Iridoid compounds with anti-inflammatory activity and preparation method and application thereof

Abstract

The present invention belongs to the field of medical technology, and specifically relates to three iridoid compounds with anti-inflammatory activity, as well as preparation methods and applications thereof. The present invention provides a preparation method for three new iridoid compounds extracted from the leaves of the rhizome of the Chinese twig. The three new iridoid compounds all exhibit excellent anti-inflammatory effects, have a significant inhibitory effect on NO production, can effectively reduce the expression of pro-inflammatory factors TNF-a and IL-6, can reduce the inflammatory response caused by the overexpression of inflammatory factors, and can be used as lead compounds for the development of new anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to three iridoid compounds with anti-inflammatory activity and preparation methods and applications thereof. Background Art

[0002] War bone (Zhan Gu) is a commonly used traditional Chinese medicine. The plant's name is Premnafulva Craib, a member of the Verbenaceae family and genus Premna. It's also known as Tubawang (Tubawang) and Chuanyunjian (Chuanyunjian). Its roots, stems, and leaves are all used medicinally, boasting benefits such as promoting blood circulation, dispelling ailments, strengthening tendons and bones, and dispelling wind and relieving pain. It's commonly used in Guangxi to treat low back pain, sprains, rheumatoid arthritis, and liver pain, making it a local Guangxi medicinal herb.

[0003] The investigation found that many products have been developed with Zhan Gu as the main raw material, such as: Jian Gu Injection, Compound Huangmao Doufu Chai Liniment, Zhan Gu Mixture, etc., which have anti-inflammatory, detumescent and analgesic effects, and are mainly used for the treatment of ankylosing spondylitis (i.e. lumbar spondylosis), periarthritis of the shoulder and other diseases.

[0004] A typical manifestation of inflammatory response is the excessive accumulation of nitric oxide (NO), and the production of NO is regulated by nitric oxide synthase. The main chemical components in the leaves of the genus Artemisia selengensis are divided into iridoid terpenes and their glycosides, which have good anti-inflammatory, analgesic, anti-osteoporosis and other activities. At present, the research on the chemical components of the genus Artemisia selengensis and its pharmacological activity has found that the main research focus of the pharmacological activity is on the rhizomes, but there are few studies on the leaves of the genus Artemisia selengensis at home and abroad. Therefore, based on literature research and the research accumulation of the research group, in order to make more full use of plant resources, the inventors studied the chemical components and activity screening of the leaves of the genus Artemisia selengensis, and provided three iridoid terpenes with anti-inflammatory activity and their preparation methods and applications. Summary of the Invention

[0005] In order to overcome the deficiencies in the existing technology for the research and development of the resources of the rhizome of scutellaria baicalensis, the present invention provides an cyclopentadiene ether terpenoid compound with excellent anti-inflammatory effect obtained from the rhizome of scutellaria baicalensis and a preparation method thereof, and provides its anti-inflammatory activity and medical use.

[0006] The first object of the present invention is to provide three new iridoid ether terpenoid compounds extracted from the leaves of the rhamnoides genus: 6-O-(2″-(3″′-O-trans-p-methoxycinnamoyl-α-L-rhamnopyranosyl)-3″-O-trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol (1); 6-O-(2″,4″-di-trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol (2); and 6-O-(3″-O-trans-m-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol (3).

[0007] A second object of the present invention is to provide a method for preparing the above-mentioned iridoid compounds 6-O-(2″-(3″′-O-trans-p-methoxycinnamoyl-α-L-rhamnopyranosyl)-3″-O-trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol (1); 6-O-(2″, 4″-di-trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol (2); and 6-O-(3″-O-trans-m-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol (3).

[0008] A third object of the present invention is to provide the use of the above-mentioned iridoid ether terpenoid compounds 6-O-(2″-(3″′-O-trans-p-methoxycinnamoyl-α-L-rhamnopyranosyl)-3″-O-trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol (1); 6-O-(2″, 4″-di-trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol (2); and 6-O-(3″-O-trans-m-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol (3) in the preparation of anti-inflammatory drugs.

[0009] The present invention is to extract and separate three new iridoid ether terpenoid compounds from the leaves of Premnafulva for the first time, namely 6-O-(2″-(3″′-O-trans-p-methoxycinnamoyl-α-L-rhamnopyranosyl)-3″-O-trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol (1), 6-O-(2″,4″-di-trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol (2), and 6-O-(3″-O-trans-m-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol (3), with molecular formulas of C 47 H 58 O 22 、C 41 H 48 O 18 、C 31 H 40 O 16 ; Its chemical structure is as follows:

[0010]

[0011] 6-O-(2″-(3″′-O-trans-p-methoxycinnamoyl-α-L-rhamnopyranosyl)-3″-O-trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol(1)

[0012]

[0013] 6-O-(2″,4″-di-trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol(2)

[0014]

[0015] 6-O-(3″-O-trans-m-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol(3)

[0016] The present invention also provides a method for preparing an iridoid compound having anti-inflammatory activity, comprising the following steps:

[0017] S1. Taking the leaves of the scutellaria baicalensis, soaking them in an alcohol solvent and filtering them, repeating this process at least once, combining them to obtain an extract, and removing the solvent from the extract to obtain a total extract;

[0018] S2. The total extract obtained in step S1 is subjected to macroporous adsorption resin column chromatography, and gradient elution is performed with a mixed eluent of alcohol and water, the gradient eluates of each gradient are collected, and the eluates are detected by TLC spot plate. Similar elution fractions are combined and concentrated to obtain 6 fractions Fr.1 to Fr.6;

[0019] S3. Subjecting the fourth fraction Fr.4 obtained in step S2 to column chromatography using a mixed eluent of alcohol and water for gradient elution, collecting the gradient eluates, detecting the fractions by TLC spot plate, combining and concentrating similar elution fractions, and collecting 8 fractions Fr.4-(1-8);

[0020] S4. The first fraction Fr.4-1 obtained in step S3 is first subjected to column chromatography with a gradient elution using a mixed eluent of alcohol and water, followed by thin-layer chromatography detection. Similar fractions are combined and concentrated to obtain subfractions Fr.4-1-(1-10). The subfraction Fr.4-1-5 is then subjected to gel column chromatography with a gradient elution using a mixed eluent of alcohol and water, followed by thin-layer chromatography detection. Similar fractions are combined and concentrated to obtain nine fractions Fr.4-1-5-(1-9).

[0021] S5. Elution of the fourth fraction Fr.4-1-5-4 obtained in step S4 by HSCCC chromatography was performed, and detection was performed by TLC spot plate. Similar elution fractions were combined and concentrated to obtain six subfractions Fr. I-VI.

