Application of geranyl hydroquinone and derivative thereof in preparation of product for relieving and / or treating rheumatoid arthritis

Abstract

The invention discloses an application of geranyl hydroquinone and a derivative thereof in preparation of a product for relieving and / or treating rheumatoid arthritis, and particularly discloses an application of a compound shown in a formula I or pharmaceutically acceptable salt thereof in preparation of a product for relieving and / or treating rheumatoid arthritis, # imgabs0, r in the formula I is selected from any one of substituted or unsubstituted C3-C5 unsaturated alkenyl and substituted or unsubstituted C3-C5 branched alkyl, and the substituted group is independently selected from any one of hydroxyl and methoxyl. The invention finds that the compound (geranyl hydroquinone and the derivative thereof) shown in the formula I has a relatively good inflammation inhibition effect and can be used for relieving and / or treating rheumatoid arthritis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of geranyl hydroquinone and its derivatives in the preparation of products for relieving and / or treating rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a common autoimmune disease characterized by erosive joint inflammation with unclear etiology and pathogenesis. The main clinical manifestations are chronic, symmetrical multi-joint involvement. Repeated and persistent attacks of RA eventually lead to destruction and deformity of articular cartilage and bone, and even loss of self-care ability. In related technologies, anti-rheumatic drugs, non-steroidal anti-inflammatory drugs, glucocorticoids and other drugs are commonly used to improve the condition. Although they can effectively control or alleviate the progression of RA patients, these drugs have many adverse reactions, which makes the degree of disease relief in patients far lower than expected.

[0003] Although the exact pathogenesis of RA is complex and vague, recent studies have confirmed that many cytokines play an important role in the occurrence and development of RA. For example, nitric oxide (NO) is a simple but unstable free radical that participates in many physiological and pathological processes. NO was found in the serum, articular cartilage and synovial fluid of RA patients to significantly increase pathological secretion, indicating that NO is involved in the formation of RA. In addition, tumor necrosis factor-α (TNF-α) can stimulate the activation of endothelial cells and leukocytes and amplify cytokine and chemokine responses. Interleukin-1β (IL-1β) is associated with synovial inflammation and synovial fibrosis in RA. Secondly, interleukin-6 (IL-6) induces the differentiation and formation of osteoclasts, promotes angiogenesis and the degradation of bone and cartilage. More importantly, IL-6 can promote the synergistic effect of TNF-α and IL-1β, further aggravate arthritis, and promote the establishment and maintenance of the synovial inflammatory environment. At the same time, a large number of studies have confirmed that these proinflammatory cytokines are abundant in the serum or synovial fluid of RA patients and are related to the disease activity of RA.

[0004] In summary, the pathogenesis of RA is complex, and the pathogenesis of joint pain is not yet clear. Related drugs are difficult to effectively relieve the pain caused by rheumatoid arthritis. Based on this, there is an urgent need for a drug that can effectively relieve the degree of inflammation in RA patients. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes the use of geranyl hydroquinone and its derivatives in the preparation of products for relieving and / or treating rheumatoid arthritis. The present invention has found through experiments that geranyl hydroquinone (hereinafter referred to as GM-1) and its derivatives have good anti-inflammatory activity (compound 1 is the best), which may reduce the inflammation and pain of rheumatoid arthritis and have good application prospects.

[0006] The first aspect of the present invention provides a use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a product for relieving and / or treating rheumatoid arthritis.

[0007]

[0008] Wherein, R in the formula I is selected from any one of substituted or unsubstituted C3-C5 unsaturated alkenyl, substituted or unsubstituted C3-C5 branched alkyl, and the substituted groups are independently selected from any one of hydroxyl and methoxy.

[0009] It is understood that the term "substituted or unsubstituted" in the present invention refers to two situations where one or more hydrogen atoms on the substituted group can be "substituted" or "unsubstituted" by one or more substituents. The number of substitutions ranges from single substitution to the maximum number of substitutions.

[0010] The application of the embodiments of the present invention has at least the following beneficial effects:

[0011] The present invention has found through experiments that the compound shown in Formula I (geranyl hydroquinone and its derivatives) has good anti-inflammatory activity, can reduce inflammation and pain in RA mice, play an analgesic and anti-inflammatory effect, and compared with the commonly used drugs for rheumatoid arthritis in the clinical frontline (such as leflunomide), has no side effects and has good application prospects.

[0012] In some embodiments of the present invention, R in Formula I is selected from any one of substituted or unsubstituted C5 unsaturated alkenyl, substituted or unsubstituted C5 branched alkyl, and the substituted groups are independently selected from any one of hydroxyl and methoxy.

