Iridoid dimer, preparation method and application

Abstract

The invention relates to the technical field of medicines, in particular to an iridoid dimer as well as a preparation method and application thereof. The iridoid dimer is separated from a swertiamarin thermal conversion product, the pharmacological activity of the iridoid dimer is verified through cell experiments, and the novel iridoid dimer compound shows an excellent anti-inflammatory effect, has an obvious inhibiting effect on NO generation, and can be used for preparing the novel iridoid dimer compound. Expression of proinflammatory factors TNF-alpha, IL-1beta and IL-6 can be effectively reduced, inflammatory reaction caused by overexpression of inflammatory factors can be reduced, and the compound can be used as a lead compound for developing novel anti-inflammatory drugs. The preparation steps of the novel iridoid dimer skeleton compound provided by the invention are easy to control, simple and rapid, and a foundation is laid for developing the medicinal value of the iridoid dimer compound.

Description

Technical Field

[0001] The present invention relates to the technical field of medical technology, and in particular to an icroid dimer, a preparation method and an application thereof. Background Art

[0002] Swertia quinata is the dried whole herb of Swertia quinata (TNHo et WLShih), a plant of the Gentianaceae family (Gentianaceae). It is also known as hepatitis grass, gallbladder medicine, bluefish gallbladder, etc. It is cold in nature, bitter and sweet in taste; it enters the liver, gallbladder, and bladder meridians; it has the functions of clearing the liver and promoting bile, clearing heat and removing dampness; it can be used to treat jaundice, red urine, hot stranguria, and damp-heat in the liver and gallbladder. At present, the medicinal material of Swertia quinata and its preparation, Swertia quinata tablets, can be used to treat hepatitis and urinary system diseases. Swertiamarin is a swertiamarin glycoside, which is generally considered to be the main active ingredient of Swertia quinata for the treatment of hepatitis. Its content in the whole plant is as high as 12%, and its content in the cold water extract exceeds 50%. The current quality standard for Swertia quinata tablets, the Chinese Pharmacopoeia (2020 Edition Part I), uses swertiamarin as an indicator component and stipulates that the content of swertiamarin should not be less than 4 mg / tablet. Referring to the preparation method of Qingyedan ​​Tablets, through calculation and comparison, it was found that the content of swertiamarin in Qingyedan ​​Tablets was far lower than the standard content of medicinal materials. Therefore, the inclusion of swertiamarin as the characteristic component and content detection component of Qingyedan ​​Tablets in the "Chinese Pharmacopoeia" over the years is unreasonable.

[0003] As is known to all, there are hemiacetal and ene ether bonds in cyclopentadienoids, which are unstable in nature and prone to chemical reactions, producing brown-black resinous polymer precipitations. Therefore, the content of such components will change significantly during the processing of traditional Chinese medicines. Under certain conditions, cyclopentadienol is easy to form dienes and dienophiles, and then the dienes and dienophiles undergo intermolecular [4+2] cyclization reactions under appropriate conditions to generate new cyclopentadienoid polymers. There are few reports on the thermal transformation products of cyclopentadienol at home and abroad, and no in-depth research has been conducted. Therefore, based on literature surveys and the research accumulation of the research team, in order to make more effective use of plant resources, this study conducted relevant research on the chemical composition and activity screening of the thermal transformation products of cyclopentadienol, which is of great significance for a comprehensive understanding of the properties, action pathways and drug development of cyclopentadienoids. Summary of the invention

[0004] In order to overcome the above technical defects, the present invention provides an cyclopentadiene dimer with excellent anti-inflammatory effect obtained from the thermal transformation product of swertiamarin, a preparation method and application thereof, and studies its anti-inflammatory activity and medical use.

[0005] In a first aspect, the present invention provides an iridoid dimer having a structure as shown in Formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0006]

[0007] In a second aspect, the present invention provides use of an iridium dimer having a structure as shown in Formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof in the preparation of an anti-inflammatory drug.

[0008] Furthermore, the following applications are also within the protection scope of the present invention:

[0009] The iridoid dimer with a structure as shown in formula I or its pharmaceutically acceptable salt, tautomer, stereoisomer is used to prepare inhibitors of NO, TNF-α, IL-6, and IL-1β.

[0010] The iridoid dimer or its pharmaceutically acceptable salt, tautomer, stereoisomer with a structure as shown in formula I can be used alone in the preparation of the food, health care product or medicine, or can be used in combination with other components with anti-inflammatory activity.

