg, and can be taken once or 2-3 times; the dosage of the children is 0.01-15 mg, preferably 0.06-5 mg/kg body weight. The above-mentioned dosages may be administered in a single dosage form or in divided dosage forms, for example, two, three or four dosage forms, which are limited by the clinical experience of the administering physician and the administration 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 beneficial technical effects are as follows:

1. the red paeony root compound has obvious antiallergic effect, firstly, can obviously inhibit the release of histamine, and has the efficacy of 5 mu mol/L. Secondly, the release of beta-hexosaminidase can be obviously inhibited, and the drug effect reaches 5 mu mol/L. And the toxicity evaluation proves that the safety of the compound I, II, III, IV, V, VI is better than that of paeoniflorin, and the pharmacological activity is stronger than that of paeoniflorin, in particular to compound II.

2. The red paeony root compound has the potential of further developing medicines for preventing and treating allergic diseases, in particular to a compound II.

Drawings

FIG. 1, preparation flow of radix Paeoniae Rubra parts CS-1, CS-2, CS-3, CS-4.

FIG. 2, effect of Compound II on histidine decarboxylase activity.

FIG. 3 effect of Compound II on degranulation of RBL-2H3 cells by anaphylaxis

FIG. 4 influence of Compound II on the cytoskeleton of RBL-2H3 cells caused by anaphylaxis

FIG. 5, influence of Compound II on the stability of the mitochondrial Membrane of RBL-2H3 cells caused by anaphylaxis

FIG. 6 influence of Compound II on IL-3, IL-4, IL-5, IL-13 in cell supernatants caused by anaphylaxis

FIG. 7 influence of Compound II on expression level of MAPK family proteins in RBL-2H3 cells

FIG. 8 molecular docking mimics the binding of Compound II to HDC in MAPK family proteins

FIG. 9, influence of Compound II on passive skin allergy in BALB/c mice

FIG. 10ELISA method for detecting the effect of Compound II on serum inflammatory factors IL-4, IL1 beta, IFN-gamma of allergic rhinitis disease model

FIG. 11ELISA method for detecting the effect of Compound II on serum inflammatory factors IL-4, IL1 beta, IFN-gamma of allergic conjunctivitis disease model

Detailed Description

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

1. Preparation method of paeoniflorin and compound I, II, III, IV, V, VI of red paeony root monomer compound

Example 1 preparation of Compound I, II, III, IV, V, VI

50kg of red paeony root decoction pieces, soaking in distilled water, and then carrying out ultrasonic extraction for 3 times, each time for 1 hour. Separating the water extract directly with macroporous adsorbent resin, eluting with 50% ethanol, and concentrating the eluate under reduced pressure. Separating the 50% ethanol eluate by MCI column, sequentially eluting with 30% ethanol and 50% ethanol, and concentrating the eluate under reduced pressure, wherein the 30% ethanol eluate is CS-3 (2000 g), and the 50% ethanol eluate is CS-4 (316 g). The residue was concentrated to no alcohol by 95% ethanol extraction, extracted 5 times with equal volume of ethyl acetate, and the extracts concentrated under reduced pressure to give ethyl acetate fraction CS-1 (580 g) and aqueous fraction CS-2 (270 g), as shown in FIG. 1.

We have found that only CS-4 in the above red peony sites has antiallergic effect by prior activity screening. Thus, the red peony site CS-4 was further extracted: dissolving radix Paeoniae Rubra CS-4 in methanol, and further extracting radix Paeoniae Rubra: dissolving the CS-4 methanol at the red peony root part, separating by a SephadexLH-20 column, and eluting with 50% methanol and methanol respectively to obtain components CS-4-1-CS-4-8. CS-4-4 (12.9 g) was isolated by reverse phase medium pressure chromatography, conditions: 20% methanol-water (40 min), 20% -100% methanol-water (230 min) and 100% methanol (60 min) to obtain components A1 and A2, wherein component A2 (8.3 g) is subjected to silica gel column chromatography (dichloromethane-methanol 50:1-10:1), and components B1-B9 are separated. B3 is separated by silica gel column chromatography (petroleum ether-acetone 10:1-2:1) to obtain D1-D11, D9 is separated by medium pressure chromatography (50% methanol-water) to obtain D9-1-D9-5, D9-1 is separated by preparative liquid chromatography (RpC, 20% acetonitrile-water, 230 nm) to obtain I (2 mg) and II (3 mg); d9-2 was separated by preparative liquid chromatography (RpC, 21% acetonitrile-water, 230 nm) to give III (2 mg); d9-3 was separated by preparative liquid chromatography (RpC, 24% acetonitrile-water, 230 nm) to give IV (6 mg), V (5 mg); d9-5 was separated by preparative liquid chromatography (RpC, 24% acetonitrile-water, 230 nm) to give VI (4 mg). The structure of the compound is analyzed and identified by UV, IR, NMR, MS and other spectroscopy means, and the compound is a caged monoterpene glycoside compound.

The above compound spectrum information is as follows:

paeoniflorin: white powder; ESI-MS m/z 481[ M+H ]] ⁺ ,503[M+Na] ⁺ ； ¹ H NMR(CD ₃ OD,600MHz)δ:8.07(2H,d,J＝7.5Hz,H-2″,H-6″),7.64(1H,t,J＝7.5Hz,H-4″),7.51(2H,t,J＝7.5Hz,H-3″,H-5″),4.78(2H,d,J＝5.4Hz,H ₂ -8),5.44(1H,s,H-9),4.55(1H,d,J＝7.6Hz,H-1′),3.87(1H,d,J＝11.8Hz,H-6′a,H-6′b),3.63(1H,dd,J＝11.8,5.8Hz),3.36(1H,m,H-5′),3.25(1H,m,H-3′,H-4′),3.22(1H,m,H-2′),2.60(1H,d,J＝6.5Hz,H-5),2.51(1H,dd,J＝11.0,6.9Hz,H-7a),2.21(1H,d,J＝12.6Hz,H-3a),1.98(1H,d,J＝11.0Hz,H-7b),1.83(1H,d,J＝12.6Hz,H-3b),1.38(3H,s,H ₃ -10)； ¹³ C NMR(CD ₃ OD,150MHz)δ:168.0(C-7″),134.4(C-4″),131.2(C-1″),130.7(C-2″,6″),129.6(C-3″,5″),106.4(C-4),102.3(C-9),100.2(C-1′),89.4(C-1),87.3(C-2),78.1(C-3′),78.0(C-5′),75.0(C-2′),72.3(C-6),71.8(C-4′),62.9(C-6′),61.7(C-8),44.6(C-3),44.0(C-5),23.4(C-7),19.6(C-10)。

