CN116333018A - Iridoid new compound with anti-inflammatory activity and preparation method and application thereof - Google Patents
Iridoid new compound with anti-inflammatory activity and preparation method and application thereof Download PDFInfo
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- CN116333018A CN116333018A CN202310122458.3A CN202310122458A CN116333018A CN 116333018 A CN116333018 A CN 116333018A CN 202310122458 A CN202310122458 A CN 202310122458A CN 116333018 A CN116333018 A CN 116333018A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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- C07H1/08—Separation; Purification from natural products
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- A—HUMAN NECESSITIES
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to three new iridoid compounds with anti-inflammatory activity, and a preparation method and application thereof. The invention provides a preparation method for extracting 3 novel iridoid compounds from a war bone leaf. Three new iridoid compounds all show excellent anti-inflammatory effect, have obvious inhibition effect on NO generation, can effectively reduce the expression of pro-inflammatory factors TNF-a and IL-6, can reduce inflammatory reaction caused by the over-expression of inflammatory factors, and can be used as lead compounds for developing novel anti-inflammatory drugs.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to three new iridoid compounds with anti-inflammatory activity, and a preparation method and application thereof.
Background
The war bone is a common strong medicine, and is named as yellow hair firewood (Premna fulva Craib) which is a plant of the genus Premna (Verbenaceae) of the family Verbenaceae, and is also named as Tuba, chuanyun arrow. The root, stem and leaf of the war bone can be used as medicines, has the effects of promoting blood circulation, dispelling lump, strengthening tendons and bones, dispelling wind and relieving pain, is commonly used for treating lumbago and skelalgia, traumatic injury, rheumatic arthritis, rheumatoid arthritis, liver region pain and the like in Guangxi folks, and is a Guangxi genuine medicinal material.
Research has found that various products have been developed using war bones as the main raw material, such as: the bone strengthening injection, compound yellow hair bean curd Chai Cha agent, war bone mixture and the like have the effects of resisting inflammation, relieving swelling and easing pain, and are mainly used for treating hypertrophic spondylitis (namely lumbar vertebra hyperosteogeny), scapulohumeral periarthritis and other diseases.
A typical manifestation of inflammatory reactions is the excessive accumulation of Nitric Oxide (NO), the production of which is regulated by nitric oxide synthase. Main chemical components in the war bone leaf are divided into iridoids and glycosides thereof, and the war bone leaf has good anti-inflammatory, analgesic, anti-osteoporosis and other activities. At present, the research on the chemical components of the war bones and the pharmacological activity thereof shows that the pharmacological activity is mainly studied in the aspect of rhizome, but the research on the war bones and leaves at home and abroad is less. Therefore, based on research and accumulation of literature research and subject groups, in order to more fully utilize plant resources, the inventor researches on chemical components and activity screening of the fight against bone leaves, and provides three iridoid novel compounds with anti-inflammatory activity, and a preparation method and application thereof.
Disclosure of Invention
In order to overcome the defects of research and development of the resources of the fight bone leaves in the prior art, the invention provides an iridoid compound with good anti-inflammatory effect obtained from the fight bone leaves, a preparation method thereof, and anti-inflammatory activity and medical application thereof.
The first object of the invention is to provide 3 new iridoid compounds 6-O- (2 ' - (3 ' -O-trans-p-methoxycinnamonyl-alpha-L-rhamnopyranosyl) -3 ' -O-trans-p-methoxycinnamonyl) -alpha-L-rhamnopyranosyl catalpol (1) extracted from the leaf of the war bone; 6-O- (2 ', 4' -di-trans-p-methoxycinnamonyl) -alpha-L-rhamnopyranosyl catalpol (2); 6-O- (3 "-O-trans-m-methoxycinnamonyl) -alpha-L-rhamnopyranosyl catalpol (3).