[0011] In a third aspect, the present invention provides a drug having anti-inflammatory activity, comprising an icroid dimer as shown in formula I, a pharmaceutically acceptable salt, tautomer, stereoisomer and pharmaceutically acceptable excipient of the icroid dimer as shown in formula I.

[0012] In particular, the pharmaceutically acceptable carriers are generally recognized for this purpose and serve as inactive ingredients of medicaments.

[0013] The carrier includes excipients, such as starch, water, etc.; lubricants, such as magnesium stearate, etc.; disintegrants, such as microcrystalline cellulose, etc.; fillers, such as lactose, etc.; binders, such as pregelatinized starch, dextrin, etc.; sweeteners; antioxidants; preservatives; flavoring agents; spices, etc.

[0014] The drug is in the form of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, and patches.

[0015] Wherein, the drug is administered via gastrointestinal and parenteral routes.

[0016] Particularly, the non-gastrointestinal administration route is selected from injection, respiratory tract administration, skin administration, mucosal administration or cavity administration.

[0017] Among them, the parenteral preparation is selected from injections, sprays, aerosols, patches and the like.

[0018] Particularly, the gastrointestinal administration preparation is selected from tablets, capsules, powders, granules, pills, solutions, emulsions or syrups.

[0019] Furthermore, the drug with anti-inflammatory activity provided by the present invention can be used in combination with other components with anti-inflammatory activity.

[0020] In a fourth aspect, the present invention provides a method for preparing an iridoid dimer having a structure as shown in Formula I, comprising the following steps:

[0021] S1. Take the swertiamarin monomer, add 3-5 times the amount of pure water, and reflux and heat until the swertiamarin completely disappears to obtain a water-soluble portion and a precipitate portion;

[0022] S2. The precipitate was subjected to silica gel column chromatography, and gradient elution was performed with dichloromethane-methanol, and the target gradient eluate was collected and concentrated under reduced pressure;

[0023] S3. The concentrated solution is then separated and purified by semi-preparative HPLC to obtain an icroterpene dimer as shown in formula I.

[0024] Furthermore, in S2, gradient elution is performed using dichloromethane-methanol with volume ratios of 200:1, 100:1, 50:1, 20:1, and 1:1, in sequence.

[0025] Furthermore, in S3, the concentrate is separated by semi-preparative HPLC, the mobile phase is a methanol aqueous solution with a volume concentration of 45%, the detection wavelength is 244 nm, the flow rate is 3 mL / min, and the injection volume is 20 μL.

[0026] Compared with the prior art, the present invention has the following beneficial effects: the present invention separates and obtains iridoid dimers from the thermal transformation products of swertiamarin, confirms the chemical structure and physicochemical properties of the iridoid dimers by modern spectroscopy methods such as HR-ESIMS, NMR, IR, HSQC, ROESY, etc., and verifies the pharmacological activity of the iridoid dimers by cell experiments; the new iridoid dimer compounds exhibit excellent anti-inflammatory effects, have obvious inhibitory effects on NO generation, can effectively reduce the expression of pro-inflammatory factors TNF-α, IL-1β and IL-6, can reduce the inflammatory response caused by excessive expression of inflammatory factors, and can be used as a lead compound for developing new anti-inflammatory drugs; the preparation steps of the new iridoid dimer skeleton compound provided by the present invention are easy to control, simple and rapid, and lay a foundation for developing the medicinal value of iridoid dimer compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The iridoid dimer of the present invention 1 H NMR spectrum;

[0028] Figure 2 The iridoid dimer of the present invention 1 C NMR spectrum;

[0029] Figure 3 is the IR spectrum of the iridoid dimer of the present invention;

[0030] Figure 4 The iridoid dimer of the present invention 1 H- 1 H COSY spectrum;

[0031] Figure 5 is the HSQC spectrum of the iridoid dimer of the present invention;

[0032] Figure 6 is the HMBC spectrum of the iridoid dimer of the present invention;

[0033] Figure 7 is the ROESY spectrum of the iridoid dimer of the present invention;

[0034] Figure 8 The effect of the iridoid dimer of the present invention on the survival rate of RAW264.7 cells;

[0035] Fig. 9 The effect of the iridoid dimer of the present invention on LPS-induced inflammatory factors;

[0036] In the figure, #### indicates significant difference compared with the blank group, P<0.0001; * indicates significant difference compared with the LPS group, P<0.01; *** indicates significant difference compared with the LPS group, P<0.001; **** indicates significant difference compared with the LPS group, P<0.0001. DETAILED DESCRIPTION

[0037] In order to make those skilled in the art better understand the technical scheme of the present invention, the present invention is described in detail below in conjunction with specific embodiments. The experimental methods for which specific conditions are not indicated in the following examples are usually carried out under normal conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar to or equal to the recorded content can all be applied to the present invention. The preferred implementation methods and materials described in the text are only for demonstration purposes.