[0013] In some embodiments of the present invention, the compound represented by formula I is selected from any one of the compounds of formula I-1 to formula I-5:

[0014]

[0015]

[0016] In some embodiments of the present invention, the pharmaceutically acceptable salt of the compound represented by Formula I includes any one of hydrochloride, nitrate, acetate, methanesulfonate, phosphate, citrate, fumarate, sulfate, succinate, tartrate, citrate, hydrobromide, hydroiodide, lactate, toluenesulfonate, cinnamate, salicylate, malonate, glutarate and malate.

[0017] In some embodiments of the present invention, the product for alleviating and / or treating rheumatoid arthritis includes a product that inhibits cellular inflammatory response.

[0018] In some embodiments of the present invention, the product is any one of a medicine, a health product, and a functional food.

[0019] The second aspect of the present invention provides a drug for preventing and / or treating rheumatoid arthritis, wherein the active substance of the drug includes the compound of formula I or a pharmaceutically acceptable salt thereof as described in any application of the first aspect, wherein the compound of formula I or a pharmaceutically acceptable salt thereof is as defined in any one of the first aspect.

[0020] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0021] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a solvent, an excipient, a diluent, a binder, a disintegrant, a dispersant, a flavoring agent, a suspending agent, a surfactant, an isotonic agent, a thickener, an emulsifier, a preservative, a glidant, and a lubricant.

[0022] In some embodiments of the present invention, the dosage form of the drug is any one of tablets, capsules, granules, injections, powder injections, eye drops, liniments, suppositories, ointments, and aerosols.

[0023] In some embodiments of the invention, the excipient comprises water.

[0024] In some embodiments of the present invention, the filler comprises at least one of starch and sucrose.

[0025] In some embodiments of the present invention, the binder includes at least one of a cellulose derivative, alginate, gelatin, and polyvinyl pyrrolidone.

[0026] In some embodiments of the invention, the humectant comprises glycerin.

[0027] In some embodiments of the present invention, the disintegrant comprises at least one of agar, calcium carbonate, and sodium bicarbonate.

[0028] In some embodiments of the invention, the absorption enhancer comprises a quaternary ammonium compound.

[0029] In some embodiments of the invention, the surfactant comprises cetyl alcohol.

[0030] In some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and bentonite.

[0031] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0032] In some embodiments of the present invention, the dosage form of the drug is any conventional dosage form in the art.

[0033] In some embodiments of the present invention, the dosage form of the drug is in the form of solid, semisolid or liquid, and can be an aqueous solution, non-aqueous solution or suspension.

[0034] In some embodiments of the present invention, the dosage form of the drug is tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, pellets, dry extracts, injections, and infusions.

[0035] It is understood that the drug of the present invention can be introduced into the body (such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue) by oral administration, injection, spraying, penetration, absorption, physical or chemical mediation; or can be introduced into the body after being mixed or encapsulated with other substances.

[0036] Other features and advantages of the present invention will be set forth in the description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0038] Figure 1 These are the mass spectrometry detection results of GM-1 prepared by the present invention and GM-1 in Ganoderma lucidum spore powder and Ganoderma lucidum fruiting body.

[0039] Figure 2 Schematic diagram of the transformation of geranylhydroquinone by endophytic fungi of Ganoderma lucidum.

[0040] Figure 3 Schematic diagram of the key HMBC signals of Alliodorol A and Alliodorol B, where A is compound 4 and B is compound 5.

[0041] Figure 4 These are the test results of cytotoxic activity of geranyl hydroquinone and its derivatives, wherein A is compound 1, B is compound 2, C is compound 3, D is compound 4, and E is compound 5.

[0042] Figure 5 These are the results of the inhibition of NO release by geranylhydroquinone and its derivatives. Note: Data are expressed as mean ± SEM, **p < 0.01, ***p < 0.001 and ****p < 0.0001, ns means no significance.

[0043] Figure 6 These are the results of the inhibition of cytokine IL-6 by geranyl hydroquinone and its derivatives. Note: Data are expressed as mean ± SEM, **p < 0.01, ***p < 0.001 and ****p < 0.0001, ns means no significance.

[0044] Figure 7 This is the result of the inhibition of TNF-α by geranyl hydroquinone and its derivatives. Note: Data are expressed as mean ± SEM, **p < 0.01, ***p < 0.001 and ****p < 0.0001, ns means no significance.

[0045] Figure 8 The results of the in vivo treatment effect experiment of GM-1, where A is the flow chart of the in vivo effectiveness verification experiment, B is the swelling degree of the hind limbs of four groups of mice treated with different methods, C is the statistical diagram of the swelling thickness of the hind limbs of four groups of mice treated with different methods, and D is the arthritis score results of the four groups of mice treated with different methods. Note: Figures C and D were tested by the normality Shapiro-Wilk test and did not conform to the normal distribution. Non-parametric one-way analysis of variance and Turkey multiple comparisons were used for testing. Compared with the blank group, #### P < 0.0001, ### P < 0.001, ## P<0.01, # P<0.05; compared with the model group, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. Different colors represent different groups. DETAILED DESCRIPTION

[0046] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0047] The words "preferably", "more preferably" and the like in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0048] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0049] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the associated listed items.