A second object of the present invention is to provide the above iridoid compound 6-O- (2 "- (3'" -O-trans-p-methoxycinnamonyl- α -L-rhamnopyranosyl) -3 "-O-trans-p-methoxycinnamonyl) - α -L-rhamnopyranosyl catalpol (1); 6-O- (2 ', 4' -di-trans-p-methoxycinnamonyl) -alpha-L-rhamnopyranosyl catalpol (2); and a process for the preparation of 6-O- (3' -O-trans-m-methoxinamoyl) -alpha-L-rhamnopyranosyl catalpol (3).
A third object of the present invention is to provide the above iridoid compound 6-O- (2 "- (3'" -O-trans-p-methoxycinnamonyl- α -L-rhamnopyranosyl) -3 "-O-trans-p-methoxycinnamonyl) - α -L-rhamnopyranosyl catalpol (1); 6-O- (2 ', 4' -di-trans-p-methoxycinnamonyl) -alpha-L-rhamnopyranosyl catalpol (2); and the application of 6-O- (3' -O-trans-m-methoxinamoyl) -alpha-L-rhamnopyranosyl catalpol (3) in preparing anti-inflammatory drugs.
The invention extracts and separates 3 new iridoid compounds from the war bone leaves (premna fulva leaves) for the first time, the name of the new iridoid compounds is 6-O- (2 '- (3' -O-trans-p-methoxycinamoyl-alpha-L-rhamnopyranosyl) -3 '-O-trans-p-methoxycinamoyl) -alpha-L-rhamnopyranosyl catalpol (1), 6-O- (2', 4 '-di-trans-p-methoxycinnamonyl) -alpha-L-rhamnopyranosyl catalpol (2), and 6-O- (3' -O-trans-m-methoxycinnamonyl) -alpha-L-rhamnopyranosyl catalpol (3), of formula C respectively 47 H 58 O 22 、C 41 H 48 O 18 、C 31 H 40 O 16 The method comprises the steps of carrying out a first treatment on the surface of the The chemical structural formula is as follows:
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)
6-O-(3″-O-trans-m-methoxycinnamoyl)-α-L-rhamnopyranosyl catalpol(3)
the invention also provides a preparation method of the novel iridoid compound with anti-inflammatory activity, which comprises the following steps:
s1, taking the war bone leaves, soaking the war bone leaves in an alcohol solvent, filtering the solution, repeating the filtering at least once, combining the solution to obtain an extracting solution, and removing the solvent in the extracting solution to obtain a total extract;
s2, subjecting the total extract obtained in the step S1 to macroporous adsorption resin column chromatography, performing gradient elution by using mixed eluent of alcohol and water, collecting gradient eluent of each gradient, detecting by using a TLC (thin layer chromatography) plate, combining similar elution fractions, concentrating, and collecting 6 fractions Fr.1-6;
s3, performing gradient elution on the fourth fraction Fr.4 obtained in the step S2 by using a mixed eluent of alcohol and water through a column chromatography method, collecting gradient eluents of each gradient, detecting through a TLC (thin layer chromatography) plate, combining similar elution fractions, concentrating, and collecting 8 fractions Fr.4- (1-8);
s4, sequentially performing gradient elution on the first fraction Fr.4-1 obtained in the step S3 by using a mixed eluent of alcohol and water through a column chromatography, detecting through a thin layer chromatography, combining similar fractions and concentrating, collecting to obtain a subfraction Fr.4-1- (1-10), performing gradient elution on the subfraction Fr.4-1-5 by using a mixed eluent of alcohol and water through a gel column chromatography, detecting through a thin layer chromatography, combining similar elution fractions and concentrating, and collecting to obtain 9 fractions Fr.4-1-5- (1-9);
s5, eluting the 4 th fraction Fr.4-1-5-4 obtained in the step S4 through HSCCC chromatography, detecting through a TLC (thin-layer chromatography) plate, combining similar elution fractions, concentrating, and collecting to obtain 6 subfractions Fr. I-VI;
s6, separating and purifying the 4 th fraction Fr. IV obtained in the step S5 through semi-preparative HPLC to obtain compounds 1 and 2 shown in the structure in the claim 1; separating and purifying the 5 th fraction Fr. V obtained in the step S5 by semi-preparative HPLC to obtain the compound 3 shown in the structure of claim 1.