[0022] S6. The fourth fraction Fr.Ⅳ obtained in step S5 is separated and purified by semi-preparative HPLC to obtain compounds 1 and 2 shown in the structure of claim 1; the fifth fraction Fr.Ⅴ obtained in step S5 is separated and purified by semi-preparative HPLC to obtain compound 3 shown in the structure of claim 1.

[0023] Specifically, in step S1:

[0024] The alcohol solvent is methanol or ethanol, preferably methanol;

[0025] The usage ratio of the scutellaria baicalensis leaf to the alcohol solvent is 15kg:(30-90)L, preferably 15kg:60L;

[0026] Soaking at room temperature; soaking time is (5-9) days, preferably 7 days;

[0027] The number of repetitions is 2-4 times, preferably 3 times.

[0028] Preferably, in step S1, the leaves of Prenmafulva Craib were collected from Guilin Botanical Garden in Yanshan District, Guilin City, Guangxi Zhuang Autonomous Region, chopped and dried in the shade indoors, and identified by researcher Tang Hui as the leaves of Prenmafulva Craib, a plant of the genus Tofu-tree of the Verbenaceae family. The certificate samples were stored in the Guangxi Key Laboratory of Plant Functional Substances and Sustainable Utilization of Resources.

[0029] Specifically, in step S2:

[0030] The macroporous adsorption resin columns are D101 column, HPD-100, and HP20SS, with D101 column being preferred. D101 type macroporous resin is a styrene type non-polar copolymer with a relatively broad application range, strong adsorption capacity, large pore size and easy elution, which is better than HPD-100 and HP20SS.

[0031] Alcohol is methanol or ethanol, preferably methanol;

[0032] The volume ratio gradient of alcohol and water is 10:90-100:0, preferably, 10:90, 20:80, 30:70, 50:50, 80:20, and 100:0.

[0033] Preferably, in step S2, the D101 column has a length of 100 cm and an inner diameter of 10 cm.

[0034] Specifically, in step S3:

[0035] Column chromatography uses C18, C8, MCI, and HP20SS, with C18 columns being preferred because C18 has a longer carbon chain than C8 and has better retention properties for separated compounds. In addition, the separation function of MCI and HP20SS materials is a molecular sieve function, and their effect on the preliminary segmentation and re-separation of crude extracts that have been pore-screened is not as good as that of C18.

[0036] Alcohol is methanol or ethanol, preferably methanol;

[0037] The volume ratio gradient of alcohol and water is 40:60-100:0, preferably, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, and 100:0.

[0038] Preferably, the C18 column in step S3 has a length of 50 cm and an inner diameter of 5 cm.

[0039] Specifically, in step S4:

[0040] Column chromatography uses MCI-GEL CHP series materials with different particle sizes (4μm-300μm), with HP 20SS columns being preferred. MCI-GELCHP20P (HP 20SS) combines the functions of a molecular sieve and a reversed-phase material, meeting the requirements for extract separation efficiency and fine separation.

[0041] Gel column chromatography uses Sephadex LH-20 and Sephadex G-25, with Sephadex LH-20 being preferred because Sephadex LH-20 has both gel filtration and reverse phase chromatography effects during the separation process, resulting in better separation results than other chromatographic columns.

[0042] Alcohol is methanol or ethanol, preferably methanol;

[0043] The volume ratio gradient of alcohol and water in column chromatography is 20:80-100:0, preferably 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, and 100:0;

[0044] The volume ratio gradient of alcohol and water in gel column chromatography is 20:80-100:0, preferably, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0.

[0045] Preferably, the HP 20SS column has a length of 50 cm and an inner diameter of 5 cm; the gel column has a length of 60 cm and an inner diameter of 4 cm.

[0046] The reason why methanol and water are used as eluents in steps S1-S4 is that methanol and water are commonly used mobile phases. Methanol mainly reduces the amount of water contacting the carrier, and water mainly adjusts the retention time. Through the adjustment of water, the peak of the component to be measured can be delayed, thereby achieving a good separation effect. The use of other organic solvents and water as eluents will encounter problems of high cost and solvent viscosity, respectively, and it is impossible to carry out a large number of experiments.

[0047] Specifically, the elution conditions in step S5 are as follows: the solvent system is a two-phase solvent system of ethyl acetate, n-butanol and water, the rotation speed is 880 rpm, the flow rate is 3.0 mL / min, the detection wavelength is 230 nm, the column temperature is 25 ° C, the elution time is 280 min, and the injection volume is 320 mg; the volume ratio gradient of ethyl acetate, n-butanol and water is 5:5:10, 7:3:10, 7.5:2.5:10, and 8:2:10, respectively.

[0048] More specifically, in step S5, the high-speed countercurrent chromatography uses a TautoTBE-300C system, equipped with 3 polytetrafluoroethylene multilayer coils (inner diameter: 1.9 mm; total volume 320 mL), a 20 mL sample loop, a DC-0506 low-temperature thermostat, a dual-wavelength UV detector to measure UV absorbance, and an Advantec CHF161RA fraction collector; Easychrom-1000 software is used for data acquisition and analysis; the elution conditions are: the solvent system is a two-phase solvent system of ethyl acetate / n-butanol / water (7.5:2.5:10 v / v) (the stationary phase is an organic phase and the mobile phase is an aqueous phase), the rotation speed is 880 rpm, the flow rate is 3.0 mL / min, the detection wavelength is 230 nm, the column temperature is 25°C, and the elution time is 280 min.

[0049] Specifically, in step S6:

[0050] Subfraction Fr.Ⅳ was separated and purified by semi-preparative HPLC;

[0051] The elution conditions were as follows: gradient elution with methanol and water as the mobile phases, a flow rate of 3.0 mL / min, and a detection wavelength of 230 nm, to obtain compound 1 and compound 2, respectively; the gradient volume ratio of methanol to water was 40:60-68:32.

[0052] The subfraction Fr.V was separated and purified by semi-preparative high performance liquid chromatography; the elution conditions were: gradient elution with methanol and water as the mobile phase, the volume ratio gradient of methanol and water was 40:60-50:50, the flow rate was 3.0 mL / min, and the detection wavelength was 230 nm to obtain compound 3.

[0053] More specifically, the semi-preparative HPLC in step S6 was performed using an Agilent 1260 system equipped with a UV detector and a ZorbaxSB-C18 column (250 mm in length, 9.4 mm in inner diameter, 5 μm); the elution conditions were: gradient elution with methanol and water as the mobile phase, a flow rate of 3.0 mL / min, and a detection wavelength of 230 nm.

[0054] The above preparation method has the following innovative features:

[0055] 1. The present invention obtains three iridoid glycoside compounds with anti-inflammatory activity from the leaves of the rhizome of scutellaria baicalensis.