[0050] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0051] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0052] Example 1: Compound GM-1

[0053] This embodiment provides a Ganoderma lucidum terpenoid compound geranylhydroquinone (hereinafter referred to as GM-1), the chemical structure of which is shown in Formula I-1:

[0054]

[0055] The compound exists in Ganoderma lucidum spore powder and Ganoderma lucidum fruiting body, but the content of GM-1 extracted from traditional Chinese medicine is low. In this embodiment, the compound is prepared by organic synthesis method. The specific preparation method is as follows:

[0056] Add 2.0g (0.0212mol) of phenol and 0.2g (0.0074mol) of aluminum powder to the reaction tube, stir in an oil bath at 170°C until the aluminum powder is completely dissolved. Take out the reaction tube from the oil bath, cool it to 40°C, add 9.36g (0.0659mol) of 1,4-phenol and 7.44ml (0.0429mol) of geraniol, and react at 180°C in an oil bath for 24h. Suspend it with 5ml of water, extract it with ethyl acetate, and concentrate it to obtain a crude sample. Separate and purify it with a silica gel column to obtain a brown oily compound 1 (2.0g).

[0057] The brown oily compound 1 was subjected to nuclear magnetic resonance analysis, and its hydrogen spectrum and carbon spectrum data were as follows:

[0058] 1 H-NMR(600MHz,CD3OD)δ:6.59(d,J=8.5Hz,1H,H-6),6.54(d,J=2.6Hz,1H,H -3),6.45(m,1H,H-5),5.31(t,J=7.4Hz,1H,H-2′),5.12(t,J=7.3Hz,1H,H- 6′),3.24(s,1H,Ha-1′),3.23(s,1H,Hb-1′),2.12(q,J=7.4Hz,2H,H-4′),2 .05(m,2H,H-5′),1.70(s,3H,H-10′),1.67(s,3H,H-8′),1.60(s,3H,H-9′).

[0059] 13 C-NMR(150MHz,CD3OD)δ:151.1(C-1),148.9(C-4),136.8(C-3′),132.2(C-7′),130.2(C-2),125.4(C-6′),124.0(C-2′) ,117.1(C-3),116.5(C-6),113.8(C-5),40.9(C-4′),29.1(C-5′),27.8(C-1′),25.9(C-8′),17.7(C-9′),16.2(C-10′).

[0060] The identification results were consistent with expectations, indicating that the brown oily compound 1 was GM-1. Further, mass spectrometry analysis was performed on GM-1 from Ganoderma lucidum spore powder and Ganoderma lucidum fruiting body and GM-1 synthesized by the present invention. The results were as follows: Figure 1 As shown, the results are consistent with expectations.

[0061] Example 2: Isoalliodorol

[0062] This embodiment provides a derivative of geranylhydroquinone (Isoalliodorol), the structural formula of the derivative is shown in Formula I-2:

[0063]

[0064] The derivative is obtained by transformation using the endophytic fungus Mucor irregularis of Ganoderma lucidum. The specific transformation flow chart is as follows: Figure 2 The specific experimental steps are as follows:

[0065] (1) The endophytic fungus Mucor irregularis was inoculated into a liquid potato culture medium (4 L) for fermentation, and the fermentation conditions were set as follows: 220 rpm, 30° C., and 4 days. After the fermentation was completed, compound 1 (final concentration of 2 mM) was used for precursor feeding for 2 days. The fermentation broth was then collected and extracted three times with an equal volume of ethyl acetate to obtain a crude extract.

[0066] (2) The fragments were separated by Sephadex LH-20 gel column (methanol) to obtain two small fragments Fr.1 and Fr.2. The Fr.1 fragment was separated by high performance liquid chromatography (HPLC) to obtain compound 2 (28.6 mg, CH3CN / H2O=31:69, flow rate of 3.0 mL / min, retention time of 15.3 min) and compound 3 (17.9 mg, CH3CN / H2O=40:60, flow rate of 3.0 mL / min, retention time of 18.0 min). Similarly, the Fr.2 fragment was separated by high performance liquid chromatography (HPLC) to obtain compound 4 (2.6 mg, CH3CN / H2O=45:55, flow rate of 3.0 mL / min, retention time of 14.3 min) and compound 5 (10.4 mg, CH3CN / H2O=60:40, flow rate of 3.0 mL / min, retention time of 15.1 min).

