Iridoid dimer, preparation method and application

Abstract

The present invention relates to the technical field of medical technology, and specifically to an iridoid dimer, a preparation method, and an application thereof. The structural formula of the iridoid dimer is shown in Formula I: The present invention separates and obtains the iridoid dimer from a thermal conversion product of swertiamarin, and verifies the pharmacological activity of the iridoid dimer through cell experiments. The new iridoid dimer compound exhibits excellent anti-inflammatory effects, has a significant inhibitory effect on NO production, can effectively reduce the expression of pro-inflammatory factors TNF-α, IL-1β, and IL-6, can reduce the inflammatory response caused by overexpression of inflammatory factors, and can be used as a lead compound for the development of 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, laying a foundation for the development of 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 iridoid dimer, a preparation method and an application thereof. Background Art

[0002] Swertiamileensis is the dried whole herb of the plant Swertia (TnHo et WLShih) of the Gentianaceae family. It is also known as hepatitis grass, gallbladder-moving medicine, and blackfish gallbladder. It is cold in nature, bitter and sweet in taste, and enters the liver, gallbladder, and bladder meridians. It has the functions of clearing the liver and promoting bile secretion, clearing heat and removing dampness, and can be used to treat jaundice, dark urine, hot stranguria, and damp-heat in the liver and gallbladder. Currently, the medicinal material Swertiamileensis and its preparation, Swertiamileensis tablets, can be used to treat hepatitis and urinary system diseases. Swertiamarin is a secoiridoid ether glycoside, which is generally considered to be the main active ingredient of Swertiamileensis 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 Swertiamileensis tablets, the "Chinese Pharmacopoeia" (Part 1, 2020 edition), uses swertiamarin as an indicator component and stipulates that the swertiamarin content should not be less than 4 mg / tablet. By referring to the preparation method of Qingyedan ​​Tablets, calculations and comparisons revealed that the content of scutellarin in Qingyedan ​​Tablets is far below the standard content of the medicinal material. Therefore, the inclusion of scutellarin as the hallmark ingredient and content detection component of Qingyedan ​​Tablets in the Chinese Pharmacopoeia over the years is unreasonable.

[0003] It is well known that iridoid compounds contain hemiacetal and ether bonds, which are unstable and prone to chemical reactions, producing brown-black resinous polymer precipitates. Therefore, the content of these components can change significantly during the processing of traditional Chinese medicines. Under certain conditions, scutellaria serrata easily forms dienes and dienophiles. Subsequently, under appropriate conditions, the dienes and dienophiles undergo intermolecular [4+2] cyclization reactions to produce new iridoid polymers. There are few reports on the thermal transformation products of scutellaria serrata at home and abroad, and no in-depth research has been conducted. Therefore, based on literature research and the research accumulated by the research team, and in order to make more effective use of plant resources, this study conducted chemical composition and activity screening of the thermal transformation products of scutellaria serrata, related to chemical and pharmacological activities. This study is of great significance for a comprehensive understanding of the properties, pathways of action, and drug development of iridoid compounds. Summary of the Invention

[0004] In order to overcome the above technical defects, the present invention provides an iridoid dimer with excellent anti-inflammatory effect obtained from the thermal conversion product of swertiamarin, a preparation method and application, 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 iridoid 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 scope of protection of the present invention:

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

[0010] The iridoid dimer having a structure as shown in Formula I or its pharmaceutically acceptable salts, tautomers, and stereoisomers can be used alone in the preparation of the food, health product, or medicine, or can be used in combination with other components having anti-inflammatory activity.

[0011] In a third aspect, the present invention provides a drug with anti-inflammatory activity, 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.

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

[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] The drug is administered via the gastrointestinal tract 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 swertiamarin monomer, add 3-5 times the amount of pure water, and reflux until swertiamarin completely disappears to obtain a water-soluble portion and a precipitate;

[0022] S2. The precipitate was subjected to silica gel column chromatography, and gradient elution was performed with dichloromethane and 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 iridoid 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 was separated by semi-preparative HPLC, the mobile phase was a methanol aqueous solution with a volume concentration of 45%, the detection wavelength was 244 nm, the flow rate was 3 mL / min, and the injection volume was 20 μL.

[0026] Compared with the prior art, the present invention has the following beneficial effects: the present invention separates and obtains an iridoid dimer from a thermal conversion product of swertiamarin, confirms the chemical structure and physicochemical properties of the iridoid dimer by modern spectroscopic methods such as HR-ESIMS, NMR, IR, HSQC, and ROESY, and verifies the pharmacological activity of the iridoid dimer by cell experiments; the new iridoid dimer compound exhibits excellent anti-inflammatory effects, has a significant inhibitory effect on NO production, 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, laying a foundation for developing the medicinal value of the iridoid dimer compound. 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 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] Figure 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 specified in the following examples are usually based on conventional conditions or 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 equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described in the text are only for demonstration purposes.

[0038] The endpoints of the ranges and any values ​​disclosed herein 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 endpoints of each range, the endpoints of each range and 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 to be specifically disclosed herein.