I: a pale yellow powder; ESI-MS m/z 509[ M+H ]] ⁺ ,531[M+Na] ⁺ ； ¹ H NMR(CD ₃ OD,600MHz)δ:8.02(2H,dd,J＝8.3,1.2Hz,H-2″,H-6″),7.59(1H,t,J＝7.8Hz,H-4″),7.47(2H,t,J＝7.8Hz,H-3″,H-5″),5.41(1H,s,H-9),4.72(2H,s,H ₂ -8),4.50(1H,d,J＝7.7Hz,H-1′),3.82(1H,d,J＝12.4Hz,H-6′a),3.67(1H,m,H-3′),3.64(3H,m,H-6′b,4-O ₂ CHCH ₃ ),3.19(3H,m,H-2′,H-4′,H-5′),2.72(1H,d,J＝6.8Hz,H-5),2.46(1H,dd,J＝11.0,6.9Hz,H-7a),2.12(1H,d,J＝12.5Hz,H-3a),1.91(1H,dd,J＝12.5,1.7Hz,H-3b),1.90(1H,d,J＝11.0Hz,H-7b),1.34(3H,s,H ₃ -10),1.13(3H,t,J＝7.1Hz,4-OCH _{2 3} CH)； ¹³ C NMR(CD ₃ OD,150MHz)δ:168.0(C-7″),134.5(C-4″),131.2(C-1″),130.6(C-2″,6″),129.7(C-3″,5″),109.3(C-4),102.5(C-9),100.2(C-1′),89.2(C-1),87.3(C-2),78.1(C-3′),78.0(C-5′),75.0(C-2′),71.8(C-6,C-4′),62.9(C-6′),61.7(C-8),60.4(4-O ₂ CHCH ₃ ),42.7(C-3),41.7(C-5),23.5(C-7),19.6(C-10),15.8(4-OCH _{2 3} CH)

II: white powder; ESI-MS m/z 585[ M+H ]] ⁺ ,607[M+Na] ⁺ ； ¹ H NMR(CD ₃ OD,500MHz)δ:7.93(4H,dd,J＝8.4,1.4Hz,H-2″,6″,2″′,6″′),7.46(2H,t,J＝7.8Hz,H-4″,4″′),7.32(4H,t,J＝7.8Hz,H-3″,5″,3″′,5″′),5.34(1H,s,H-9),4.62(2H,s,H ₂ -8),4.56(1H,brd,J＝11.5Hz,H-6′a),4.49(1H,d,J＝8.0Hz,H-1′),4.41(1H,dd,J＝11.5,7.0Hz,H-6′b),3.73(1H,ddd,J＝9.5,7.0,2.0Hz,H-5′),3.37(1H,dd,J＝9.0,9.0Hz,H-3′),3.33(1H,dd,J＝9.0,9.5Hz,H-4′),3.25(1H,dd,J＝8.0,9.0Hz,H-2′),2.45(1H,d,J＝6.5Hz,H-5),2.37(1H,dd,J＝11.0,6.5Hz,H-7a),1.75(1H,d,J＝12.5Hz,H-3a),1.64(1H,d,J＝11.0Hz,H-7b),1.60(1H,d,J＝12.5Hz,H-3b),1.18(3H,s,H ₃ -10),； ¹³ C NMR(CD ₃ OD,125MHz)δ:167.9(C-7″),167.6(C-7″′),131.2(C-1″,C-1″′),131.0(C-4″′,C-4″),130.5(C-2″′,C-6″′,C-2″,C-6″),129.7(C-3″,C-5″,C-3″′,C-5″′),106.2(C-4),102.1(C-9),99.9(C-1′),89.2(C-1),87.0(C-2),77.7(C-3′),75.0(C-5′),74.8(C-2′),72.0(C-6),71.8(C-4′),65.2(C-6′),61.6(C-8),44.4(C-3),43.7(C-5),23.0(C-7),19.7(C-10)。

III: white powder; ESI-MS m/z 607[M+Na] ⁺ ,623[M+K] ⁺ ,619[M+Cl] ^- ； ¹ H NMR((CD ₃ ) ₂ CO,600MHz)δ:2.18(1H,d,J＝12.6Hz,H-3a),1.78(1H,dd,J＝12.6,1.2Hz,H-3b),2.57(1H,d,J＝7.2Hz,H-5),2.61(1H,dd,J＝10.4,7.2Hz,H-7a),1.96(1H,d,J＝10.4Hz,H-7b),4.78(1H,d,J＝12.0Hz,H-8a),4.74(1H,d,J＝12.0Hz,H-8b),5.40(1H,s,H-9),1.35(3H,s,H ₃ -10),4.76(1H,d,J＝8.0Hz,H-1′),5.01(1H,dd,J＝8.0,9.0Hz,H-2′),3.76(1H,dd,J＝9.0,9.0Hz,H-3′),3.41(1H,dd,J＝9.0,8.4Hz,H-4′),3.58(1H,m,H-5′),3.66(1H,dd,J＝12.0,2.4Hz,H-6′a),3.62(1H,dd,J＝12.0,6.0Hz,H-6′b),8.04(2H,dd,J＝8.3,1.2Hz,H-2″,6″),7.54(2H,t,J＝7.8Hz,H-3″,5″),7.66(1H,t,J＝7.8Hz,H-4″),8.07(2H,d,J＝8.3,1.2Hz,H-2″′,6″′),7.54(2H,t,J＝7.8Hz,H-3″′,5″′),7.66(1H,t,J＝7.8Hz,H-4″′)； ¹³ C NMR((CD ₃ ) ₂ CO,150MHz)δ:89.27(C-1),86.2(C-2),44.6(C-3),105.8(C-4),44.0(C-5),71.8(C-6),23.4(C-7),61.5(C-8),101.8(C-9),19.8(C-10),100.0(C-1′),76.0(C-2′),75.6(C-3′),73.1(C-4′),75.2(C-5′),62.4(C-6′),131.3(C-1″),130.5(C-2″,6″),129.6(C-3″,5″),134.2(C-4″),167.0(C-7″),131.3(C-1″′),130.6(C-2″′,6″′),129.5(C-3″′,5″′),134.2(C-4″′),166.5(C-7″′)。