[0067] (3) A nuclear magnetic resonance spectrum analysis was performed on one of the products extracted from the conversion, and the hydrogen spectrum and carbon spectrum data were as follows:

[0068] 1 H NMR (600MHz, CD3OD)δ H:6.59(1H,d,J=8.6Hz,H-6),6.54(1H,d,J=3.0Hz,H-3),6.44(1H,dd,J=3.0and 8.5Hz,H-5),5.30(1H,t,J=7.4Hz,H-2′),5.25(1H,m,H-6′),4.01(2H,s,H-9′),3.23(2H,d,J =7.4Hz,H-1′),2.17(2H,m,H-4′),2.04(2H,m,H-5′),1.73(3H,s,H-10′),1.68(3H,s,H-8′).

[0069] 13 C NMR (150MHz, CD3OD)δ C :151.0(C-4),148.8(C-1),136.5(C-3′),135.6(C-7′),130.1(C-2),128.6(C-6′),124.12(C-2′),117.1(C- 3),116.5(C-6),113.8(C-5),61.4(C-9′),41.0(C-4′),29.1(C-5′),27.1(C-1′),21.5(C-8′),16.2(C-10′).

[0070] The identification results showed that the obtained product was Isoalliodorol (Compound 2).

[0071] Example 3: Alliodorol

[0072] This embodiment provides another derivative of geranyl hydroquinone (Alliodorol), the structural formula of the derivative is shown in Formula I-3:

[0073]

[0074] The derivative is obtained by transformation of the endophytic fungus Mucor irregularis of Ganoderma lucidum (refer to Example 2 for the specific method). The transformed and extracted product is subjected to nuclear magnetic resonance analysis, and its hydrogen spectrum and carbon spectrum data are as follows:

[0075] 1 H NMR (600MHz, CD3OD)δ H:6.63(d,J=8.5Hz,1H,H-6),6.62(d,J=3.0Hz 1H,H-3),6.54(dd,J=8.5,3.0Hz,1H,H-5),5.41(t,J=7.0Hz,1H,H-2′),5 .00(t,J=7.4Hz,1H,H-6′),4.04(s,2H,H-8′),3.28(d,2H,H-1′),2.45(br s,1H,Ha-4′),2.16-2.29(m,3H,Hb-4′and 5′),1.66(s,3H,H-10′),1.64(s,3H,H-9′).

[0076] 13 C NMR (150MHz,CDCl3)δ C :150.1(C-4),147.4(C-1),137.3(C-3′),134.8(C-7′),128.2(C-2),125.1(C-6′),122.3(C-2′),116.3(C-3 ), 116.1(C-6), 113.5(C-5), 68.4(C-8′), 39.1(C-4′), 28.5(C-5′), 24.9(C-1′), 15.4(C-9′), 13.6(C-10′).

[0077] The results showed that the obtained product was Alliodorol (Compound 3).

[0078] Example 4: Alliodorol A

[0079] This embodiment provides another geranyl hydroquinone derivative (Alliodorol A), the structural formula of the derivative is shown in Formula I-4:

[0080]

[0081] The derivative is obtained by transformation of the endophytic fungus Mucor irregularis of Ganoderma lucidum (refer to Example 2 for the specific method). The nuclear magnetic resonance analysis of the transformed and extracted product shows that the hydrogen spectrum and carbon spectrum data are as follows:

[0082] 1 H NMR (600MHz, CD3OD)δ H:6.59(d,J=8.5Hz,1H,H-6),6.55(d,J=3.0Hz 1H,H-3),6.44(dd,J=8.5,3.0Hz,1H,H-5),5.38(tq,J=7.3,1.4Hz,1H,H-2′) ,3.41(dd,J=7.3Hz,1H,H-6′),3.26(s,1H,Ha-1′),3.25(s,1H,Hb-1′),3.19( s,3H,H-11′),2.30(m,1H,Ha-4′),2.09(m,1H,Hb-4′),1.73(s,3H,H-10′),1 .68(m,1H,Ha-5′),1.39(m,1H,Hb-5′),1.14(s,3H,H-8′),1.10(s,3H,H-9′).

[0083] 13 C NMR (150MHz, CD3OD)δ C :151.1(C-1),148.9(C-4),136.7(C-3′),130.2(C-2),124.3(C-2′),117.2(C-3),116.5(C-6),113.8(C-5),78.7( C-7′), 76.5(C-6′), 49.5(C-11′), 37.8(C-4′), 30.5(C-5′), 29.2(C-1′), 21.3(C-8′), 20.6(C-9′), 16.2(C-10′).

[0084] The results showed that the obtained product was Alliodorol A (Compound 4).