[0038] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0039] The present invention aims to provide an cyclopentadiene dimer, which is obtained by separating and screening the thermal transformation products of swertiamarin. The compound has a significant inhibitory effect on NO generation, can effectively reduce the expression of pro-inflammatory factors TNF-α, IL-1β and IL-6, can reduce the inflammatory response caused by excessive expression of inflammatory factors, and has good anti-inflammatory activity.

[0040] In some specific embodiments, an iridoid dimer having a structure as shown in Formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof is provided:

[0041]

[0042] In some specific embodiments, provided is the use of an icroid dimer having a structure as shown in Formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof in the preparation of an anti-inflammatory drug.

[0043] In some examples of this embodiment, the iridoid dimer having a structure as shown in Formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof is used to prepare an inhibitor of NO, TNF-α, IL-6, or IL-1β.

[0044] In some examples of this embodiment, the cyclopentadiene dimer or pharmaceutically acceptable salt, tautomer, stereoisomer thereof as shown in formula I can be used alone in the preparation of the food, health care product or medicine, or can be used in combination with other components having anti-inflammatory activity.

[0045] In another specific embodiment, a drug with anti-inflammatory activity is provided, comprising an iridoid dimer as shown in Formula I, a pharmaceutically acceptable salt, tautomer, stereoisomer and pharmaceutically acceptable excipient of the iridoid dimer as shown in Formula I.

[0046] In some examples of this embodiment, other components with anti-inflammatory activity are also included.

[0047] In some examples of this embodiment, it is an oral dosage form or an injection dosage form, and a controlled release or sustained release dosage form known in the modern pharmaceutical industry can also be used.

[0048] In another specific embodiment, a method for preparing an iridoid dimer having a structure as shown in Formula I is provided, comprising the following steps:

[0049] S1. Take the swertiamarin monomer, add 3-5 times the amount of pure water, and reflux and heat until the swertiamarin completely disappears to obtain a water-soluble portion and a precipitate portion;

[0050] S2. The precipitate was subjected to silica gel column chromatography, and gradient elution was performed with dichloromethane-methanol, and the target gradient eluate was collected and concentrated under reduced pressure;

[0051] S3. The concentrated solution is then separated and purified by semi-preparative HPLC to obtain an icroterpene dimer as shown in formula I.

[0052] In some examples of this embodiment, in S2, gradient elution is performed using dichloromethane-methanol with volume ratios of 200:1, 100:1, 50:1, 20:1, and 1:1, in sequence.

[0053] In some examples of this implementation mode, in S3, the concentrate is separated by semi-preparative HPLC, the mobile phase is a methanol aqueous solution with a volume concentration of 45%, the detection wavelength is 244nm, the flow rate is 3mL / min, and the injection volume is 20μL.

[0054] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0055] Example 1 Extraction and separation of iridoid dimers

[0056] 1000 g of swertiamarin monomer was added with 5 L of pure water, and refluxed for more than 7 h until swertiamarin completely disappeared, and concentrated under reduced pressure to obtain 632 g of a water-soluble portion and 237 g of a precipitate portion; the precipitate portion was mixed with 1 times the sample amount of column chromatography silica gel, and 8 times the sample amount of column chromatography silica gel (W / W) was dry-column-loaded, and gradient eluted with dichloromethane-methanol in a volume ratio of 200:1, 100:1, 50:1, 20:1, and 1:1, respectively. After TLC detection, the same portions were combined, and the extract of dichloromethane-methanol = 100:1 was concentrated under reduced pressure to collect; the extract of dichloromethane-methanol = 100:1 was passed through a semi-preparative high performance liquid chromatograph, and the Agilent ZorbaxSB-C18 (9.4×150nm, 5μm, 3mL / min) was used for separation, and the mobile phase volume ratio was 45:55 methanol / water for isocratic elution. The injection volume was 20μL and the UV detection wavelength was 244nm to obtain 5g of colorless cubic crystals named Swertialactone A.