[0039] The present invention aims to provide an iridoid dimer obtained by separating and screening the thermal conversion products of swertiamarin. This compound has a significant inhibitory effect on NO production, can effectively reduce the expression of pro-inflammatory factors TNF-α, IL-1β and IL-6, can reduce the inflammatory response caused by the overexpression 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 iridoid 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 inhibitors of NO, TNF-α, IL-6, or IL-1β.

[0044] In some examples of this embodiment, the iridoid dimer or pharmaceutically acceptable salt, tautomer, or stereoisomer thereof as shown in Formula I can be used alone in the preparation of the food, health 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, the dosage form 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 swertiamarin monomer, add 3-5 times the amount of pure water, and reflux until swertiamarin completely disappears to obtain a water-soluble portion and a precipitate;

[0050] S2. The precipitate was subjected to silica gel column chromatography, and gradient elution was performed with dichloromethane and 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 iridoid 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 embodiment, 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.

[0054] The technical solutions of the present invention are 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 scutellaria glycoside monomer was added with 5 L of pure water, and refluxed for more than 7 h until scutellaria glycoside completely disappeared, and concentrated under reduced pressure to obtain 632 g of a water-soluble portion and 237 g of a precipitate; the precipitate 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-loaded onto the column, and eluted with a dichloromethane-methanol gradient with a volume ratio of 200:1, 100:1, 50:1, 20:1, and 1:1, respectively. After TLC detection, the same fractions were combined and concentrated under reduced pressure to collect an extract with dichloromethane-methanol = 100:1; the extract with dichloromethane-methanol = 100:1 was passed through a semi-preparative high performance liquid chromatograph using an Agilent The product was separated by ZorbaxSB-C18 (9.4×150 nm, 5 μm, 3 mL / min) and isocratic elution was performed with a mobile phase volume ratio of methanol / water of 45:55. The injection volume was 20 μL and the UV detection wavelength was 244 nm to obtain 5 g of colorless cubic crystals named Swertialactone A.

[0057] Example 2 Structural Characterization of Iridoid Dimers

[0058] The mass spectrometry and nuclear magnetic resonance characterization results of Swertialactone A (such as Figure 1-2 and Table 1), it can be confirmed that the molecular formula of the iridoid 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 was -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 ) in 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) and constructed the A and E rings; 1 H- 1 Related signals of H COSY spectrum: [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')] deduced the connected fragments C-1' / C-9' / C-8' / C-10' / C-3 in the structure, according to H-1 to C-3 (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 mode of the A ring and the B ring was determined by the correlation from H-1 to C-5 and H-3 to C-4; the connection mode of the A ring and the B ring was determined by the correlation from H-1 to C-8'(δ C 72.9), the C ring can be identified from the correlation between H-3 and C-8', as well as from H-10' and C-9'; the connection between the B ring and the C ring can be determined based on the correlation between H-1 and C-8', and H-3 and C-8'; the existence of the D ring can be known from the correlation between H-3' and 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, the planar structure of Swertialactone A is deduced to be an iridoid dimer containing two α-,β-unsaturated δ-lactones, with a 6 / 6 / 6 / 6 / 6 / 6 six-membered ring fused to an epoxy ring. Crystallographic data of Swertialactone A (CCDC 2204368) confirm the planar structure and clarify the relative configuration of Swertialactone A as 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 96-well plates, 100 μL per well, and cultured in a 37°C, 5% CO2 incubator for 24 hours. A control group (normally cultured RAW264.7 cells) and a drug-treated 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 hours of culture, 10 μL of CCK-8 was added to each well and incubated for 1.5 hours. The OD was measured using a microplate reader. 450 , the results are as follows Figure 8 As shown, it is shown that the iridoid dimer prepared by the present invention has no cytotoxic effect.

[0065] Example 4 Investigating the Effects 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 96-well plates 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-treated 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. Figure 9 shown.

[0067] It can be seen that compared with the control group, the levels 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 cell culture supernatant were reduced, indicating that the iridoid 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 various similar expressions without violating the purpose and claims of the present invention. Such changes fall within the scope of protection of the present invention.

Claims

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

2. Use of the iridoid dimer of formula I or a pharmaceutically acceptable salt thereof in the preparation of an anti-inflammatory drug.

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 shown in formula I or a pharmaceutically acceptable salt thereof is used alone in the preparation of medicines, or is used in combination with other components having anti-inflammatory activity.

5. A composition having anti-inflammatory activity, characterized in that The invention comprises an iridoid dimer as shown in formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

6. The composition according to claim 5, characterized in that Other components with anti-inflammatory activity are also included.

7. The composition according to claim 5 or 6, which is in an oral dosage form or an injectable dosage form, or a controlled-release or sustained-release dosage form.

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

9. The preparation method according to claim 8, characterized in that In the S2, gradient elution was 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 the above-mentioned S3, the concentrate was separated by semi-preparative HPLC, the mobile phase was a methanol aqueous solution with a volume concentration of 45%, the detection wavelength was 244 nm, the flow rate was 3 mL / min, and the injection volume was 20 μL.

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