[0085] Example 5: Alliodorol B

[0086] This embodiment provides another derivative of geranyl hydroquinone (Alliodorol B), the structural formula of which is shown in Formula I-5:

[0087]

[0088] The derivative is obtained by transformation of the endophytic fungus Mucor irregularis of Ganoderma lucidum (refer to Example 2 for the specific method). The nuclear magnetic resonance analysis of the transformed and extracted product shows that the hydrogen spectrum and carbon spectrum data are as follows:

[0089] 1 H NMR (600MHz, CD3OD)δ H:6.59(d,J=8.5Hz,1H,H-6),6.55(d,J=3.0Hz 1H,H-3),6.44(dd,J=8.5,3.0Hz,1H,H-5),5.38(tq,J=7.4,1.4Hz,1H,H-2′),3.27(dd,J=7.3Hz,1H,H-6′),3.25(s,2H,H-1′),2.31( m,1H,Ha-4′),2.09(m,1H,Hb-4′),1.73(s,3H,H-10′),1.77(m,1H,Ha-5′),1.40(m,1H,Hb-5′),1.17(s,3H,H-8′),1.14(s,3H,H-9′).

[0090] 13 C NMR (150MHz, CD3OD)δ C :151.1(C-1),148.9(C-4),136.9(C-3′),130.2(C-2),124.1(C-2′),117.2(C-3),116.5(C-6),113.8(C-5) ,79.0(C-6′),73.8(C-7′),37.9(C-4′),30.7(C-5′),29.1(C-1′),25.7(C-8′),24.9(C-9′),16.2(C-10′).

[0091] The identification results were consistent with the expectation, indicating that the obtained product was Alliodorol B (Compound 5).

[0092] In addition, the present invention also conducted two-dimensional nuclear magnetic resonance data analysis on the derivatives of geranyl hydroquinone, Alliodorol A and Alliodorol B. The test results showed that the derivatives of geranyl hydroquinone, Alliodorol A and Alliodorol B, were new compounds, among which the key HMBC signals of compounds 4 and 5 were as follows: Figure 3 As shown, A is Alliodorol A and B is Alliodorol B, showing that the methoxy H3-11′ in compound 4 is associated with C-7′, H-2′ and H-1′ are associated with C-2, H-4′ is associated with C-2′, H-5′ is associated with C-3′, and H3-8′ and H3-9′ are associated with C-7′. The above HMBC-related signals well explain the structural correctness of compound 4. Similarly, the HMBC-related signals in compound 5 show that H3-8′ and H3-9′ are associated with C-6′ and C-7′, revealing that the position of the hydroxyl group in compound 5 is the same as that in compound 4, except that there is one less methoxy group.

[0093] Test Example 1: Cytotoxic Activity Test

[0094] In order to detect whether the above compounds 1 to 5 have real anti-inflammatory activity rather than cytotoxic activity, this test example uses GM-1 and its derivatives as test objects to detect their cytotoxic activity. The specific CCK-8 detection method is as follows:

[0095] The tested drugs were designed into 1μM, 10μM, 30μM, and 50μM concentration groups, with 4 replicate wells in each concentration group, and a control group was set up. RAW 264.7 cells with good growth status were taken and inoculated into 96-well plates with 5×10 cells per well. 3 After the cells adhered to the wall, different concentrations of drug-containing culture medium were added and the cells were cultured in a cell incubator (37°C, 5% CO2) for 72 hours. The culture medium was removed and the cells were washed three times with PBS solution, and then a PBS solution containing 10% CCK-8 solution was added and incubated for 1 hour. The absorbance (OD) at 450 nm was measured using an enzyme reader.

[0096] Cytotoxic activity test results Figure 4 The results showed that geranyl hydroquinone and its derivatives (compounds 1 to 5) had no cytotoxic effect on normal RAW 264.7 cells, and could even promote cell proliferation at certain concentrations, indicating that they were non-toxic to mouse RAW264.7 cells.

[0097] Test example 2: NO release inhibition activity test

[0098] In order to explore whether the geranyl hydroquinone and its derivatives prepared in Examples 1 to 5 above have the effect of reducing the inflammation of rheumatoid arthritis, this test example used LPS-induced RAW 264.7 cells to detect their anti-inflammatory activity. The specific detection method includes the following steps:

[0099] (1) Take RAW 264.7 cells in the logarithmic growth phase, remove the old culture medium and wash twice with 1 mL PBS, add 1 mL high-glucose DMEM medium (containing 10% fetal bovine serum, 1% penicillin and streptomycin mixture), gently blow off the cells with a pipette and transfer them to a 5 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, remove the supernatant, add 2 mL complete culture medium, mix by pipetting, and make a single cell suspension;

[0100] (2) Place the above cell suspension in a cell counting plate and count the cells under an inverted microscope to adjust the cell concentration to 1×10 6 100 μL of each cell was inoculated into a 96-well plate and placed in a 37°C, 5% CO2 constant temperature incubator for 24 hours to allow the cells to fully adhere to the wall and grow.

[0101] (3) The old culture medium in the 96-well plate was discarded, and a blank group, a model group, and an experimental group were set up, with 4 replicate wells in each group. 200 μL of DMEM medium was added to each well of the blank group and the model group, and 200 μL of DMEM medium containing the test compound (final concentration of 40 μM or 80 μM) was added to each well of the experimental group. LPS (final concentration of 1 μg / mL) was added to both the model group and the experimental group. The plates were cultured in a constant temperature incubator at 37°C and 5% CO2 for 24 hours and set aside.