IV: white amorphous powder; HRESIMS m/z 669.2514[ M+Na ]] ⁺ ； ¹ H NMR((CD ₃ ) ₂ CO,600MHz)δ:8.07(2H,dd,J＝8.4,1.2Hz,H-2″/6″),7.64(1H,t,J＝7.8Hz,H-4″),7.51(2H,t,J＝7.8Hz,H-3″/5″),5.44(1H,s,H-9),4.77(1H,d,J＝12.6Hz,H-8a),4.74(1H,d,J＝12.6Hz,H-8b),4.62(1H,dd,J＝11.8,1.8Hz,H-6′a),4.58(1H,d,J＝7.7Hz,H-1′),4.09(1H,dd,J＝11.9,6.0Hz,H-6′b),3.57(1H,dd,J＝10.6,7.8Hz,H-10″′a),3.50(1H,dd,J＝10.6,7.8Hz,H-10″′b),3.47(1H,ddd,J＝9.0,6.0,1.8Hz,H-5′),3.36(1H,t,J＝9.0Hz,H-3′),3.30(1H,d,J＝9.0Hz,H-4′),3.23(1H,dd,J＝7.7,9.0Hz,H-2′),2.76(1H,ddd,J＝6.6,6.0,1.8Hz,H-1″′),2.55(1H,dd,J＝10.9,6.9Hz,H-7a),2.27(1H,m,H-2″′),2.12(2H,m,H-7″′a),1.96(1H,m,H-4″′b),1.91(1H,d,J＝10.8Hz,H-7b),1.83(1H,dd,J＝12.6,1.5Hz,H-3b),1.51(1H,m,H-3″′b),1.33(3H,s,H-10),1.27(3H,s,H-9″′),1.05(1H,d,J＝9.6Hz,H-7″′b)； ¹³ C NMR(CD ₃ OD,150MHz)δ:180.0(C-8″′),168.1(C-7″),134.6(C-4″),131.4(C-1″),130.8(C-2″/6″),129.8(C-3″/5″),106.4(C-4),102.4(C-9),100.2(C-1′),89.6(C-1),87.2(C-2),78.0(C-3′),75.6(C-5′),75.1(C-2′),72.3(C-6),71.7(C-4′),67.7(C-10″′a/10″′b),65.0(C-6′a),61.8(C-8a/8b),52.1(C-6″′),44.9(C-2″′),44.7(C-3a/3b),42.8(C-1″′),41.6(C-5″′),35.1(C-7″′a/7″′b),26.0(C-4″′a/4″′b),23.3(C-7a/7b),19.9(C-10),19.6(C-3a/3b),18.9(C-9″′).

V is white powder; ESI-MS m/z 467[ M+H ]] ⁺ ,489[M+Na] ⁺ ； ¹ H NMR(CD ₃ OD,600MHz)δ:8.04(2H,dd,J＝8.3,1.4Hz,H-2″,6″),7.61(1H,m,H-4″),7.49(2H,m,3″,5″),4.79(1H,d,J＝8.5Hz,H-9a),4.74(1H,d,J＝11.3Hz,H-8a),4.66(1H,d,J＝11.3Hz,H-8b),4.49(1H,d,J＝7.7Hz,H-1′),4.17(1H,dd,J＝7.8,3.8Hz,H-4),3.85(1H,dd,J＝11.9,1.8Hz,H-6′a),3.78(1H,d,J＝8.6Hz,H-9b),3.61(1H,m,H-6′b),3.35(1H,m,H-3′),3.23(3H,m,H-2′,4′,5′),2.60(2H,m,H-5,7a),2.42(1H,dd,J＝15.5,7.9Hz,H-3a),1.84(1H,dd,J＝15.6,1.1Hz,H-3b),1.73(1H,d,J＝9.7Hz,H-7b),1.34(3H,s,H ₃ -10)； ¹³ C NMR(CD ₃ OD,150MHz)δ:168.2(C-7″),134.3(C-4″),131.3(C-1″),130.6(C-2″,6″),129.6(C-3″),100.1(C-1′),91.3(C-2),87.8(C-1),78.1(C-5′),77.9(C-3′),75.1(C-2′),71.8(C-4′),70.4(C-9),69.9(C-4),67.2(C-8),62.9(C-6′),59.0(C-6),45.5(C-3),40.5(C-5),28.2(C-7),21.6(C-10)。

VI, white amorphous powder; HRESIMS m/z 471.1639[ M+Na ]] ⁺ ； ¹ H NMR(CD ₃ OD,600MHz)δ:8.06(2H,dd,J＝8.3,1.2Hz,H-2″),7.64(1H,t,J＝7.5Hz,H-4″),7.51(2H,t,J＝7.5Hz,H-3″/5″),5.76(1H,s,H-3),5.07(1H,d,J＝11.8Hz,H-8a),4.59(2H,m,H-1′/8b),3.85(1H,dd,J＝11.9,2.2Hz,H-6′a),3.66(1H,dd,J＝11.9,5.4Hz,H-6′b),3.42(2H,m,H-3′/4′),2.88(1H,dd,J＝7.0,2.6Hz,H-5),2.62(1H,d,J＝9.6Hz,H-7b),2.16(3H,s,H-10),1.24(3H,s,H-9)； ¹³ C NMR(CD ₃ OD,150MHz)δ:204.3(C-4),176.0(C-2),168.1(C-7″),134.6(C-4″),131.4(C-1″),130.7(C-2″/6″),129.9(C-3″/5″),121.9(C-3),100.3(C-1′),84.6(C-1),78.2(C-3′),78.2(C-5′),75.3(C-2′),71.8(C-4′),68.7(C-8a/8b),63.4(C-6′a/6′b),62.9(C-6),48.9(C-5),44.6(C-7a/7b),20.4(C-10),16.5(C-9).

2. Pharmacological Activity experiment of radix Paeoniae Rubra part

Experimental example 1

Cell toxicity exploration of red peony part

RBL-2H3 cells in the logarithmic growth phase are taken out of 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 medium ⁵ Inoculating 200 mu L of each well into a 96-well plate, uniformly mixing three wells for 24 hours, removing the supernatant, adding medicines to be screened with different concentrations (0.2 mu g/ml, T2 is 2 mu g/ml and T3 is 20 mu g/ml) prepared by fresh culture medium, adding 3 compound wells of each group, adding normal groups (cell blank holes without medicines) and zeroing holes (cell blank holes without inoculation), culturing for 24 hours, removing the supernatant, adding 200 mu L of MTT solution (serum-free culture medium: 5mg/ml: MTT=1:10) prepared by serum-free, incubating for 4 hours, centrifuging for 400g/5 minutes, removing the supernatant, adding 150 mu L of DMSO, and fully shaking to dissolve crystals After the solution, the OD value of each well was measured at 570 nm.

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

The results are shown in Table 1. As shown in Table 1, the viability of the RBL-2H3 cells was higher than 95% after the administration of each drug, and thus, the cytotoxicity of the red peony portion was small, and it was considered that the red peony portion was not cytotoxic at the administration dose of 20. Mu.g/ml. From the above results, the red peony root has low cytotoxicity and high safety. The dose selection range of 0.2-20 mug/ml can be selected as the dosing range, and the influence of the part on antigen-induced RBL-2H3 cell activation degranulation HIS and beta-HEX release rate can be further discussed.