Specifically, in step S1:
the alcohol solvent is methanol or ethanol, preferably methanol;
the dosage ratio of the war bone leaf to the alcohol solvent is 15kg (30-90) L, preferably 15kg:60L;
soaking at room temperature; the soaking time is (5-9) d, preferably 7d;
the number of repetitions is 2-4, preferably 3.
Preferentially, in step S1, the war bone leaves are collected from Guangxi plant autonomous region Gui Linshi, wild mountain area Guilin plant garden, cut up, dried in the shade and dried in the air, identified by Tang Hui researchers as the leaves of Premna microphylla Prenma fulva Craib of Premna microphylla of Verbenaceae, and the voucher samples are stored in Guangxi plant functional materials and resource continuous utilization key laboratories.
Specifically, in step S2:
the macroporous adsorption resin column is a D101 column, HPD-100 and HP20SS, preferably a D101 column, the D101 macroporous resin is a styrene type nonpolar copolymer, the application range is wider, the adsorption capacity is strong, the aperture is large, the elution is easy, and the application is better than HPD-100 and HP20 SS.
The alcohol is methanol or ethanol, preferably methanol;
the volume ratio gradient of the alcohol to the water is 10:90-100:0, preferably 10:90, 20:80, 30:70, 50:50, 80:20 and 100:0 in sequence.
Preferably, in step S2, the length of the D101 column is 100cm, and the inner diameter is 10cm.
Specifically, in step S3:
column chromatography uses C18, C8, MCI, HP20SS, preferably a C18 column, because C18 is longer than the carbon chain of C8, with better retention properties for the isolated compounds; in addition, the separation function of MCI and HP20SS materials is molecular sieve function, and the separation effect of the pre-stage segmentation and the re-separation of the crude extract subjected to pore size screening is not as good as that of C18.
The alcohol is methanol or ethanol, preferably methanol;
the volume ratio gradient of alcohol to water is 40:60-100:0, preferably 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 100:0 in sequence.
Preferably, in step S3, the C18 column is 50cm long and 5cm in inner diameter.
Specifically, in step S4:
the column chromatography adopts MCI-GEL CHP series materials with different particle sizes (4 μm-300 μm), preferably HP20SS column. The MCI-GELCHP20P (HP 20 SS) has the functions of a molecular sieve and a reverse phase material, so that the requirements of extract separation efficiency and fine separation are met;
the gel column chromatography adopts Sephadex LH-20 and Sephadex G-25, preferably Sephadex LH-20, because Sephadex LH-20 has gel filtration effect and reverse phase chromatography effect in the separation process, and has better separation effect than other chromatographic columns;
the alcohol is methanol or ethanol, preferably methanol;
the volume ratio gradient of alcohol to water in the 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, 100:0 in sequence;
the volume ratio gradient of alcohol to 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, 100:0 in sequence.
Preferably, the HP20SS column is 50cm long and 5cm in inner diameter; the gel column was 60cm long and the inner diameter was 4cm.
The reason that the eluent of methanol and water is adopted in the steps S1-S4 is that the methanol and the water are commonly used mobile phases, the methanol mainly reduces a large amount of water to contact with the carrier, the water mainly adjusts the retention time, the components to be detected can be separated from the peak late through the adjustment of the water, a good separation effect is achieved, and the eluent of other organic solvents and the water respectively encounters the problems of overhigh cost and solvent viscosity, so that a large amount of experiments cannot be carried out.
Specifically, the elution conditions in step S5 are: the solvent system is a two-phase solvent system of ethyl acetate, n-butanol and water, the rotating speed is 880rpm, the flow rate is 3.0mL/min, the detection wavelength is 230nm, the column temperature is 25 ℃, the elution time is 280min, and the sample injection amount is 320mg; the volume ratio gradient of ethyl acetate, n-butanol and water is 5:5:10, 7:3:10, 7.5:2.5:10, 8:2:10 in sequence.