[0057] Example 2 Structural Characterization of Iridoid Dimers

[0058] The mass spectrometry and NMR characterization results of Swertialactone A (such as Figure 1-2 and Table 1), it can be confirmed that the molecular formula of the cyclopentadiene dimer is C 19 H 18 O7,

[0059] Table 1

[0060]

[0061]

[0062] According to the HRESIMS mass spectrum data, the ion peak m / z 359.1121 [M+H] + indicates that its molecular formula is C19H18O7 (calculated value C 19 H 19 O7,359.1125), the unsaturation is 11; combined with infrared spectrum ( Figure 3 ) can be seen that the ester carbonyl (1699cm -1 ) and double bond (1627cm -1 ), optical rotation value is -8.60 (c 0.1, MeOH); 13 C-NMR spectrum ( Figure 2 ) shows that 19 carbon signals are given, including 7 quaternary carbons, 6 methines, and 6 methylenes. Among them, 2 carbonyl carbon atoms [δ C =164.7ppm(C-11'),δ C =162.2ppm(C-11)] and 4 double bond carbons [δ C =158.5ppm(C-3'),δ C =145.3ppm(C-4),δ C =133.3ppm(C-5),δ C =113.2 ppm (C-4′)] could be determined. 1 H- 1 H COSY spectrum ( Figure 4 ) shows that [δ H 4.38,td,J=11.3,4.2Hz(H-7) / δ H 2.13,m(H-6)],[δ H 4.44,m(H-7') / δ H 2.23,m(H-6')], indicating that there are two key fragments (C-7 / C-6) and (C-7' / C-6'); combined with the HMBC spectrum ( Figure 6 ) from H-7 to C-4 (δ C145.3) and C-11 (δ C 162.2), H-6 to C-4, C-5 (δ C 133.3) and C-7 (δ C 66.1), H-7' to C-5' (δ C 35.5) and C-11'(δ C 164.7); H-6' to C-4' (δ C 113.2), C-5' and C-7' (δ C 65.1), the A ring and the E ring were constructed; 1 H- 1 H COSY spectrum related signals: [δ H 4.82,m(H-1) / δ H 2.31,m(H-9')],[δ H 4.10,d,J=6.8Hz(H-3) / δ H 2.31,m(H-10')],[δ H 2.31,m(H-9') / δ H 3.97,m(H-8') / δ H 2.31,m(H-10')] to deduce the connected fragments C-1' / C-9' / C-8' / C-10' / C-3 in the structure. According to H-1 to C3 (δ C 70.7) and C-5 (δ C 133.3); H-3 to C-1 (δ C 64.1) and C-4 to construct the B ring; the connection between the A ring and the B ring was determined by the correlation between H-1 to C-5 and H-3 to C-4; the correlation between H-1 to C-8' (δ C 72.9), the C ring can be identified from the correlations from H-3 to C-8', and from H-10' and C-9'; the connection between the B ring and the C ring can be determined from the correlations from H-1 to C-8' and H-3 to C-8'; the existence of the D ring can be known from the correlations from H-3' to C-5' and C-8'. H 3.36(H-8) / δ H 2.49(H-10)], combined with the HMBC spectrum ( Figure 6 ):H-6' to C-10(δ C33.6), the position of the F ring was determined based on the correlations of H-9' to C-9 and H-10 to C-9, and the interconnection of the D, E, and F rings was determined based on the correlations of H-3' to C-8', H-3' to C-5', H-3' to C-11', H-6' to C-9', H-6' to C-10, H-10 to C9, and H-9' to C-9. C =54.6ppm,H-8δ H =3.36ppm,C-9δ C =54.3ppm], it can be deduced that the G ring is an epoxy ring. Therefore, it is deduced that the planar structure of Swertialactone A is a cyclopentadiene dimer containing two α, β-unsaturated δ-lactones with a 6 / 6 / 6 / 6 / 6 / 6 six-membered ring fused to an epoxy ring. The crystallographic data of Swertialactone A (CCDC 2204368) confirmed the planar structure and clarified that the relative configuration of Swertialactone A is 1R*, 3S*, 5'R*, 8'S*, 9'S*, 8S*, 9R*.