TABLE 1 cytotoxic Effect of red peony on RBL-2H3 cells (mean.+ -. Standard deviation, n=6)

Experimental example 2

Study of the Effect of radix Paeoniae Rubra on the degranulation of RBL-2H3 cells caused by anaphylaxis

Digesting cells in logarithmic growth phase, and adjusting cell density to 1×10 ⁵ And each ml. 200. Mu.L/well was added to a 96-well plate, and zeroing wells, blank wells, total enzyme wells, and each dosing well were set. The drug delivery wells were divided into model control groups, T1, T2 and T3 groups, wherein the final concentration of T1 drug delivery was 0.2. Mu.g/ml, T2 was 2. Mu.g/ml, and T3 was 20. Mu.g/ml. Incubating overnight, adding complete medium into zeroing well, normal well and total enzyme well for normal culture, adding 200 μl of anti-DNP-IgE prepared from complete medium with final concentration of 750ng/mL into model group, adding 200 μl of anti-DNP-IgE prepared from each concentration of drug and final concentration of 750ng/mL into administration well, incubating for 24 hr, centrifuging, adding improved desk liquid, cleaning to no residual medium, adding 200 μl of blank improved desk liquid into zeroing well and blank control well, adding 200 μl of 1% Triton X-100 lysate into total enzyme well, adding 200 μl of DNP-BSA prepared from improved desk liquid into administration group and model control group, centrifuging 3000r/5min after culturing for 2 hr to obtain supernatant, and measuring histamine and beta Aminoglycosidase release and observing the cell morphology microscopically. The histamine release amount measurement method was 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 reaction, stabilizing for 15min, and measuring fluorescence values of each group at the excitation wavelength of 355nm and the emission wavelength of 460 nm. From the measured fluorescence values of each group, the histamine release rate was calculated as follows:

histamine release rate (%) = (sample supernatant fluorescence value-zeroed supernatant fluorescence value)/(total enzyme well fluorescence value-zeroed supernatant fluorescence value) ×100%

The method for measuring the release amount of the beta-aminoglycosidase comprises the following steps: the cell supernatant was taken at 50. Mu.L, a substrate of beta-aminoglycosidase was added, incubated at 37℃for 45min, stop reaction was stopped at 200. Mu.L by adding stop solution NaHCO3/Na2CO3, and absorbance of each well was measured at 405 nm. From the OD values measured in each group, the beta-aminoglycosidase release rate was calculated according to the following formula

Beta-aminoglycosidase release (%) = (sample supernatant value-zeroing value)/(total enzyme Kong Zhi-zeroing value) ×100%

As shown in Table 2, the results of the histamine and beta-aminoglycosidase release rate assays indicate that 750ng/mL of anti-DNP-IgE stimulation, 1. Mu.g/mL of DNP-BAS stimulation can significantly increase the content of histamine and beta-aminoglycosidase in cell culture supernatant (P <0.001 ), while the above red peony compound can inhibit the release of histamine and beta-aminoglycosidase at 20. Mu.g/mL (P <0.001 ).

TABLE 2 Effect of red peony on cell degranulation due to anaphylaxis (0.2. Mu.g/mL, 2. Mu.g/mL, 20. Mu.g/mL, mean.+ -. Standard deviation, n=6)

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

As is clear from the results in Experimental example 2, CS-1, CS-2 and CS-3 had no antiallergic effect at each of the doses administered. Only CS-4 at the red peony site significantly inhibited the release of histamine and beta-aminoglycosidase (P < 0.001) in degranulation of cells by allergic reactions at a dose of 20. Mu.g/mL. Thus, the CS-4 site was further extracted and isolated.

3. Pharmacological Activity experiment of Red peony root monomer Compound

The research at home and abroad shows that common allergens in nature such as pollen, dust, catkin, animal fur, lampblack, food, medicine and the like appear in various links of life, and the allergens can cause organisms to be allergic or anaphylactic reaction. The pharmacological experiment shows that the compound I, II, III, IV, V, VI has obvious effect of inhibiting histamine and beta-hexosaminidase from releasing, and can be used for preparing medicines for preventing or treating allergic diseases.

Experimental example 3

Red peony compound cytotoxicity search

RBL-2H3 cells in the logarithmic growth phase are taken out of 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 medium ⁵ Inoculating 200 mu L of each well into a 96-well plate, uniformly mixing three wells per well, culturing for 24 hours, discarding the supernatant, adding drugs to be screened with different concentrations (0.08, 0.2, 4, 10 and 50 mu M) prepared by fresh culture medium, respectively setting a normal group (cell blank hole without drug) and a zeroing hole (cell blank hole without drug) in each group of 3 compound wells, discarding the supernatant after culturing for 24 hours, adding 200 mu L of MTT solution (serum-free culture medium: 5mg/ml: MTT=1:10) prepared by serum-free culture, incubating for 4 hours, centrifuging for 400g/5 minutes, discarding the supernatant, adding 150 mu L of DMSO, sufficiently shaking to dissolve crystals, and measuring the OD value of each hole at 570 nm.

Cell viability (%) = (drug group OD value-zeroing group OD value)/(normal group OD value-zeroing group OD value)

×100％

The results are shown in tables 3 and 4. As shown in Table 3, after paeoniflorin acts on RBL-2H3 cells, the survival rate of the cells at a dose of 100 mu M is 67.00%, and the survival rate of the cells at a dose of 20 mu M is 73.87%, so that paeoniflorin has certain cytotoxicity on RBL-2H3 cells. After the compounds I-VI act on RBL-2H3 cells, the survival rate of the cells is higher than 80%, so that the red paeony root compounds I-VI have low cytotoxicity and high safety. The dosage selection range of 0.08-50 mu M can be selected as the administration range, and the influence of each drug on antigen-induced RBL-2H3 cell activation degranulation HIS and beta-HEX release rate is further discussed.

TABLE 3 cytotoxic Effect of paeoniflorin on RBL-2H3 cells (mean.+ -. Standard deviation, n=6)

TABLE 4 cytotoxicity of red peony compounds I-VI on RBL-2H3 cells (mean.+ -. Standard deviation, n=6)

Experimental example 4

Investigation of the Effect of Red peony root Compounds on the degranulation of RBL-2H3 cells by anaphylaxis