[0056] 2. Because iridoid glycosides have similar structures, traditional separation requires repeated column chromatography (up to six times or more), which is time-consuming and requires the use of large amounts of solvent. The present invention first uses column chromatography (three times) to crudely separate the extract, then uses high-speed countercurrent chromatography (liquid-liquid separation chromatography with high sample reproducibility and high recovery), preferably using a separation solvent system, to efficiently enrich the iridoid glycosides. Finally, preparative high-performance liquid chromatography is used to separate the individual iridoid glycosides.

[0057] The present invention also provides the use of the three new iridoid compounds for preparing anti-inflammatory drugs.

[0058] The iridoid compounds described in the present invention are new chemical components discovered by the inventors from the leaves of the rhizome ... 1 H-NMR, 13 C-NMR, HSQC, HMBC, 1 H- 1 The compounds isolated by the above method were structurally identified by H COSY and HR-ESI-MS, confirming the chemical structure and physicochemical properties of the three iridoid compounds. The pharmacological activities of the three new iridoid compounds were verified by cell experiments. The three new iridoid compounds all showed excellent anti-inflammatory effects, with a significant inhibitory effect on NO production, and can effectively reduce the expression of pro-inflammatory factors TNF-a and IL-6, reducing the inflammatory response caused by the overexpression of inflammatory factors. They can serve as lead compounds for the development of new anti-inflammatory drugs.

[0059] The iridoid compounds of the present invention are derived from the leaf part of the war bone and can be developed into medicines. The experimental steps of the preparation method are easy to control, simple and rapid, making the preparation of the iridoid compounds easier. This not only expands the utilization of the medicinal parts and medicinal resources of the war bone, but also reduces the environmental pollution problem caused by resource waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 1H-NMR (500 MHz, DMSO-d6) spectrum of compound 1.

[0061] Figure 2 This is the 13C-NMR (125 MHz, DMSO-d6) spectrum of compound 1.

[0062] Figure 3 1H-NMR (500 MHz, DMSO-d6) spectrum of compound 2.

[0063] Figure 4This is the 13C-NMR (125 MHz, DMSO-d6) spectrum of compound 2.

[0064] Figure 5 This is the 1H-NMR (500 MHz, DMSO-d6) spectrum of compound 3.

[0065] Figure 6 This is the 13C-NMR (125 MHz, DMSO-d6) spectrum of compound 3.

[0066] Figure 7 The effect of compounds 1, 2, and 3 on LPS-induced NO release in RAW264.7 cells.

[0067] Figure 8 This is a graph showing the effects of compounds 1 and 3 on the production of the inflammatory factor tumor necrosis factor.

[0068] Figure 9 This is a graph showing the effects of compounds 1, 2, and 3 on the production of the inflammatory factor interleukin. DETAILED DESCRIPTION

[0069] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified.

[0070] Liquid chromatograph (LC-2030C, Shimadzu Enterprise Management (China) Co., Ltd.); electronic analytical balance (XS225A-SCS, Priss International Trade (Shanghai) Co., Ltd.); high-speed countercurrent chromatograph TBE-300C (Shanghai Tongtian Biotechnology Co., Ltd.); automatic receiver (CHF161RA, Advantec, Japan); vacuum centrifugal concentrator (miVac, GeneVac, UK); LC-MS / IT-TOF liquid chromatography-mass spectrometer (Shimadzu, Japan); superconducting nuclear magnetic resonance spectrometer (Brucker Avance 500 MHz, Brucker, Germany); N-1100 rotary evaporator (Eyela, Japan); D101 macroporous adsorption resin (Cangzhou Baoen Adsorption Material Technology Co., Ltd.); Sephadex LH-20 gel column (GE Healthcare Bio-science AB, Switzerland); C18 chromatography column (Fuji Silyia Chemical, Japan) Ltd.); HP-20SS chromatography column (Mitsubishi Chemical Co., Ltd., Japan); silica gel thin layer plate F 254(thickness of 0.2 mm, Merck, Germany); methanol used in HPLC analysis was chromatographically pure (Tedia, USA); acetone, petroleum ether, methanol, ethanol, ethyl acetate, and n-butanol used in extraction and separation were all analytically pure (Guangdong Guanghua Technology Co., Ltd.).

[0071] Example 1 Extraction and separation of iridoid compounds from the leaves of the genus Zanthoxylum bunge

[0072] The leaves of Craib used in the experiment were collected from Guilin Botanical Garden in Yanshan District, Guilin City, Guangxi Zhuang Autonomous Region on August 25, 2020. After being chopped, they were dried in the shade indoors. They were identified by researcher Tang Hui as leaves of Prenmafulva Craib, a plant of the genus Tofu-tree in the Verbenaceae family. The voucher samples are preserved in the Guangxi Key Laboratory of Plant Functional Substances and Sustainable Utilization of Resources.

[0073] 1. Experimental Methods

[0074] 1. Extraction and separation of compounds

[0075] 15.0 kg of dried scutellaria baicalensis leaves were soaked in 60 L of methanol at room temperature for 7 days and then filtered. This process was repeated three times. The filtrates were combined and the methanol was recovered to obtain 1.2 kg of scutellaria baicalensis leaf methanol extract.

[0076] D101 column chromatography: Take the methanol extract (1.2 kg) and perform gradient elution with methanol-water (volume ratio gradient is 10:90, 20:80, 30:70, 50:50, 80::20, 100:0), collect the gradient eluate of each gradient, detect by thin layer chromatography, combine similar fractions and concentrate, and collect a total of 6 fractions (Fr.1~6).

[0077] Fraction Fr.4 (305.00 g) was loaded on a C18 column and gradient eluted with methanol-water (volume ratio gradient of 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 100:0), with 2 column volumes for each gradient elution. After thin layer chromatography detection, similar fractions were combined and concentrated to obtain a total of 8 subfractions (Fr.4-(1-8)); subfraction Fr.4-1 (24.32 g) was purified by HP20 The column was packed with SS and gradient eluted with methanol-water (volume ratio gradient of 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 100:0) with 2 column volumes for each gradient elution. After thin layer chromatography detection, similar fractions were combined and concentrated to obtain a total of 10 subfractions (Fr.4-1-(1-10)). Subfraction Fr.4-1-5 (12.58 g) was loaded onto a gel column and gradient eluted with methanol-water (volume ratio gradient of 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0) for 2 column volumes per gradient elution. After thin-layer chromatography (TLC), similar fractions were combined and concentrated to yield a total of nine subfractions (Fr.4-1-5-(1-9)). Subfraction Fr.4-1-5-4 (2.81 g) was separated by high-speed countercurrent chromatography using a Tauto TBE-300C system equipped with three polytetrafluoroethylene multilayer coils (1.9 mm inner diameter; total volume 320 mL), a 20 mL sample loop, a DC-0506 cryostat, and a dual-wavelength UV detector for UV absorbance measurement. Advantec CHF161RA fraction collector; Easychrom-1000 software was used for data acquisition and analysis; the elution conditions were as follows: the solvent system was a two-phase solvent system of ethyl acetate / n-butanol / water (7.5:2.5:10 v / v) (the stationary phase was an organic phase, the mobile phase was an aqueous phase), the rotation speed was 880 rpm, the flow rate was 3.0 mL / min, the detection wavelength was 230 nm, the column temperature was 25°C, the elution time was 280 min, and the injection volume was 320 mg), and the TLC spot plate detection was performed. Similar elution fractions were combined and concentrated to collect 6 subfractions Fr.Ⅰ-Ⅵ.