More specifically, the high-speed countercurrent chromatography in step S5 employs a TautoTBE-300C system equipped with 3 polytetrafluoroethylene multi-layer coils (inner diameter: 1.9mm; total volume: 320 mL), a 20mL sample ring, a DC-0506 cryostat, a dual-wavelength ultraviolet detector to measure ultraviolet absorbance, and an Advantec CHF161RA fraction collector; adopting easy chrom-1000 software to collect and analyze data; the elution conditions were: the solvent system was a two-phase solvent system (stationary phase is organic phase, mobile phase is aqueous phase) of ethyl acetate/n-butanol/water (7.5:2.5:10 v/v), the rotational speed was 880rpm, the flow rate was 3.0mL/min, the detection wavelength was 230nm, the column temperature was 25 ℃, and the elution time was 280min.
Specifically, in step S6:
separating and purifying the subfraction Fr. IV by semi-preparative high performance liquid chromatography;
the elution conditions were: gradient elution is carried out by taking methanol and water as mobile phases, the flow rate is 3.0mL/min, the detection wavelength is 230nm, and the compound 1 and the compound 2 are respectively obtained; the volume ratio gradient of methanol to water is 40:60-68:32.
Separating and purifying the subfraction Fr. V by semi-preparative high performance liquid chromatography; the elution conditions were: gradient elution is carried out by taking methanol and water as mobile phases, the volume ratio gradient of the methanol to the water is 40:60-50:50, the flow rate is 3.0mL/min, and the detection wavelength is 230nm to the compound 3.
More specifically, the semi-preparative high performance liquid chromatography in step S6 employs an Agilent 1260 system equipped with an ultraviolet detector and a ZorbaxSB-C18 column (250 mm long, 9.4mm inside diameter, 5 μm); the elution conditions were: the methanol and water are used as mobile phase for gradient elution, the flow rate is 3.0mL/min, and the detection wavelength is 230nm.
The preparation method has the following innovation points:
1. the invention obtains 3 new iridoid glycoside compounds with anti-inflammatory activity from the war bone leaves.
2. Because the iridoid glycoside compounds have the characteristic of similar structures, repeated column chromatography (more than 6 times) is needed for traditional separation, and the time is long and a large amount of solvent is needed. The invention firstly adopts column chromatography (3 times) to carry out coarse separation on the extract, then adopts high-speed countercurrent chromatography (liquid-liquid separation chromatography with high sample repetition and high recovery rate), and preferably adopts a solvent system for separation to obtain the iridoid glycoside compound through high-efficiency enrichment. Finally, separating by adopting a preparative high performance liquid chromatography technology to obtain each iridoid glycoside compound.
The invention also provides application of the 3 novel iridoid compounds in preparing anti-inflammatory drugs or health care products.
The iridoid compound disclosed by the invention is a novel chemical component found by the inventor in the war bone leaves. The inventor adopts physicochemical properties and modern spectroscopy methods 1 H-NMR、 13 C-NMR、HSQC、HMBC、 1 H- 1 H COSY and HR-ESI-MS) to verify the chemical structure and physical and chemical properties of the three iridoid compounds, and to verify the pharmacological activity of the three new iridoid compounds through cell experiments, wherein the three new iridoid compounds all show excellent anti-inflammatory effect, have obvious inhibiting effect on NO generation, can effectively reduce the expression of pro-inflammatory factors TNF-a and IL-6, can reduce inflammatory reaction caused by the over-expression of inflammatory factors, and can be used as lead compounds for developing novel anti-inflammatory drugs.
The iridoid compound is derived from the leaf part of the war bone, can be developed into health-care food or medicine, and each experimental step of the preparation method is easy to control, simple and quick, so that the preparation of the iridoid compound is easier, the utilization of medicinal parts and medicinal resources of the war bone is enlarged, and the problem of environmental pollution caused by resource waste is solved.
Drawings
FIG. 1 is a 1H-NMR (500 MHz, DMSO-d 6) spectrum of compound 1.
FIG. 2 is a 13C-NMR (125 MHz, DMSO-d 6) spectrum of compound 1.
FIG. 3 is a 1H-NMR (500 MHz, DMSO-d 6) spectrum of compound 2.
FIG. 4 is a 13C-NMR (125 MHz, DMSO-d 6) spectrum of compound 2.