Digesting cells in logarithmic growth phase, and adjusting cell density to 1×10 ⁵ And each ml. 200. Mu.L/well was added to a 96-well plate, and zeroing wells, blank wells, total enzyme wells, and each dosing well were set. The administration holes are divided into model control group and T ₁ 、T ₂ And T ₃ Group, wherein T ₁ The final concentration of administration is 50 mu M, T ₂ 25 mu M, T ₃ 5. Mu.M. Incubating overnight, adding complete medium into zeroing well, normal well and total enzyme well for normal culture, adding 200 μl of anti-DNP-IgE prepared from complete medium with final concentration of 750ng/mL into model group, adding 200 μl of anti-DNP-IgE prepared from each concentration of drug and final concentration of 750ng/mL into administration well, incubating for 24 hr, centrifuging, adding improved desk liquid, cleaning to no residual medium, adding 200 μl of blank improved desk liquid into zeroing well and blank control well, adding 200 μl of 1% Triton X-100 lysate into total enzyme well, adding 200 μl of DNP-BSA prepared from improved desk liquid into administration group and model control group, adding 1 μg/mL of DNP-BSA prepared from improved desk liquid, and culturing for 2 hr, 3000 The supernatant was centrifuged at r/5min, and the release amounts of histamine and beta-hexosaminidase were determined and the cell morphology was observed microscopically. The histamine release amount measurement method was 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 reaction, stabilizing for 15min, and measuring fluorescence values of each group at the excitation wavelength of 355nm and the emission wavelength of 460 nm. From the measured fluorescence values of each group, the histamine release rate was calculated as follows:

histamine release rate (%) = (sample supernatant fluorescence value-zeroed supernatant fluorescence value)/(total enzyme well fluorescence value-zeroed supernatant fluorescence value) ×100%

The method for measuring the release amount of the beta-hexosaminidase comprises the following steps: taking 50 mu L of cell supernatant, adding beta-hexosaminidase substrate, incubating for 45min at 37 ℃, adding stop solution NaHCO ₃ /Na ₂ CO ₃ The reaction was stopped at 200. Mu.L and the absorbance of each well was measured at 405 nm. From the OD values measured in each group, the release rate of beta-hexosaminidase was calculated according to the following formula

Beta-hexosaminidase release (%) = (sample supernatant value-zeroing value)/(total enzyme Kong Zhi-zeroing value) ×100%

Microscopic observation results show that the RBL-2H3 cells of the normal group are long fusiform, complete in edge and compact in structure. The cell volume of the model group is increased, the edge is irregular, a large number of vacuoles or particle-like structures appear, most cell membranes are broken, and particle-like substances are exuded. The cellular state of the red paeony root compound is obviously improved, the vacuole-like structure is obviously reduced, and the red paeony root compound is suggested to be capable of effectively protecting the cellular form to be perfect and inhibiting the exudation of the particle-like substances.

As shown in Table 5, the results of the histamine and beta-aminohexosaminidase release rate assays indicate that 750ng/ml of anti-DNP-IgE stimulation, 1. Mu.g/ml of DNP-BAS stimulation significantly increased the histamine and beta-aminohexosaminidase content (P<0.001,P<0.001 Red peony compounds I-VI selectively significantly inhibited the release of histamine or β -hexosaminidase in the 5-50 μm dose range. Wherein histamine: compounds I-VI each significantly reduced their release at 25. Mu.M dose (P<0.001，P<0.001，P<0.001，P<0.001，P<0.01，P<0.001 A) is provided; at a dose of 5. Mu.M, compounds I-IV and VI significantly reduced their release levels (P)<0.001，P<0.001，P<0.001，P<0.05，P<0.001). For beta-hexosaminidase: compounds I-VI each significantly reduced their release at 25. Mu.M dose (P<0.001，P<0.001，P<0.001，P<0.001，P<0.05，P<0.001 A) is provided; at a dose of 5. Mu.M, compounds I-IV significantly reduced their release (P<0.001，P<0.001，P<0.01，P<0.05). Paeoniflorin can significantly inhibit the release of histamine or beta-hexosaminidase in the dosage range of 25-50 mu M, but does not exhibit the effect of inhibiting the release of histamine and beta-hexosaminidase at the dosage of 5 mu M, and the corresponding EC ₅₀ The value is higher than that of the red paeony root compounds I-VI.

TABLE 5 Effect of Red peony root Compounds on cell degranulation due to anaphylaxis (5. Mu.M, 25. Mu.M, 50. Mu.M, mean.+ -. Standard deviation, n=3)

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

From the results of experimental example 4, it was found that paeoniflorin and compound I, II, III, IV, V, VI significantly inhibited the release of histamine and beta-hexosaminidase from degranulation of cells caused by anaphylaxis (P < 0.001) at a dose of 50 μm. At the administration dose of 5 μm, paeoniflorin and compound V showed no significant inhibition, compound VI showed only a relatively significant antihistamine effect, but compound I, II, III, IV still had a relatively strong antiallergic effect. The compound II with better antiallergic effect is screened out, and the generation of histamine can be obviously reduced, so that the antiallergic activity and the action mechanism thereof are further explored.

Experimental example 5

Effect of Compound II on histidine decarboxylase Activity

Histidine can generate histamine under the action of histidine decarboxylase (Histidine decarboxylase, HDC), and the histamine can stimulate organisms to generate allergic reaction, and gram negative bacteria Klebsiella pneumoniae (BeNa Culture Collection, kunshan, china) which can highly express the HDC is selected as a source of the HDC and used for preliminary research on inhibiting the HDC by the compound II. The activity of HDC was quantified by the amount of histamine synthesis. Klebsiella pneumoniae was uniformly dispersed in a liquid medium containing L-histidine HCl (pH 5.3). DMSO was diluted to a final concentration of 0.1% with bacterial broth and compound VI was dissolved to final concentrations of 5, 25 and 100 μm. 200 μl of bacterial broth with or without compound II was added to 96-well plates. After 24h of incubation, the bacterial culture was collected and filtered (bacteria removed). HDC activity was quantified by the relative content of HIS in the supernatant after filtration. The HIS release rate was calculated and expressed as relative percentage of the control group.

As shown in fig. 2, the effect of compound II on histidine decarboxylase activity indicates that compound II at a final concentration of greater than 25 μm significantly inhibited HDC activity (P <0.05 vs. blank, data corresponding to this is shown in table 6).

TABLE 6 influence of Compound II on the ammonia release rate

* P <0.05vs blank group

Experimental example 6

Effect of Compound II on the degranulation of RBL-2H3 cells by anaphylaxis

Taking sterile 6-hole plate, placing into cell climbing sheet, taking cells in logarithmic phase, and adjusting cell density to 1.0X10 ⁴ Each mL was inoculated into a 6-well plate at an inoculation volume of 1mL and cultured overnight. With different concentrations of Compound II (5, 25, 100, 200) and anti-DNP-IgE (750 ng/mL)And (5) cell management. After 24h incubation, the modified desktop liquid was added and washed until no residual medium was present, 1mL of blank modified desktop liquid was added to the normal wells, 1mL of DNP-BSA was added to the final concentration of 1. Mu.g/mL of modified desktop liquid prepared for the administration group and model group, and the slides were removed after 1h of stimulation.

The slide was immediately placed in a 95% ethanol solution. Subsequently, toluidine blue staining solution was added for staining. The cell morphology was directly observed under a microscope.

As shown in fig. 3, we found that by incubating the cells from a lower concentration (5 μm) to a higher concentration (200 μm) of compound II, it can dose-dependently inhibit cell degranulation. The model group showed significant grain-like exudation, which was significantly reduced after administration.