[0078] Subfraction Fr.Ⅳ (73.80 mg) was separated and purified by semi-preparative high performance liquid chromatography (using an Agilent 1260 system equipped with a UV detector and a ZorbaxSB-C18 column (length 250 mm, inner diameter 9.4 mm, 5 μm); elution conditions were: methanol-water as the mobile phase for gradient elution (40:60-68:32, v / v), a flow rate of 3.0 mL / min, a detection wavelength of 230 nm, and an injection volume of 50 μL) to give compound 1 (17.60 mg) and compound 2 (8.60 mg).

[0079] Subfraction Fr.V (64.60 mg) was separated and purified by semi-preparative high performance liquid chromatography (using an Agilent 1260 system equipped with a UV detector and a ZorbaxSB-C18 column (length 250 mm, inner diameter 9.4 mm, 5 μm); elution conditions were: methanol-water as the mobile phase for gradient elution (40:60-50:50, v / v), a flow rate of 3.0 mL / min, a detection wavelength of 230 nm, and an injection volume of 50 μL) to give compound 3 (8.10 mg).

[0080] Example 2 Structural Identification of Iridoid Compounds

[0081] 1. Structural identification of compound 1

[0082] Compound 1 is a white powder, and high-resolution mass spectrometry HR-ESI-MS gives m / z: 1019.2628

[0083] [M+HCOO] - The quasi-molecular ion peak of 47 H 58 O 22 The degree of unsaturation is 19. In the UV spectrum (methanol solution), compound 1 has a maximum absorption value at a wavelength of 230 nm.

[0084] exist 1 In the H-NMR spectrum, the four trans-olefin protons δ H 6.51, 7.63 (d, J=16.0Hz), δ H 5.87, 6.95 (d, J = 12.8 Hz), and four pairs of ortho-coupled aromatic protons δ H 7.79 (d, J = 8.8 Hz, H-2″″, 6″″), 6.98 (d, J = 8.8 Hz, H-3″″, 5″″), 7.72 (d, J = 9.0 Hz, H-2″″′, 6″″′), 6.93 (d, J = 9.0 Hz, H-3″″′, 5″″′) and two aromatic methoxy groups δ HThe signals at 3.79 (3H, s) and 3.80 (3H, s) indicate that compound 1 contains two trans-methoxycinnamoyl units; the DEPT-135 spectrum of compound 1 also confirms the presence of two methoxycinnamic acids. 1 H- 1 In the HCOSY spectrum, the proton signal δ H 3.65 (1H, d, J = 2.7 Hz), 3.92 (1H, dd, J = 8.1, 2.1 Hz), 2.29 (1H, m), 2.37 (1H, m), 4.97 (1H, m) are sequentially correlated, suggesting that they come from the same spin coupling system. Combined with the relevant information provided by the HSQC spectrum, its carbon skeleton δ C 57.2 (C-7), 81.6 (C-6), 35.5 (C-5), 41.8 (C-9), 93.1 (C-1). In the HMBC spectrum, the proton signal δ H 2.37 (H-9) and 4.97 (H-1) are consistent with the carbon signal δ C 65.4 (C-8) is related, suggesting that C-8 is connected to C-9. In addition, δ H 2.37(H-9) hydrogen signal and δ C 57.2 (C-7) carbon signal, indicating that C-7 and C-8 are connected to form a five-membered ring structure. H 3.92) / C-8(δ C 65.4), H-7(δ H 3.65) / C-9(δ C 41.8) further confirmed the existence of the five-membered ring. C 57.2 (C-7), 65.4 (C-8), it can be inferred that C-7, C-8 form an epoxy three-membered ring with oxygen; combined with the relevant information provided by the HSQC spectrum, the carbon skeleton δ C 57.2 (C-7), 65.4 (C-8), 58.7 (C-10). Olefin hydrogen signal δ H 6.43 (1H, dd, J=4.9, 1.9Hz, H-3) and δ C The olefinic carbon signal of 102.2 (C-4) is HMBC-correlated, suggesting that C-3 and C-4 are connected in the structure; combined with the relevant information provided by the HSQC spectrum, the carbon skeleton δ C 141.0 (C-3), 102.2 (C-4). In addition, in the HMBC spectrum, the olefinic hydrogen proton H-3 (δ H 6.43) and C-5(δ C 35.5) related, H-5(δ H2.29) are respectively related to C-3(δ C 141.0), C-4(δ C 102.2), suggesting that C-4 is connected to C-5. H 6.43) and C-1(δ C 93.1) HMBC correlation suggests that C-1 and C-3 are connected by a heteroatom (oxygen atom). Thus, a six-membered ring structure is obtained. Thus, the skeleton structure of iridoid terpenoid is obtained. H Two anomeric proton signals were observed at 4.95 (m, H-1″), 4.94 (m, H-1″′), and at δ H Two proton resonance signals were observed at 1.17 (3H, d, J = 6.2 Hz) and 1.21 (3H, d, J = 6.2 Hz). C Two carbon signals were observed at 68.5 (C-5″), 68.5 (C-5″′), which were attributed to two α-rhamnosyl groups; in addition, at δ H 4.59 (d, J=7.9Hz, H-1′) and δ C There is an anomeric proton and carbon signal at 97.8 (C-1′), and H There are other signals in the 3.21-3.85 region, indicating the presence of a β-glucose group. 1 H- 1 The HCOSY spectrum can be used to infer that the 2-position hydrogen signal and the 3-position hydrogen signal of rhamnose A are δ H 3.71 and δ H 5.01, the terminal hydrogen of rhamnose B δ H 4.94(H-1″′) and rhamnose A 2 carbon δ C 68.8 (C-2″) has a long-range correlation, suggesting that rhamnose B is connected to the C-2 position of rhamnose A; the 3-position hydrogen signal of rhamnose A δ H 5.01 and the carbonyl carbon δ on the trans-p-methoxycinnamoyl A group C 166.1 (C-α-C=O) has a long-range correlation, suggesting that the trans-p-methoxycinnamoyl A group is attached to the C-3 position of rhamnose. 1 H- 1 The HCOSY spectrum can be used to infer that the 3-position hydrogen signal of rhamnose B is δ H 5.03, and the 3-position hydrogen signal of rhamnose B δ H 5.03 and the carbonyl carbon δ on the trans-p-methoxycinnamoyl B group C165.3 (C-α′-C=O) has a long-range correlation, suggesting that the trans-p-methoxycinnamoyl B group is connected to the C-3 position of rhamnose B. The terminal hydrogen of glucose has a long-range correlation with the carbon 1 position of the iridoid skeleton, indicating that glucose is connected to the C-1 position of the iridoid skeleton; the terminal hydrogen of rhamnose A has a long-range correlation with the carbon 6 position of the iridoid skeleton, indicating that rhamnose A is connected to the C-6 position of the iridoid skeleton. The relevant H and C data can be obtained by 1 H-NMR, 13 C-NMR, 1 H- 1 H COSY, HMBC, HSQC, HR-ESI-MS and other spectra were assigned (see Figure 1-2 ). Combining the above data and literature analysis, 1 H and 13 C NMR is shown in Table 1, which proves that the structure of compound 1 (6-O-(2″-(3″′-O-trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl-3″-O-trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol) is as shown in Formula Ⅰ and is an iridoid ether terpenoid compound.