FIG. 5 is a 1H-NMR (500 MHz, DMSO-d 6) spectrum of compound 3.
FIG. 6 is a 13C-NMR (125 MHz, DMSO-d 6) spectrum of compound 3.
FIG. 7 shows the effect of compounds 1,2,3 on LPS-induced release of NO by RAW264.7 cells.
FIG. 8 is a graph showing the effect of compounds 1 and 3 on the production of inflammatory factor tumor necrosis factor.
FIG. 9 is a graph showing the effect of compounds 1, 2and 3 on the production of interleukin.
Detailed Description
The principles and features of the present invention are described below, and the examples are provided for illustration only and are not intended to limit the scope of the invention.
Liquid chromatograph (LC-2030C, shimadzu corporation (china); electronic analytical balance (XS 225A-SCS, pr Li Saisi International trade (Shanghai) Co., ltd.); high-speed countercurrent chromatography TBE-300C (Shanghai Tongtian Biotechnology Co., ltd.); automatic receiver (CHF 161RA, advantec, japan); vacuum centrifugal concentrator (miVac, geneVac Co., UK); LC-MS/IT-TOF liquid chromatography mass spectrometry (Shimadzu, japan); superconducting nuclear magnetic resonance spectrometer (Brucker)Avance 500MHz, brucker company, germany); an N-1100 type rotary evaporator (Eyela, japan); d101 macroporous adsorbent resin (baen adsorbent materials technologies, inc.); sephadex LH-20 gel column (GE Healthcare Bio-science AB, switzerland); c18 column (japan Fuji Silyia Chemical Ltd corporation); HP-20SS column (Japanese Mitsubishi Chemical Co.); silica gel thin layer board F 254 (thickness 0.2mm, merck, germany); the methanol used in the HPLC analysis is chromatographic pure (Tedia, U.S.); the reagents used for extraction and separation, namely acetone, petroleum ether, methanol, ethanol and ethyl acetate, and n-butanol are all analytically pure (Guangdong Guanghua technology Co., ltd.).
EXAMPLE 1 extraction and separation of the Ether terpenoids from the Flores Miquel She Huanxi
The experimental war bone leaves are collected from Guangxi plant autonomous region Gui Linshi Yanshan Guilin plant garden in 8 months and 25 days in 2020, cut up, dried in the shade and dried in the air, identified by Tang Hui researchers as the leaves of the Bean curd firewood plant war bone Prenma fulva Craib in Verbenaceae, and the voucher samples are stored in Guangxi plant functional substances and resource continuous utilization key laboratories.
1. Experimental method
1. Extraction and separation of compounds
15.0kg of dried war bone leaf, soaking in 60L of methanol at room temperature for 7d, filtering, repeating for 3 times, mixing filtrates, and recovering methanol to obtain 1.2kg of war bone leaf methanol extract.
D101 column chromatography: taking methanol extract (1.2 kg), carrying out gradient elution by methanol-water (volume ratio gradient is 10:90, 20:80, 30:70, 50:50, 80:20, 100:0), collecting gradient eluent of each gradient, detecting by thin layer chromatography, combining similar fractions, concentrating, and collecting 6 fractions (Fr.1-6).