[0063] Example 3 Investigation of the cytotoxicity of iridoid dimers

[0064] Take the RAW264.7 cell suspension in the logarithmic growth phase and use 1×10 5 / mL density was seeded in a 96-well plate, 100 μL per well, and cultured in a 37°C, 5% CO2 incubator for 24 h. A control group (normally cultured RAW264.7 cells) and a drug group (normally cultured RAW264.7 cells were added with 25, 50, 100, and 200 μmol / L of Swertialactone A, respectively) were set up. After 24 h of culture, 10 μL of CCK-8 was added to each well and incubated for 1.5 h. The OD was measured with an enzyme reader. 450 , the results are as follows Figure 8 As shown, it is indicated that the iridoid dimer prepared by the present invention has no cytotoxic effect.

[0065] Example 4 Investigating the effect of iridoid dimers on the expression of inflammatory factors NO, TNF-α, IL-6 and IL-1β

[0066] RAW264.7 cells in the logarithmic growth phase were inoculated into a 96-well plate and cultured in an incubator for 24 hours. Four groups were set up: control group: RAW264.7 cells cultured normally; model group: lipopolysaccharide (LPS) at a concentration of 1 μg / mL; positive drug group: dexamethasone (DEX) at a concentration of 25 μmol / L; drug administration group: Swertialactone A at concentrations of 25, 50, and 100 μmol / L, respectively. After incubation for 24 hours, the supernatant was collected and centrifuged to remove residual cells. The corresponding contents were determined according to the instructions of the NO content detection kit and the ELISA kit for TNF-α, IL-6, and IL-1β detection. The results are shown in the figure. Fig. 9 shown.

[0067] It can be seen that compared with the control group, the contents of NO, TNF-α, IL-6, and IL-1β in the LPS group were increased (P < 0.0001), indicating that the model was successfully replicated; compared with the LPS group, 24 hours after administration, the levels of NO, TNF-α, IL-6, and IL-1β in the supernatant of the cell culture fluid were reduced, indicating that the cyclopentadiene dimer prepared by the present invention has a certain anti-LPS-induced RAW264.7 cell damage activity.

[0068] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Under the guidance of the present invention, those skilled in the art can make a variety of similar expressions without violating the purpose and claims of the present invention, and such changes all fall within the protection scope of the present invention.

Claims

1. An iridoid dimer having a structure as shown in Formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

2. Use of the iridoid dimer having a structure as shown in formula I in claim 1 or its pharmaceutically acceptable salt, tautomer, stereoisomer in the preparation of anti-inflammatory drugs.

3. The use according to claim 2, characterized in that: Used to prepare inhibitors of NO, TNF-α, IL-6, and IL-1β.

4. The use according to claim 2, characterized in that The iridoid dimer or its pharmaceutically acceptable salt, tautomer, stereoisomer as shown in formula I can be used alone in the preparation of the food, health care product or medicine, or can be used in combination with other components having anti-inflammatory activity.

5. A drug having anti-inflammatory activity, characterized in that The invention comprises an iridoid dimer as shown in formula I, and pharmaceutically acceptable salts, tautomers, stereoisomers and pharmaceutically acceptable excipients of the iridoid dimer as shown in formula I.

6. The composition according to claim 5, characterized in that Other anti-inflammatory active ingredients are also included.

7. The composition according to claim 6 or 7 is in oral dosage form or injection form, and can also adopt controlled release or sustained release forms known in the modern pharmaceutical industry.

8. The method for preparing the iridoid dimer having a structure as shown in Formula I in claim 1, characterized in that: The following steps are involved: S1. Take the swertiamarin monomer, add 3-5 times the amount of pure water, and reflux and heat until the swertiamarin completely disappears to obtain a water-soluble portion and a precipitate portion; S2. The precipitate was subjected to silica gel column chromatography, and gradient elution was performed with dichloromethane-methanol, and the target gradient eluate was collected and concentrated under reduced pressure; S3. The concentrated solution is then separated and purified by semi-preparative HPLC to obtain an icroterpene dimer as shown in formula I.

9. The preparation method according to claim 8, characterized in that: In the S2, gradient elution is performed using dichloromethane-methanol with volume ratios of 200:1, 100:1, 50:1, 20:1, and 1:1 in sequence.

10. The preparation method according to claim 8, characterized in that: In S3, the concentrate is separated by semi-preparative HPLC, the mobile phase is a methanol aqueous solution with a volume concentration of 45%, the detection wavelength is 244 nm, the flow rate is 3 mL / min, and the injection volume is 20 μL.

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