[0085]

[0086] 2. Structural identification of compound 2

[0087] Compound 2 is a white powder. High-resolution mass spectrometry HR-ESI-MS gives m / z: 873.6626 [M+HCOO] - The quasi-molecular ion peak of 41 H 48 O 18 The degree of unsaturation is 18. In the UV spectrum (methanol solution), compound 2 has a maximum absorption value at a wavelength of 230 nm.

[0088] exist 1 In the H-NMR spectrum, two groups of typical trans double bond olefin hydrogen proton signals δ H 5.88and 6.95 (d, J=12.8Hz), δ H 6.52 and 7.63 (d, J = 16.0 Hz), and four pairs of ortho-coupled aromatic protons δ H7.74 (d, J = 8.8 Hz, H-2″′, 6″′), 6.98 (d, J = 8.8 Hz, H-3″′, 5″′), 7.70 (d, J = 8.8 Hz, H-2″″, 6″″), 6.94 (d, J = 8.8 Hz, H-3″″, 5″″) and two aromatic methoxy groups (δ H 3.79, 3.80) can clearly indicate that the acyl moiety is two trans-p-methoxycinnamoyl units. 13 C NMR confirmed the presence of two trans-p-methoxycinnamoyl groups. 1 H- 1 In the H COSY spectrum, the proton signal δ H 3.64 (1H, d, J = 3.8 Hz), 3.91 (1H, d, J = 8.3 Hz), 2.28 (1H, m), 2.38 (1H, dd, J = 7.4, 2.5 Hz), 4.96 (1H, d, J = 4.6 Hz) are sequentially correlated, suggesting that they come from the same spin coupling system. Combined with the relevant information provided by the HSQC spectrum, its carbon skeleton δ C 57.4 (C-7), 81.8 (C-6), 35.6 (C-5), 41.8 (C-9), 93.1 (C-1). In the HMBC spectrum, the proton signal δ H 2.38 (H-9) and 4.96 (H-1) are consistent with the carbon signal δ C 65.3 (C-8) is related, suggesting that C-8 is connected to C-9. In addition, δ H 2.38 (H-9) hydrogen signal and δ C 57.4 (C-7) carbon signal, indicating that C-7 and C-8 are connected to form a five-membered ring structure. H 3.91) / C-8(δ C 65.3), H-7(δ H 3.64) / C-9(δ C 41.8) further confirmed the existence of the five-membered ring. C 57.4 (C-7), 65.3 (C-8), it can be inferred that C-7, C-8 form an epoxy three-membered ring with oxygen; combined with the relevant information provided by the HSQC spectrum, the carbon skeleton δ C 57.4 (C-7), 65.3 (C-8), 58.7 (C-10). Olefin hydrogen signal δ H 6.44 (1H, m, H-3) and δ CThe olefinic carbon signal of 102.2 (C-4) is HMBC-correlated, suggesting that C-3 and C-4 are connected in the structure; combined with the relevant information provided by the HSQC spectrum, the carbon skeleton δ C 141.0 (C-3), 102.2 (C-4). In addition, in the HMBC spectrum, the olefinic hydrogen proton H-3 (δ H 6.44) and C-5(δ C 35.6) related, H-5(δ H 2.28) are respectively related to C-3(δ C 141.0), C-4(δ C 102.2), suggesting that C-4 is connected to C-5. H 6.44) and C-1(δ C 93.1) HMBC correlation suggests that C-1 and C-3 are connected by a heteroatom (oxygen atom). Thus, a six-membered ring structure is obtained. Thus, the skeleton structure of iridoid terpenoid is obtained. H An anomeric proton signal was observed at 4.89 (d, J = 5.7 Hz, H-1″) and at δ H A proton resonance signal was observed at 1.06 (3H, d, J = 6.2 Hz), and at δ C A carbon signal was observed at 66.4 (C-5″), which was assigned to an α-rhamnosyl group; in addition, H 4.59 (d, J=7.9Hz, H-1′) and δ C There is an anomeric proton and carbon signal at 97.8 (C-1′), and H There are other signals in the 3.21-3.85 region, indicating the presence of a β-glucose group. 1 H- 1 The HCOSY spectrum can be used to infer that the 2-position hydrogen signal and the 4-position hydrogen signal of rhamnose are δ H 3.74 and δ H 4.94, the 2-hydrogen signal of rhamnose δ H 3.74 and the carbonyl carbon δ on the trans-p-methoxycinnamoyl A group C 165.4 (C-α-C=O) has a long-range correlation, suggesting that the trans-p-methoxycinnamoyl A group is connected to the C-2 position of rhamnose; and the hydrogen signal at the 4th position of rhamnose δ H 4.94 and the carbonyl carbon δ on the trans-p-methoxycinnamoyl B group C166.2 (C-α′-C=O) has a long-range correlation, suggesting that the trans-p-methoxycinnamoyl B group is connected to the C-4 position of rhamnose. The terminal hydrogen of glucose has a long-range correlation with the carbon 1 position of the iridoid skeleton, indicating that glucose is connected to the C-1 position of the iridoid skeleton; the terminal hydrogen of rhamnose has a long-range correlation with the carbon 6 position of the iridoid skeleton, indicating that rhamnose A is connected to the C-6 position of the iridoid skeleton. The relevant H and C data can be obtained by 1 H-NMR, 13 C-NMR, 1 H- 1 H COSY, HMBC, HSQC, HR-ESI-MS and other spectra were assigned (see Figure 3-4 ).

[0089] Combining the above data and literature analysis, 1 H and 13 C NMR is shown in Table 1, which proves that the structure of compound 2 (6-O-(2",4"-di-trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol) is as shown in Formula II and is an iridoid compound.