Experimental example 7

Influence of Compound II on the cytoskeleton of RBL-2H3 cells caused by anaphylaxis

Taking sterile 6-hole plate, placing into cell climbing sheet, taking cells in logarithmic phase, and adjusting cell density to 1.0X10 ⁴ Each mL was inoculated into a 6-well plate at an inoculation volume of 1mL and cultured overnight. Cells were treated with different concentrations of Compound II (5, 25, 100, 200) and anti-DNP-IgE (750 ng/mL). After 24h incubation, the modified desktop liquid was added and washed until no residual medium was present, 1mL of blank modified desktop liquid was added to the normal wells, 1mL of DNP-BSA was added to the final concentration of 1. Mu.g/mL of modified desktop liquid prepared for the administration group and model group, and the slides were removed after 1h of stimulation. Cells were washed 2 times with 37℃pre-warmed PBS, cell fixation was performed with 4% formaldehyde solution, and after 10min PBS washing was repeated for a total period of 20-30min. Cells were treated with Triton X-100 (0.5%) for 5min after washing. The PBS washing operation was repeated, 200. Mu.L of the phalloidin working solution was added to each cell slide, and incubation time was 30min in the absence of light. After continuing to repeat the PBS wash operation, the slide was placed on a slide with the caplet and DAPI dropped. Excess formulation was wiped off with paper towel and photographed using a microscope containing TRITC excitation/emission filter and DAPI excitation/emission filter, excitation wavelength/emission wavelength=540/570 nm and excitation wavelength/emission wavelength=364/454 nm.

As shown in fig. 4, compound II showed significant cytoskeletal protection, model group cytoskeletal curl, and curl was significantly improved after dosing.

Experimental example 8

Effect of Compound II on stability of mitochondrial Membrane of RBL-2H3 cells caused by anaphylaxis

Taking sterile 6-well plate, and adjusting cell density to 1.0X10 ⁵ Each mL was inoculated into a 6-well plate at an inoculation volume of 1mL and cultured overnight. Cells were treated with different concentrations of Compound II (5, 25, 100, 200) and anti-DNP-IgE (750 ng/mL). After 24h incubation, the cells were incubated for 10min in the absence of light, trypsinized, and cell density was adjusted to 1X 10 with cell culture medium by adding 1mL of modified desktop liquid to normal wells, adding 1mL of DNP-BSA to 1. Mu.g/mL of modified desktop liquid to the dosing and model groups, stimulating for 1h, adding rhodamine 123 (1. Mu.g/mL), incubating for 10min in the absence of light ⁶ 1 drop per mL was taken on a slide, covered with a cover slip, observed under a microscope and photographed (excitation wavelength/emission wavelength=488 nm/530 nm)

As shown in fig. 5, compound II showed a significant effect of enhancing mitochondrial membrane stability, with most of the visual field mitochondrial membrane of the model group being incomplete and most of the visual field mitochondrial membrane remaining intact after administration.

Experimental example 9

Effect of Compound II on IL-3, IL-4, IL-5, and IL-13 in cell supernatant caused by anaphylaxis by taking cell digestion in logarithmic growth phase, and adjusting cell density to 1×10 ⁵ And each ml.500 μl/well was added to the 24-well plate, and zeroing wells, blank wells, and each dosing well were set. The administration holes are divided into model control group and T ₁ 、T ₂ And T ₃ Group, wherein T ₁ The final concentration of administration is 100 mu M, T ₂ 25 mu M, T ₃ 5. Mu.M. Incubating overnight, adding complete culture medium into zeroing well, normal well and total enzyme well for normal culture, adding 200 μl of anti-DNP-IgE prepared from complete culture medium with final concentration of 750ng/ml into model group, adding 200 μl of each concentration of drug and total 200 μl of anti-DNP-IgE with final concentration of 750ng/ml into administration well, incubating for 24h, centrifuging, adding improved desk liquid, cleaning to no residual culture medium, adding 200 μl of blank improved desk liquid into zeroing well and blank control well,DNP-BSA with the final concentration of 1 mug/mL is added into the administration group and the model control group, after 2 hours of culture, the supernatant is centrifugally taken out at 3000r/5min, and the content of inflammatory factors TNF-alpha and IL-4 in the supernatant is detected according to the instruction method of the reagent kit of rats TNF-alpha and IL-4 of Jiangsu Jingmei biotechnology Co.

As shown in FIG. 6, the results of the detection of the release of inflammatory mediators IL-3, IL-4, IL-5 and IL-13 showed that 750ng/ml of anti-DNP-IgE stimulation and 1. Mu.g/ml of DNP-BAS stimulation significantly increased the content of IL-3, IL-4, IL-5 and IL-13 in the cell culture supernatant (P<0.05,P<0.05，P<0.05,P<0.05 And red peony compound VI can significantly inhibit the release of IL-3, IL-4, IL-5 and IL-13 at 50 mu M dose (P<0.05). Wherein, in addition to IL-3, compound II can still significantly inhibit the content of the compound II in supernatant (P) under the action of a dose of 5 mu M<Blank control group of 0.05vs ^# P<The 0.05vs model control group, its corresponding specific values are shown in Table 7).

TABLE 7 detection of the release of the inflammatory mediators IL-3, IL-4, IL-5, IL-13 shows

#P <0.05vs blank; * P <0.05vs model control group

Experimental example 10

Effect of Compound II on MAPK family protein expression in RBL-2H3 cells

Since the expression of IL-3, IL-4, IL-5 and IL-13 is affected by the regulation of MAPK family proteins, the amount of MAPK family protein expression was investigated. RBL-2H3 cells were removed from the incubator and the cell density was adjusted to 1X 10 ⁶ Each mL is inoculated into a culture flask containing complete culture medium, and after being evenly mixed, the culture flask is placed into an incubator with 5 percent CO2 and 37 ℃ for incubation. After 24h incubation, cells were washed 3 times with PBS and randomized into normal, model, and compound VI dosing groups. Adding complete culture medium into normal group, adding anti-DNP IgE 1mL prepared from complete culture medium with final concentration of 750ng/mL into model group, and adding compound VI (50 μm) and anti-DNP-IgE mixed solution with final concentration of 750ng/mL into administration group And total 1mL. After 24h incubation, the modified desktop liquid was added and washed until no residual medium was present, 1mL of blank modified desktop liquid was added to the normal wells, 1mL of DNP-BSA was added to the final concentration of 1 μg/mL of modified desktop liquid prepared for administration and model sets, and 1h was stimulated. The cells were extracted and lysed on ice for 30min, protein concentration was measured using BCA protein assay kit, and expression of MAPK family proteins p-p38 (Thr 180/Tyr 182), p38, p-ERK (Thr 202/Tyr 204), ERK, p-JNK (Thr 183/Tyr 185), JNK was detected by western Blot method, and changes in protein were detected using ECL chromogenic assay.