Fractions fr.4 (305.00 g) were packed with C18, gradient eluted with methanol-water (volume ratio gradients 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 100:0), 2 column volumes per gradient eluted, detected by thin layer chromatography, the similar fractions were combined and concentrated, collecting a total of 8 subfractions (fr.4- (1-8)); subfractions Fr.4-1 (24.32 g) were packed with HP20SS and eluted with a gradient of methanol-water (volume ratio gradients 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 100:0), 2 column volumes each eluted, detected by thin layer chromatography, and similar fractions were combined and concentrated to give a total of 10 subfractions (Fr.4-1- (1-10)). Subfractions Fr.4-1-5 (12.58 g) were packed with gel and eluted with a gradient of methanol-water (volume ratio gradients 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0), 2 column volumes per gradient eluted, detected by thin layer chromatography, and similar fractions were combined and concentrated to give a total of 9 subfractions (Fr.4-1-5- (1-9)). The subfractions Fr.4-1-5-4 (2.81 g) were subjected to high-speed countercurrent chromatography (TautoTBE-300C system, 3 polytetrafluoroethylene multi-layer coils (inner diameter: 1.9mm; total volume: 320 mL), 20mL sample ring, DC-0506 cryostat, dual-wavelength UV detector for measuring UV absorbance, advantec CHF161RA fraction collector, data acquisition and analysis by Easychrom-1000 software, eluting with a two-phase solvent system (organic phase stationary phase, aqueous phase) of ethyl acetate/n-butanol/water (7.5:2.5:10 v/v) at a rotation speed of 880rpm, a flow rate of 3.0mL/min, a detection wavelength of 230nm, a column temperature of 25deg.C, an elution time of 280min, a sample size of 320 mg), detection by TLC plate, combining similar elution fractions and concentrating to obtain 6 subfractions Fr. I-VI.
The subfraction Fr. IV (73.80 mg) was separated and purified by semi-preparative high performance liquid chromatography (Agilent 1260 system, equipped with UV detector and ZorbaxSB-C18 column (length 250mm, inner diameter 9.4mm,5 μm), under conditions of gradient elution (40:60-68:32, v/v) with methanol-water as mobile phase, flow rate of 3.0mL/min, detection wavelength of 230nm, sample injection amount of 50. Mu.L) to give Compound 1 (17.60 mg) and Compound 2 (8.60 mg).
The subfraction Fr. V (64.60 mg) was purified by semi-preparative high performance liquid chromatography (Agilent 1260 system, equipped with UV detector and ZorbaxSB-C18 column (250 mm long, 9.4mm inner diameter, 5 μm) under conditions of gradient elution (40:60-50:50, v/v) with methanol-water as mobile phase, flow rate of 3.0mL/min, detection wavelength of 230nm, and sample injection amount of 50. Mu.L) to give compound 3 (8.10 mg).
Example 2 structural identification of iridoids
1. Structural identification of Compound 1
[M+HCOO] - The molecular weight of the quasi-molecular ion peak of (2) is 974.3420, and the molecular formula is C 47 H 58 O 22 . The unsaturation was 19. In the UV spectrum (methanol solution), compound 1 has a maximum absorbance at a wavelength of 230nm.
At the position of 1 In the H-NMR spectrum, four trans-olefin protons delta H 6.51、7.63(d，J＝16.0Hz)，δ H 5.87, 6.95 (d, j=12.8 Hz), 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 delta s H 3.79 The signal of (3H, s) 3.80 (3H, s) indicates that compound 1 contains two trans-methoxycinnamoyl units; the DEPT-135 spectrum of compound 1 also confirms the presence of two methoxy cinnamic acids. Compound 1 in 1 H- 1 Proton signal delta in the H COSY spectrum H 3.65 (1 h, d, j=2.7 Hz), 3.92 (1 h, dd, j=8.1, 2.1 Hz), 2.29 (1 h, m), 2.37 (1 h, m), 4.97 (1 h, m) are related in sequence, suggesting that they are from the same spin coupling system. Combining the related information provided by the HSQC spectrum to obtain the carbon skeleton delta C 57.2 (C-7), 81.6 (C-6), 35.5 (C-5), 41.8 (C-9), 93.1 (C-1). In HMBC spectra, proton signal delta H 2.37 (H-9) and 4.97 (H-1) are both identical to the carbon signal delta C 