[0102] (4) The nitric oxide production rate was determined using the Bio-Tech S0021S Griess Reagent Nitric Oxide Detection Kit as follows: first, take out Griess Reagent I and II from a 4°C refrigerator and wait for them to return to room temperature; then, add the cell supernatant to a 96-well plate at 100 μL / well; then, add room temperature Griess Reagent I and Griess Reagent II to each well at 50 μL / well and mix well; finally, use a multifunctional microplate reader to measure the absorbance at λ=540 nm.

[0103] The results of anti-inflammatory activity test were as follows Figure 5 As shown, it is shown that the geranyl hydroquinone and its derivatives of the present invention can reduce the inflammatory response to a certain extent, among which compound 1 (GM-1) has the best effect and can significantly reduce the release of NO in LPS-induced raw264.7 cells. Compound 2 (Isoalliodorol) and compound 3 (Alliodorol) are second, showing better anti-inflammatory activity. Although compound 4 (Alliodorol A) and compound 5 (Alliodorol B) did not show the expected excellent anti-inflammatory activity, they still showed a little anti-inflammatory activity relative to the model group. It can be seen that the introduction of different oxidized groups at the terminal position of compound 1 has a certain anti-inflammatory effect.

[0104] Detection Example 3: Detection of Inhibition of Cytokine IL-6 Expression Activity

[0105] In order to explore whether the geranyl hydroquinone and its derivatives prepared in Examples 1 to 5 above have the effect of reducing the inflammation of rheumatoid arthritis, this test example used LPS-induced RAW 264.7 cells to detect their inhibitory cytokine IL-6 activity. The specific ELISA detection method includes the following steps:

[0106] (1) Take out the plates required for the experiment, add the standard and specimen universal diluent to the blank wells, and add the specimen and different concentrations of standard 1000, 500, 250, 125, 62.5, 31.25, 15.6, 0pg / ml to the other corresponding wells, 100μL per well. Cover the reaction wells with adhesive tape and incubate in a 37℃ incubator for 90 minutes.

[0107] (2) Prepare the biotinylated antibody working solution 20 minutes in advance and use the biotinylated antibody diluent to dilute the 30x concentrated biotinylated antibody into a 1x working solution. Wash the plate 5 times.

[0108] (3) Add biotinylated antibody diluent to the blank wells and add biotinylated antibody working solution (100 μL / well) to the remaining wells. Seal the reaction wells with new sealing tape and incubate in a 37°C incubator in the dark for 60 minutes.

[0109] (4) Prepare the enzyme conjugate working solution 20 minutes in advance and dilute the 30x concentrated enzyme conjugate into 1x working solution with the enzyme conjugate diluent. Place at room temperature (22-25°C) away from light. Wash the plate 5 times.

[0110] (5) Add enzyme conjugate diluent to the blank wells and enzyme conjugate working solution (100 μL / well) to the remaining wells. Seal the reaction wells with sealing glue and incubate in a 37°C incubator in the dark for 30 minutes. Wash the plate 5 times.

[0111] (6) Add 100 μL / well of chromogenic substrate and incubate in a 37°C incubator in the dark for 15 minutes. Add 100 μL / well of stop solution and immediately measure the OD value using a full-wavelength microplate reader set to 450 nm.

[0112] The results of the inhibition of cytokine IL-6 activity test were as follows Figure 6 As shown, compared with the LPS model group, the geranyl hydroquinone and its derivatives (at a concentration of 40 μM) of the present invention can significantly reduce the IL-6 expression level in RAW264.7 cells, and the differences are statistically significant (P<0.0001), which further explains that geranyl hydroquinone and its derivatives are beneficial to reducing the inflammation and pain symptoms of rheumatoid arthritis in mice, among which GM-1 has the best inhibitory activity on IL-6 expression.

[0113] Test example 4: Detection of inhibition of cytokine TNF-α expression activity

[0114] In order to explore whether the geranyl hydroquinone and its derivatives prepared in Examples 1 to 5 above have the effect of reducing the inflammation of rheumatoid arthritis, this test example used LPS-induced RAW 264.7 cells to detect their inhibitory cytokine TNF-α activity. The specific ELISA detection method includes the following steps:

[0115] (1) Dilute the capture antibody to a concentration of 1 μg / mL with PBS before coating, immediately coat the 96-well plate, and add 100 μL of the diluted antibody to each well. Seal and incubate at 4°C overnight.

[0116] (2) Wash each well with at least 300 μl of wash buffer for three times. After the last wash, invert the 96-well plate and remove any remaining buffer with a clean wipe.

[0117] (3) Add 300 μL of blocking buffer to each well. Incubate at room temperature for at least 1 hour. Then repeat step 2.