[0090]

[0091] 3. Structural identification of compound 3

[0092] Compound 3 was an amorphous powder. High-resolution mass spectrometry HR-ESI-MS gave m / z: 713.2298 [M+HCOO] - The quasi-molecular ion peak of 31 H 40 O 16 The degree of unsaturation is 12. In the UV spectrum (methanol solution), compound 3 has a maximum absorption value at a wavelength of 230 nm.

[0093] exist 1 In the H-NMR spectrum, a group of typical trans double bond olefin hydrogen proton signals δ H 6.50 and 7.64 (d, J = 16.0 Hz), and the four aromatic protons δ H 6.94 (d, J = 2.2 Hz, H-2″′), 6.99 (dd, J = 8.8, 2.2 Hz, H-4″′), 7.69 (d, J = 8.8 Hz, H-5″′), 7.79 (dd, J = 8.8, 2.2 Hz, H-6″′) and one aromatic methoxy group δ HThe signal at 3.79 (3H, s) clearly indicates that the acyl group is a trans-m-methoxycinnamoyl unit. In addition, the HMBC spectrum of compound 3 shows that the δ H 3.80(s) / δ C 161.1 (C-3″′) correlation confirmed the presence of trans-m-methoxycinnamoyl. 1 H- 1 In the HCOSY spectrum, the proton signal δ H 3.64 (1H, m), 3.93 (1H, dd, J = 8.1, 1.2 Hz), 2.31 (1H, dd, J = 4.2, 1.8 Hz), 2.39 (1H, dd, J = 9.7, 7.6 Hz), 4.97 (1H, s) are sequentially correlated, suggesting that they come from the same spin coupling system. Combined with the relevant information provided by the HSQC spectrum, its carbon skeleton δ C 57.4 (C-7), 81.6 (C-6), 35.5 (C-5), 41.9 (C-9), 93.1 (C-1). In the HMBC spectrum, the proton signal δ H 2.39 (H-9) and 4.97 (H-1) are consistent with the carbon signal δ C 65.3 (C-8) is related, suggesting that C-8 is connected to C-9. In addition, δ H 2.39(H-9) hydrogen signal and δ C 57.4 (C-7) carbon signal, indicating that C-7 and C-8 are connected to form a five-membered ring structure. H 3.93) / C-8(δ C 65.3), H-7(δ H 3.64) / C-9(δ C 41.9) further confirmed the existence of the five-membered ring. C 57.4 (C-7), 65.3 (C-8), it can be inferred that C-7, C-8 form an epoxy three-membered ring with oxygen; combined with the relevant information provided by the HSQC spectrum, the carbon skeleton δ C 57.4 (C-7), 65.3 (C-8), 58.7 (C-10). Olefin hydrogen signal δ H 6.44 (1H, dd, J = 6.0, 1.7 Hz, H-3) and δ C The olefinic carbon signal at 102.3 (C-4) is HMBC-correlated, suggesting that C-3 and C-4 are connected in the structure. Combined with the relevant information provided by the HSQC spectrum, the carbon skeleton δ C 140.9 (C-3), 102.3 (C-4). In addition, in the HMBC spectrum, the olefinic hydrogen proton H-3 (δ H6.44) and C-5(δ C 35.5) related, H-5(δ H 2.31) are respectively related to C-3(δ C 140.9), C-4(δ C 102.3), suggesting that C-4 is connected to C-5. H 6.44) and C-1(δ C 93.1) HMBC correlation suggests that C-1 and C-3 are connected by a heteroatom (oxygen atom). Thus, a six-membered ring structure is obtained. Thus, the skeleton structure of iridoid terpenoid is obtained. H An anomeric proton signal was observed at 4.85 (d, J = 1.7 Hz, H-1″) and at δ H A proton resonance signal was observed at 1.20 (3H, d, J = 6.3 Hz), and at δ C A carbon signal was observed at 68.1 (C-5″), which was assigned to an α-rhamnosyl group; in addition, H 4.60 (d, J=7.9Hz, H-1′) and δ C There is an anomeric proton and carbon signal at 97.8 (C-1′), and H There are other signals in the 3.21-3.85 region, indicating the presence of a β-glucose group. 1 H- 1 The HCOSY spectrum can be used to infer that the 3-hydrogen signal of rhamnose is δ H 4.89, the 3-position hydrogen signal of rhamnose δ H 4.89 and the carbonyl carbon δ on the trans-m-methoxycinnamoyl group C 166.2 (C-α-C=O) has a long-range correlation, suggesting that the trans-m-methoxycinnamoyl group is connected to the C-3 position of rhamnose. The terminal hydrogen of glucose has a long-range correlation with the carbon 1 position of the iridoid skeleton, indicating that glucose is connected to the C-1 position of the iridoid skeleton; the terminal hydrogen of rhamnose has a long-range correlation with the carbon 6 position of the iridoid skeleton, indicating that rhamnose A is connected to the C-6 position of the iridoid skeleton. The relevant H and C data can be obtained by 1 H-NMR, 13 C-NMR, 1 H- 1 H COSY, HMBC, HSQC, HR-ESI-MS and other spectra were assigned (see Figure 5-6 ).

[0094] Combining the above data and literature analysis, 1 H and 13C NMR is shown in Table 1, which proves that compound 3 (6-O-(3″-O-trans-m-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol) has the structural formula shown in Formula III and is an iridoid compound.

[0095]

[0096] Table 1 Compound 1-3 (DMSO-d6) 1 H spectrum and 13 C spectrum data

[0097]

[0098] Example 3 Anti-inflammatory activity test of iridoid compounds

[0099] In this example, the anti-inflammatory activity of compounds 6-O-(2″-(3″′-O-trans-p-methoxycinnamoyl-α-L-rhamnopyranosyl)-3″-O-trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol (1), 6-O-(2″,4″-di-trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol (2), and 6-O-(3″-O-trans-m-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol (3) on LPS-induced RAW 264.7 cells in vitro was studied.

[0100] 1. Experimental Materials and Instruments

[0101] Clean bench (Suzhou Antai Air Technology Co., Ltd.); constant temperature CO2 cell culture incubator (Thermo Fisher Scientific, USA); electric constant temperature water bath (Shanghai Boxun Industrial Co., Ltd.); inverted microscope (LEICA, Germany); multifunctional microplate reader (TECAN, Switzerland); refrigerated centrifuge (Zhuhai Dark Horse Company); electronic balance (Shanghai Tianmei Balance Instrument Co., Ltd.); vortex shaker (Haimen Qilin Bell Company); PBS phosphate buffer (Beijing Solebold Company); nitric oxide test kit (Biyuntian Biotechnology); Mouse TNF-α ELISA kit and Mouse IL-6 ELISA kit (Wuhan Yilirui Company); Cell Counting Kit-8 (Med Chem Express, USA); LPS (Sigma, USA); DMEM (Sigma, USA); DMSO (Shanghai McLean Biochemical Technology Co., Ltd.); indomethacin (Sigma, USA).