As shown in FIG. 7, western blot analysis shows that the expression levels of P-P38, P-ERK1/2 and P-JNK in RBL-2H3 cells in a model group are obviously and obviously increased compared with a control group, but the phosphorylation expression condition (#P) of the protein can be obviously reduced when the compound II is incubated with the cells<Blank control group of 0.05vs ^* P<The 0.05vs model control group, the specific values of which are shown in Table 8).

Table 8 numerical display of MAPK family protein expression levels in RBL-2H3 cells by Compound II

Blank control group with #p <0.05 vs. model control group with #p <0.05vs

Experimental example 11

Molecular docking mimics the binding of Compound II to HDC in MAPK family proteins

In order to gain insight into the binding characteristics of compound II to HDC and MAPKs from the standpoint of ligand-enzyme interactions, we obtained the crystal structures of HDC (PDB code: 4 ELO), ERK (PDB code: 5 UMO) (since ERK1 is highly homologous to ERK2, we extracted the structure of ERK2 for analysis), p38 (PDB code: 4 LOO) and JNK1 (PDB code: 4 LOO) from the PDB database. The structure of the protein was treated by a 100 step minimization process of adding hydrogen atoms, deleting water molecules, assigning AMBER7 FF99 charge, and using Sybyl X2.1 prior to the docking process. The molecular structure is generated by a Sybyl/Sketch module, the molecular structure is optimized by a Powell method under a Tripos force field, and a convergence criterion is set as follows Charge distribution is performed by->The method. Other parameters remain default values. Molecular docking was performed by Autodock.

The results showed that the free energy of binding of compound II to the target protein was low, indicating that compound II binds tightly to the target protein (table 9). Compound II bound well to HDC and occupied the enzyme catalytic site (as in fig. 8), resulting in the inability of L-histidine to be catalyzed to HIS. Meanwhile, compound II can well occupy the ATP binding sites of ERK, p38, JNK (see fig. 8). Moreover, the binding patterns are matched with the binding patterns of classical ERK, p38, JNK inhibitors occupying active pockets. In addition, the C4-OH and glycosyl moieties of Compound II form more strong hydrogen bonding interactions with amino acid residues of MAPKs at the ATP-active site. The benzoyl moiety forms a hydrophobic interaction with the hydrophobic portion of the pocket. This binding pattern reasonably accounts for the inhibition of HDC and ERK, JNK, p38 kinase by compound II.

TABLE 9 binding of Compound II to HDC and ERK, JNK, p kinase

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Experimental example 12

2.9 Effect of Compound II on passive skin allergy in BALB/c mice

Passive skin allergy (Passive cutaneous anaphylaxis reaction, PCA) in mice is a common model for in vivo evaluation of allergic symptoms. Mice (6 weeks old, SPF grade) were randomly divided into Control group (Control group), DNPBAS-stimulated group (Model group), administration group 1 (45 mg/kg), administration group 2 (90 mg/kg), administration group 3 (180 mg/kg) according to body weight, and 6 mice each were weighed and numbered. The control group was given PBS by subcutaneous injection at the ear, 15. Mu.L of PBS was injected into the ear, and the other groups were given different concentrations of Compound 5 in PBS after 23h of priming stimulation at 50. Mu.g/mL of anti-DNP-IgE, and the control group was given PBS by lavage at 10 mL/kg. After 1h of action, the control group was injected with PBS according to 10mL/kg tail vein, and the other groups were respectively injected with 0.4% Evans blue solution containing 1.25 mg/mL. The left ear was observed for 30min, cut along the root of the ear, minced, and placed in 800. Mu.L of acetone-physiological saline (7:3) solution. The supernatants were centrifuged at 5000r/10min at 65℃for 12h and transferred to blank 96-well plates with a transfer volume of 200. Mu.L for each sample. Absorbance values were measured at 620 nm.

As shown in fig. 9, the experimental results show that the left ear permeability of mice in the model group is significantly increased, and the evans blue exudation is gradually reduced after administration, which suggests that compound II can inhibit the occurrence of passive skin allergy (×p) in mice<Blank control group of 0.05vs ^# P<The 0.05vs model control group, its corresponding specific values are shown in Table 10).

TABLE 10 Effect of Compound II on passive skin allergy in BALB/c mice

Blank control group with #p <0.05 vs. model control group with #p <0.05vs

As is clear from experimental example 4, the anti-allergic activity of the red peony compound II is strongest. From experimental examples 5 to 12, it is known that the red peony compound II can reduce HIS release by combining with HDC, thereby alleviating symptoms related to allergic reaction; and inhibit the release of inflammatory factors by blocking ERK1/2, JNK and p38 expression in the MAPK signaling pathway. In addition, compound VI can also enhance the cytoskeletal stability of RBL-2H3 cells and the stability of the mitochondrial membrane. Molecular docking simulations then provide the reason for the inhibition of allergic reactions by compound VI from the point of view of ligand-enzyme interactions. In addition, in vivo experiments also demonstrate that compound VI can alleviate the passive skin allergy of BALB/c mice caused by IgE. Thus, compound II can be considered a promising dual HDC and MAPK signaling pathway inhibitor of natural origin. As is clear from example 3 and Experimental example 1, the above red peony site and red peony compound are high in safety. As is clear from the results in Experimental example 2 and Experimental example 4, the concentration of the catalyst was 20. Mu.g/mL The red peony site can significantly inhibit the release of histamine and beta-aminoglycosidase in cell degranulation caused by anaphylaxis (P<0.001). I. II, III, IV, V, VI the lowest concentration of the significant difference produced by the red peony monomer compounds I-IV was 5. Mu.M/L and the lowest concentration of the significant difference produced by the compounds V-VI was 25. Mu.M/L. The relative molecular weights of the 6 red peony root compounds are 508.52, 584.57, 584.57, 646.69, 466.48 and 448.47 respectively, and after the transformation unit, the concentrations of the compounds are 2.54 mug/mL, 2.92 mug/mL, 3.23 mug/mL, 11.67 mug/mL and 11.21 mug/mL respectively, and the concentrations are smaller than the dosage of the administration part of 20 mug/mL, so the antiallergic and anaphylactic-like effects of the red peony root monomer compounds are stronger than those of the red peony root part (CS-4). The minimum dose of paeoniflorin production is 25 mu M/L, the relative molecular weight is 480.45, the corresponding concentration after transformation unit is 12.011 mu g/mL, the concentration is lower than the administration dose of the red peony root part but higher than the administration dose of other red peony root compounds, and the EC thereof ₅₀ The values are also higher than for compounds I-VI.

Therefore, the experimental results show that the caged monoterpene glycoside red paeony root compounds I-VI have higher safety, good antiallergic and anti-inflammatory activities, and the red paeony root monomers have stronger activity compared with the parts, and the antiallergic effect of the compounds I-VI is stronger than that of paeoniflorin.