[0118] (4) Prepare TNF-α standard with 1 mL of dilution buffer at 6000, 3000, 1500, 750, 375, 187.5, 93.75, and 0 pg / mL. Add 100 μL of sample or standard dilution buffer to each well. Seal the culture dish and incubate at room temperature for 2 hours. Then repeat step 2.

[0119] (5) Dilute the detection antibody to a concentration of 0.15 μg / mL with dilution buffer. Add 100 μL of detection antibody to each well and dilute with dilution buffer. Seal the culture dish and incubate at room temperature for 1 hour. Repeat step 2.

[0120] (6) Add 200 μL of substrate solution to each well and incubate at room temperature for 20 minutes, keeping away from light.

[0121] (7) Add 50 μL of blocking solution to each well. Gently tap the plate to ensure thorough mixing. Immediately measure the OD value using a full-wavelength microplate reader set to 450 nm.

[0122] Test results such as Figure 7 As shown, compared with the LPS model group, 40μM GM-1 and compound 2 can reduce the expression level of TNF-α to varying degrees, which is statistically significant (P<0.0001), while the TNF-α expression levels of compounds 3 to 5 are not statistically significant compared with the model group. Therefore, compounds 1 to 5 can improve LPS-induced RAW264.7 cell inflammation, and its mechanism may be to inhibit the release of NO, TNF-α and IL-6 inflammatory factors in cells, thereby exerting an anti-inflammatory effect. It can be concluded that compounds 1 to 5 have the activity of improving inflammation and have a positive effect on most diseases caused by inflammation, such as rheumatoid arthritis, pneumonia, hepatitis and other diseases.

[0123] Test Example 5: In vivo therapeutic effect experiment

[0124] In this test example, DBA / 1 male mice were used to establish a collagen-induced rheumatoid arthritis CIA model, and compound 1 (GM-1) was used as an example to detect its therapeutic effect in vivo. The specific experimental process is as follows.

[0125] 1. Construction of DBA / 1 male mouse CIA model:

[0126] The method for constructing the DBA / 1 male mouse CIA model in this test example is as follows:

[0127] Modeling by two immunization injections: the first injection was CFA (equal volume collagen solution and complete Freund's adjuvant) emulsion injection on the first day, and the second injection was IFA (equal volume collagen solution and incomplete Freund's adjuvant) emulsion injection on the 21st day for booster immunization. Injection method: The mouse was captured and fixed in the mouse holder, exposing the tail. The tail was disinfected with an alcohol cotton ball, and the tail was slightly tightened. The needle was inserted into the subcutaneous blood vessel 2 cm away from the base of the tail. Draw back to see if the injection site has blood return. If there is no blood return, it means that the injection site is correct. If there is blood return, consider puncturing the blood vessel site, which is likely to cause vascular embolism and lead to the death of the mouse. Each mouse was injected with a total of 100 μL of 1% DMSO aqueous solution, positive drug and therapeutic drug GM-1. After the injection, press the injection site with a cotton ball for 5 to 10 seconds to prevent overflow. The injection was effortless, and the injection point could see a white skin bump or the subcutaneous whitening of the entire tail was considered a successful injection.

[0128] Criteria for judging the success of the CIA mouse model: The CIA mice were scored for arthritis index and the morphological indicators of the ankle joints were observed. After inflammation was induced, the lesions of the limb joints of the mice were observed and recorded. The arthritis index (AI) was calculated based on the cumulative score of the lesions of the limb joints.

[0129] The scoring criteria are: no swelling or redness in the joints, 0 points; mild redness and swelling of the ankle and wrist joints or obvious redness and swelling of one toe / fingers, 1 point; mild redness and swelling of the ankle and wrist joints or obvious redness and swelling of two toes / fingers; mild redness and swelling of the ankle and wrist joints or obvious redness and swelling of three toes / fingers, 3 points; the palm surface of the entire ankle or wrist joint is completely swollen like a steamed bun, the epidermis is slightly red and translucent, or four toes / fingers are obviously red and swollen, 4 points. The four toes of each mouse are scored simultaneously according to the standard, and the score is the sum of the four feet, with a maximum of 16 points.

[0130] 2. Drug administration:

[0131] In this test case, DBA / 1 male mice were used to establish a collagen-induced rheumatoid arthritis CIA model, which was divided into a blank group, a model group, a positive drug group, and a treatment group (GM-1). After the model was successfully established on day 21, the blank group and the model group were gavaged with 1% DMSO aqueous solution every other day, the positive drug group was gavaged with 2 mg / kg leflunomide (LFM) every other day, and the treatment group was gavaged with 10.0 mg / kg GM-1 every other day. The specific dosing time of the treatment process is referred to Figure 8 As shown in A.