[0102] 2. Test methods

[0103] 1. Cytotoxicity and anti-inflammatory activity detection

[0104] RAW264.7 cells in the logarithmic growth phase were seeded into 96-well cell culture plates, and 100 μL of culture medium was added to each well to make the cell density reach 2×10 5 The cells were incubated in an incubator for 24 hours. The experiment was divided into a blank group and a drug-addition group. No drug was added to the blank group. The drug-addition group was incubated with compounds 1, 2, and 3 diluted to different concentrations in DMEM medium for 24 hours, with three replicates per group. After 24 hours, the 96-well plate was removed from the incubator and 10 μL of CCK-8 was added to each well. After incubation at 37°C for 1 hour, the absorbance (A) was measured at a wavelength of 450 nm and the cell viability was calculated. The experiment was repeated three times.

[0105] RAW264.7 cells in the logarithmic growth phase were seeded into 96-well cell culture plates, and 100 μL of culture medium was added to each well to make the cell density reach 2×10 5 / mL, placed in an incubator and cultured for 24 hours. The experimental groups were blank group, model group, positive drug group, and drug-added group. Different concentrations of compounds 1, 2, and 3 were added to the drug group, and LPS (1μg / mL) was added to stimulate the cells for 24 hours. Indomethacin was used as a positive control, and each group repeated 3 wells. After 24 hours, each group aspirated 50μL of supernatant into a 96-well cell culture plate, and then 50μL of Griess reagent I and Griess reagent II were added in turn. The plates were gently shaken several times to mix them thoroughly. The absorbance A was measured at a wavelength of 540nm using an enzyme reader, and the NO inhibition rate was calculated according to the standard curve. The fitting curves were obtained using GraphPad Prism 8 software to obtain the IC of compounds 1, 2, and 3 for inhibiting NO release. 50 value.

[0106] 2. ELISA detection of inflammatory factors

[0107] RAW264.7 cells in the logarithmic growth phase were seeded into 96-well cell culture plates, and 100 μL of culture medium was added to each well to make the cell density reach 2×10 5Cells were cultured in an incubator at 400 μg / mL for 24 hours. The experimental groups were blank, model, positive drug, and drug-added groups. The drug groups were treated with different concentrations of compounds 1, 2, and 3, and then LPS (1 μg / mL) was added 1 hour later to stimulate the cells for 24 hours. Indomethacin was used as a positive control, with triplicate wells per group. After 24 hours, the cell supernatant was collected and transferred to an EP tube, labeled. The supernatant was centrifuged at 1000 × g for 20 minutes at 4°C to remove particles and aggregates. The supernatant was aspirated and aliquoted into new EP tubes. Three biological replicates were set for each sample. Procedure: 1. Set up standard wells, blank wells, and sample wells. Add 100 μL of serially diluted standard to the standard wells, 100 μL of standard and sample dilution to the blank wells, and 100 μL of the test sample to the remaining wells (it is recommended that all test samples and standards be set up in duplicate). The ELISA plate was coated and incubated at 37°C for 90 minutes. Tips: When adding samples, add the sample to the bottom of the ELISA plate, try not to touch the wall of the well, shake gently to mix, and avoid bubbles. The sample addition time should be controlled within 10 minutes. 2. Shake off the liquid in the wells without washing. Add 100μL of biotinylated antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 1 hour. 3. Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350μL of washing solution to each well, soak for 1 minute, absorb or shake off the liquid in the ELISA plate, and pat dry. Repeat this washing step 3 times. Proceed to the next step immediately after washing, and do not let the microplate dry. 4. Add 100μL of enzyme conjugate working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 30 minutes. 5. Shake off the liquid in the wells and wash the plate 5 times using the same method as step 3. 6. Add 90 μL of substrate solution (TMB) to each well, cover the ELISA plate with film, and incubate at 37°C in the dark for about 15 minutes. Tip: Shorten or extend the time as appropriate according to the actual color development, but do not exceed 30 minutes. When a clear gradient appears in the standard well (a clear blue gradient appears in the first 4 color development wells), the reaction can be terminated. Turn on the enzyme reader 15 minutes in advance to preheat. 7. Add 50 μL of stop solution to each well to terminate the reaction. Tip: The order of adding the stop solution should be as consistent as possible with the order of adding the substrate solution. 8. Immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an enzyme reader.

[0108] 3. Experimental Results

[0109] 1. Effects of compounds on cytotoxicity and anti-inflammatory activity

[0110] RAW 264.7 cells were treated with compounds 1, 2, and 3 at 40, 80, and 160 μM, respectively, and incubated for 24 hours. The results showed that compounds 1, 2, and 3 had no toxic effects on cells at 40, 80, and 160 μM.

[0111] The results of inhibiting NO release at different concentrations are shown in the attached Figure 7As can be seen from the figure, the NO release in normal RAW 264.7 cells is low. After LPS induction, the NO content in the cell supernatant increased significantly to 14.09 μM, indicating that the model group was successfully established. When treated with different concentrations of compounds 1, 2, and 3, the NO content in the cell supernatant of the drug-treated group decreased. The IC values ​​of compounds 1, 2, and 3 as well as the positive drug indomethacin for inhibiting NO release were significantly higher than those of the control group. 50 The concentrations of compounds 1, 2 and 3 were 45.80, 51.17, 68.42 and 100.33 μM respectively. Compounds 1, 2 and 3 showed good anti-inflammatory effects, and the anti-inflammatory effects of compounds 1, 2 and 3 were better than those of positive drugs.

[0112] Compounds 1, 2, and 3 inhibited LPS-induced NO production in a concentration-dependent manner without affecting the cell viability of RAW264.7 macrophages. The NO production levels of the 80 μM groups of compounds 1, 2, and 3 were 6.83 μM, 7.45 μM, and 6.38 μM, respectively, indicating that compounds 1, 2, and 3 could significantly inhibit the NO release of cells.