And (3) injection: the allergic reaction is divided into two stages, a stimulation stage and a stimulation stage, and in the experiment, the allergic reaction positive medicine is divided into an exciting agent and a stimulation, wherein the exciting agent is anti-DNP-IgE, and the Chinese name is anti-dinitrophenol IgE monoclonal antibody. The excitant is DNP-BSA, and the Chinese name is dinitrophenol-bovine serum albumin.

Experimental example 13

Effect of Compound II on allergic rhinitis in BALB/c mice

SPF-class BALB/c mice, 50, male, body weight: 18-20 g, randomly divided into a blank group, a model group and a paeoniflorin group (30 mg.kg) ^-1 ) Compound II low dose administration group (30 mg.kg) ^-1 ) High dose administration group of Compound II (90 mg kg) ^-1 ) Each group had 10 mice. After one week of acclimation, other than the blank control groupAnd (5) performing molding. Basic sensitization: 1, the method comprises the following steps: ratio 1 OVA (ovalbumin, 0.5 mg.ml) ^-1 ) And aluminum hydroxide gel (40 mg.ml) ^-1 ) Evenly mixing, except for a blank control group, injecting 0.2ml of mixed solution into the abdominal cavity of each group of mice, once every two days, and continuously carrying out 14 days; the blank control group was injected with an equal amount of physiological saline in the same way. Strengthening sensitization: after basal sensitization, nasal drops were applied on both sides of the blank control group with 1% OVA saline solution, 10 μl each time per nostril, once daily for 7 consecutive days. Nasal drops were applied on both sides with 2% OVA saline solution, 10 μl each time per nostril, twice daily for 7 consecutive days. The blank control group was nasal-dropped bilaterally with an equal amount of physiological saline in the same manner. After the last stimulation, a score standard was set according to the number of nasal scratches and the degree of sneezing and nasal discharge of the mice, and the score was recorded, and the observation time was 30min. Score criteria: number of nasal flexible times: the single limb lightly rubs the nose for 1 minute, the two limbs lightly rubs the nose for 2 minutes, and the two limbs frequently rubs the nose for 3 minutes; sneezing: 1 to 3 times of 1 minute, 4 to 10 times of 2 minutes and more than 10 times of 3 minutes; degree of runny nose: the flow to the anterior nares is 1 minute, the flow to the anterior nares is 2 minutes, and the flow to the face is 3 minutes. The total score was recorded by the superposition method.

Table 11 morphological scoring of compound II on allergic rhinitis disease model

After successful molding, paeoniflorin group (30 mg.kg) ^-1 ) Mouse lavage administration of paeoniflorin, compound II Low dose administration group (30 mg.kg) ^-1 ) Compound II was administered by gavage, and compound II was administered in high dose (90 mg·kg ^-1 ) Compound II was administered by gavage, normal and model groups were given physiological saline by gavage, and the dose: 2 ml/kg ^-1 The administration was continued for 6 days, and fasted for 8 hours after the last administration.

After the abdominal aorta is taken, the centrifuge is set at 4 ℃ and 3000 r.min ^-1 And (3) centrifuging for 10min, and accurately detecting according to the specification of the detection kit product after separating serum. Serum inflammatory factors (IL-4, IL-1. Beta., IFN-. Gamma.) were measured by ELISA as follows:

table 12ELISA method to examine the effect of compound V on serum inflammatory factors of allergic rhinitis disease model (pg/mL, mean ± standard deviation, n=8)

Experimental example 14

Effect of Compound II on allergic conjunctivitis in BN rats

50 male BN rats of 6-8 weeks are randomly divided into a blank control group, a model group and a paeoniflorin group (30 mg.kg) ^-1 ) Compound II low dose administration group (30 mg.kg) ^-1 ) High dose administration group of Compound II (90 mg kg) ^-1 ) Each group had 10 rats. After one week of acclimation, molding was performed except for the blank control group. Rat left posterior plantar injection containing OVA 100 μg and adjuvant Al (OH) ₃ 35mg of Phosphate Buffer (PBS) 0.2ml; on day 7, 0.2ml of PBS containing 100 μg of OVA was intraperitoneally injected; on days 14 and 16, each eye had an OVA of 150. Mu.g/. Mu.l ^-1 5 μl of PBS for eye-drop excitation; the blank group was given an equal amount of PBS solution in the same manner. After 20min of final sensitization, the ocular features were observed with a double-blind slit-lamp microscope (both right eyes were observed). Scoring and recording according to a Magone scoring standard. Scoring criteria: conjunctival edema: mild limiting conjunctival edema is 1 minute, diffuse edema, dome affected is 2 minutes, conjunctival edema causes conjunctival sac to be submerged in 3 minutes; conjunctival congestion: mild diffuse vascular hyperemia is 1 minute, diffuse hyperemia is obviously 2 minutes near the fornix, and hyperemia is 3 minutes along with subconjunctival hemorrhage; congestion and edema of eyelid: mild eyelid congestion edema is 1 minute, eye diffuse congestion edema lid split is 2 minutes, eye severe congestion edema is 3 minutes, and delay difficulty is 3 minutes; tear scoring criteria: the tear accumulation in the caruncle portion was 1 minute, the tear overflow lid was 2 minutes, the tear flow was more than 3 minutes, and the total score was recorded by the superposition method.

Table 13 morphological scoring of compound II on allergic conjunctivitis disease model

After successful modeling, 30 mg.kg of paeoniflorin mice are used ^-1 Eye drop of paeoniflorin 50 mu L and 30 mg/kg of compound II low dose administration group mice ^-1 Compound II50 μl eye drop, 90 mg/kg for mice in high dose group of compound II ^-1 Compound II was instilled in 50 μl, normal and model mice were instilled with 50 μl of physiological saline twice daily for 6 days. After the abdominal aorta is taken, the centrifuge is set at 4 ℃ and 3000 r.min ^-1 And (3) centrifuging for 10min, and accurately detecting according to the specification of the detection kit product after separating serum. The ELISA method is used for measuring inflammatory factors (IL-4, IL-1 beta and IFN-gamma). The results were as follows: table 14ELISA method to examine the effect of compound II on serum inflammatory factors of allergic conjunctivitis disease model (pg/mL, mean ± standard deviation, n=8)

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Claims (4)

1. The application of cage-shaped monoterpene glycoside compounds and pharmaceutically acceptable salts thereof in preparing medicines for preventing, relieving and/or treating allergic diseases is characterized in that the compounds are as follows:

2. the use according to 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. Use of a pharmaceutical composition for the manufacture of a medicament for the prevention, alleviation and/or treatment of allergic diseases, characterized in that the pharmaceutical composition comprises a therapeutically effective amount of a compound as follows, 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 form selected from the group consisting of tablets, capsules, pills, granules, oral liquids, eye drops or inhalants.