[0132] Among them, the positive drug used, leflunomide, is the first-line clinical drug for rheumatoid arthritis (RA). It has a good effect in relieving arthritis pain in RA disease, but its side effects are more obvious, including fever, rash, hair loss, gastrointestinal discomfort, nausea and diarrhea.

[0133] 3. Experimental results:

[0134] (1) General condition of mice: During the experiment, the blank group mice were in normal spirit and eating state, with shiny fur, no swelling on the soles of feet and toes, and could walk and climb normally, with good mobility of limbs. The model group mice were in poor spirit, with reduced food intake, dull fur, obvious swelling on the soles of feet, slow walking speed or lameness, reduced mobility of limbs, and liked to curl up in corners. Compared with the model group mice, the spirit state, food intake, fur glossiness, swelling of soles of feet, and mobility of limbs of the positive drug group and GM-1 group were improved to varying degrees (e.g. Figure 8 In addition, compared with the positive drug group, the GM-1 group did not show any adverse symptoms such as fever, rash, hair loss, gastrointestinal discomfort, nausea and diarrhea.

[0135] (2) Changes in foot swelling thickness and arthritis index (AI) score: The mice did not show symptoms of joint redness and swelling after the initial immunization, but began to develop the disease 3-5 days after the booster immunization. Specifically, during the experiment, there was no obvious change trend in the thickness of foot swelling of the blank group mice. Compared with the blank group, the thickness of foot swelling of the model group mice increased significantly, and the overall trend was gradually increasing, and the difference was statistically significant (P<0.05). Due to the large difference in foot swelling thickness within the model group, the foot swelling thickness of the GM-1 group was not statistically significant compared with the model group on days 22-30 after the initial immunization, but was statistically different from the model group on day 32 (P<0.01). The foot swelling thickness of the positive drug group was reduced compared with the model group, and there was a statistical difference on day 26 (P<0.05) and day 32 (P<0.001) (e.g. Figure 8 as shown in C in the figure).

[0136] In addition, during the experiment, the joints of the mice in the blank group did not swell, so the arthritis index (AI) scores were all 0. Compared with the blank group, the scores of the model group were significantly increased, and the difference was statistically significant from the 24th day (P<0.01). Compared with the model group, the AI ​​scores of the positive drug group and the GM-1 group showed a synchronous trend of increasing from the 22nd to the 26th day. From the 28th day, the AI ​​scores of the GM-1 and positive drug groups decreased to varying degrees (such as Figure 8 The difference was statistically significant (P<0.0001).

[0137] In summary, the paws of CIA mice were swollen significantly, the thickness increased, and the AI ​​score increased after successful modeling. Compared with the mice in the CIA model group, GM-1 and the positive drug leflunomide can reduce the thickness of joint swelling and AI score of mice, indicating that GM-1 has good application prospects in the treatment of rheumatoid arthritis.

[0138] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a product for relieving and / or treating rheumatoid arthritis, in, In the formula I, R is selected from any one of substituted or unsubstituted C3-C5 unsaturated alkenyl and substituted or unsubstituted C3-C5 branched alkyl, and the substituted groups are independently selected from any one of hydroxyl and methoxy.

2. The use according to claim 1, characterized in that: In the formula I, R is selected from any one of substituted or unsubstituted C5 unsaturated alkenyl and substituted or unsubstituted C5 branched alkyl, and the substituted groups are independently selected from any one of hydroxyl and methoxy.

3. The use according to claim 1, characterized in that: The compound represented by formula I is selected from any one of the compounds of formula I-1 to formula I-5:

4. The use according to claim 1, characterized in that: Pharmaceutically acceptable salts of the compound represented by Formula I include any one of hydrochloride, nitrate, acetate, methanesulfonate, phosphate, citrate, fumarate, sulfate, succinate, tartrate, citrate, hydrobromide, hydroiodide, lactate, toluenesulfonate, cinnamate, salicylate, malonate, glutarate, and malate.

5. The use according to any one of claims 1 to 4, characterized in that: The products for alleviating and / or treating rheumatoid arthritis include products for inhibiting cellular inflammatory response.

6. The use according to claim 1, characterized in that: The product is any one of medicine, health care product and functional food.

7. A drug for preventing and / or treating rheumatoid arthritis, characterized in that: The active substance of the drug includes the compound of formula I or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 6, wherein the compound of formula I or a pharmaceutically acceptable salt thereof is as defined in any one of claims 1 to 4.

8. The drug according to claim 7, characterized in that: Pharmaceutically acceptable excipients are also included.

9. The drug according to claim 8, characterized in that: The pharmaceutically acceptable excipients include at least one of solvents, excipients, diluents, binders, disintegrants, dispersants, flavoring agents, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, glidants, and lubricants.

10. The drug according to any one of claims 7 to 9, characterized in that: The dosage form of the drug is any one of tablets, capsules, granules, injections, powder injections, eye drops, liniments, suppositories, ointments, and aerosols.

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