[0113] 2. Effects of compounds on the secretion of inflammatory factors TNF-α and IL-6 in cells

[0114] RAW264.7 cells in the logarithmic growth phase were seeded into 96-well cell culture plates, and 100 μL of culture medium was added to each well to make the cell density reach 2×10 5Cells were cultured in an incubator at 400 μg / mL for 24 hours. The experimental groups were blank, model, positive drug, and drug-added groups. The drug groups were treated with different concentrations of compounds 1, 2, and 3, and then LPS (1 μg / mL) was added 1 hour later to stimulate the cells for 24 hours. Indomethacin was used as a positive control, with triplicate wells per group. After 24 hours, the cell supernatant was collected and transferred to an EP tube, labeled. The supernatant was centrifuged at 1000 × g for 20 minutes at 4°C to remove particles and aggregates. The supernatant was aspirated and aliquoted into new EP tubes. Three biological replicates were set for each sample. Procedure: 1. Set up standard wells, blank wells, and sample wells. Add 100 μL of serially diluted standard to the standard wells, 100 μL of standard and sample dilution to the blank wells, and 100 μL of the test sample to the remaining wells (it is recommended that all test samples and standards be set up in duplicate). The ELISA plate was coated and incubated at 37°C for 90 minutes. Tips: When adding samples, add the sample to the bottom of the ELISA plate, try not to touch the wall of the well, shake gently to mix, and avoid bubbles. The sample addition time should be controlled within 10 minutes. 2. Shake off the liquid in the wells without washing. Add 100μL of biotinylated antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 1 hour. 3. Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350μL of washing solution to each well, soak for 1 minute, absorb or shake off the liquid in the ELISA plate, and pat dry. Repeat this washing step 3 times. Proceed to the next step immediately after washing, and do not let the microplate dry. 4. Add 100μL of enzyme conjugate working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 30 minutes. 5. Shake off the liquid in the wells and wash the plate 5 times using the same method as step 3. 6. Add 90 μL of substrate solution (TMB) to each well, cover the ELISA plate with film, and incubate at 37°C in the dark for about 15 minutes. Tip: Shorten or extend the time as appropriate according to the actual color development, but do not exceed 30 minutes. When a clear gradient appears in the standard well (a clear blue gradient appears in the first 4 color development wells), the reaction can be terminated. Turn on the enzyme reader 15 minutes in advance to preheat. 7. Add 50 μL of stop solution to each well to terminate the reaction. Tip: The order of adding the stop solution should be as consistent as possible with the order of adding the substrate solution. 8. Immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an enzyme reader.

[0115] The results are as attached Figure 8-9 As shown, after pre-treating cells with compounds 1, 2, and 3 at concentrations of 10, 40, and 160 μM for 1 hour and 24 hours of LPS stimulation, the concentrations of inflammatory factors TNF-α and IL-6 released by macrophages in the model group were significantly higher than those in the blank group, indicating that the inflammatory cell model group was successfully established. After compound treatment, the corresponding production levels of compounds 1, 2, and 3 were lower than those in the model group, and all of them downregulated the expression levels of TNF-α and IL-6 proteins, indicating that the compounds have good anti-inflammatory activity.

[0116] In summary, the iridoid compounds 1, 2, and 3 isolated from the leaves of the rhizome of scutellaria baicalensis can inhibit the production of NO and the expression of inflammatory factors TNF-α and IL-6, and have good anti-inflammatory activity.

[0117] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An iridoid compound having anti-inflammatory activity, characterized in that: Their structural formulas are as follows:

2. A method for preparing an iridoid compound with anti-inflammatory activity according to claim 1, characterized in that: The following steps are involved: S1. Taking the leaves of the scutellaria baicalensis, soaking them in an alcohol solvent and filtering them, repeating this process at least once, combining them to obtain an extract, and removing the solvent from the extract to obtain a total extract; S2. The total extract obtained in step S1 is subjected to macroporous adsorption resin column chromatography, and gradient elution is performed with a mixed eluent of alcohol and water, the gradient eluates of each gradient are collected, and the eluates are detected by TLC spot plate. Similar elution fractions are combined and concentrated to obtain 6 fractions Fr.1 to Fr.6; S3. Subjecting the fourth fraction Fr.4 obtained in step S2 to column chromatography using a mixed eluent of alcohol and water for gradient elution, collecting the gradient eluates, detecting the fractions by TLC spot plate, combining and concentrating similar elution fractions, and collecting 8 fractions Fr.4-(1-8); S4. The first fraction Fr.4-1 obtained in step S3 is first subjected to column chromatography with a gradient elution using a mixed eluent of alcohol and water, followed by thin-layer chromatography detection. Similar fractions are combined and concentrated to obtain subfractions Fr.4-1-(1-10). The subfraction Fr.4-1-5 is then subjected to gel column chromatography with a gradient elution using a mixed eluent of alcohol and water, followed by thin-layer chromatography detection. Similar fractions are combined and concentrated to obtain nine fractions Fr.4-1-5-(1-9). S5. Elution of the fourth fraction Fr.4-1-5-4 obtained in step S4 by HSCCC chromatography was performed, and detection was performed by TLC spot plate. Similar elution fractions were combined and concentrated to obtain six subfractions Fr. I-VI. S6. Separate and purify the fourth fraction Fr.IV obtained in step S5 by semi-preparative HPLC to obtain compound 1 shown in the structure of claim 1.

3. The preparation method according to claim 2, characterized in that In step S1: Alcohol solvents include methanol and ethanol; The dosage ratio of the leaves of the war bone to the alcohol solvent is 15kg:30-90L; Soak at room temperature for 5-9 days; Repeat 2-4 times.

4. The preparation method according to claim 2, characterized in that In step S2: The macroporous adsorption resin columns were D101 column, HPD-100, and HP20; Alcohols include methanol and ethanol; The volume ratio gradient of alcohol and water was 10:90-100:

0.

5. The preparation method according to claim 2, characterized in that In step S3: Column chromatography used C18, C8, MCI, and HP20SS columns; Alcohols include methanol and ethanol; The volume ratio gradient of alcohol to water is 40:60-100:

0.

6. The preparation method according to claim 2, characterized in that In step S4: Column chromatography used HP 20SS column, C18, MCI; Gel column chromatography used Sephadex LH-20 and Sephadex G; Alcohols include methanol and ethanol; The volume ratio gradient of alcohol and water in column chromatography was 20:80-100:0; The volume ratio gradient of alcohol and water in gel column chromatography is 20:80-100:

0.

7. The preparation method according to claim 2, characterized in that The elution conditions in step S5 are as follows: the solvent system is a two-phase solvent system of ethyl acetate, n-butanol and water, the rotation speed is 880 rpm, the flow rate is 3.0 mL / min, the detection wavelength is 230 nm, the column temperature is 25 ° C, the elution time is 280 min, and the injection volume is 320 mg; the solvent concentration volume ratios of ethyl acetate, n-butanol and water are 5:5:10, 7:3:10, 7.5:2.5:10, and 8:2:

10.

8. The preparation method according to claim 2, characterized in that In step S6: Subfraction Fr.Ⅳ was separated and purified by semi-preparative high performance liquid chromatography; the elution conditions were: gradient elution with methanol and water as the mobile phase, the gradient elution program was: 0-35 min, the v / v ratio of methanol:water was 40:60-68:32; the flow rate was 3.0 mL / min, the detection wavelength was 230 nm, and compound 1 was obtained in 24 min.

9. A use of the iridoid compound with anti-inflammatory activity according to claim 1, characterized in that: Used to prepare anti-inflammatory drugs.

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