65.4 (C-8) is related, suggesting that C-8 is attached to C-9. In addition, delta H 2.37 Hydrogen signal and delta of (H-9) C 57.2 The carbon signals of (C-7) are related, indicating that C-7 is linked to C-8 to form a five membered ring structure. In HMBC, H-6 (delta) H 3.92)/C-8(δ C 65.4)、H-7(δ H 3.65)/C-9(δ C 41.8 The presence of the above five-membered ring is further confirmed by the correlation peak. According to the carbon signal delta C 57.2 (C-7), 65.4 (C-8), it can be deduced that the C-7, C-8 sites are identical to each otherOxygen forms an epoxy three-membered ring; combining the related information provided by the HSQC spectrum to obtain the carbon skeleton delta C 57.2 (C-7), 65.4 (C-8), 58.7 (C-10). Alkene hydrogen signal delta H 6.43 (1H, dd, J=4.9, 1.9Hz, H-3) and delta C 102.2 The ethylenic carbon signal of (C-4) is HMBC-related, suggesting that C-3 is linked to C-4 in the structure; combining the related information provided by the HSQC spectrum to obtain the carbon skeleton delta C 141.0 (C-3), 102.2 (C-4). Furthermore, in HMBC spectra the olefinic hydrogen protons H-3 (delta H 6.43 And C-5 (delta) C 35.5 Related to H-5 (delta) H 2.29 Respectively with C-3 (delta) C 141.0)、C-4(δ C 102.2 A) the hint that C-4 is linked to C-5. H-3 (delta) H 6.43 And C-1 (delta) C 93.1 For example, it is suggested that C-1 and C-3 are linked through a heteroatom (oxygen atom). Thereby obtaining a six-membered ring structure. Thus, the skeleton structure of iridoid was obtained. At delta H 4.95 (m, H-1 '), two anomeric proton signals are observed at 4 (m, H-1') at delta H 1.17 (3 h, d, j=6.2 Hz), two proton resonance signals were observed at 1.21 (3 h, d, j=6.2 Hz), at δ C 68.5 (C-5 '), two carbon signals are observed at 68 (C-5') and are ascribed to two alpha-rhamnosyl groups. Further, at delta H 4.59 (d, J=7.9 Hz, H-1') and delta C 97.8 (C-1') has an anomeric proton and a carbon signal at delta H The region 3.21-3.85 has other signals indicating the presence of a β -glucosyl group. In HMBC spectra, according to 1 H- 1 HCOSY spectrum can be used for deducing that the 2-position hydrogen signal and the 3-position hydrogen signal of rhamnose A are delta respectively H 3.71 and delta H 5.01 terminal hydrogen delta of rhamnose B H 4.94 (H-1') with rhamnose A at carbon delta at position 2 C 68.8 (C-2') has a remote association suggesting that rhamnose B is linked to rhamnose A at the C-2 position; rhamnose a 3-position hydrogen signal delta H 5.01 carbonyl carbon delta on trans-p-methoxy cinnamoyl A group C 166.1 There was a remote correlation of (C- α -c=o), suggesting that the trans-p-methoxycinnamoyl a group is attached at the C-3 position of rhamnose. According to 1 H- 1 The HCOSY spectrum can deduce that the 3-hydrogen signal of rhamnose B is delta H 5.03, and the hydrogen signal delta at position 3 of rhamnose B H 5.03 on the B radical of trans-p-methoxy cinnamoylCarbonyl carbon delta C 165.3 There is a remote correlation of (C- α' -c=o), suggesting that the trans-p-methoxycinnamoyl B group is attached at the C-3 position of rhamnose B. The terminal hydrogen of glucose is remotely related to the carbon at the 1-position of the iridoid skeleton, which indicates that glucose is connected to the C-1 position of the iridoid skeleton; the terminal hydrogen of rhamnose A is remotely related to carbon at position 6 of the iridoid skeleton, indicating that rhamnose A is linked at position C-6 of the iridoid skeleton. Each associated H, C data can be passed through 1 H-NMR、 13 C-NMR、 1 H- 1 The spectra of H COSY, HMBC, HSQC, HR-ESI-MS, etc. were assigned (see FIGS. 1-2). In combination with the above data and literature analysis, 1 h and 13 c NMR is shown in Table 1, and it can be confirmed that compound 1 (6-O- (2 ' - (3 ' -O-trans-p-methoxycinamoyl) -alpha-L-rhamnofuranosyl-3 ' -O-trans-p-methoxycinamoyl) -alpha-L-rhamnopyranosyl catalpol) has a structural formula shown in formula I, and is an iridoid compound.
2. Structural identification of Compound 2
At the position of 1 In the H-NMR spectrum, two groups of typical trans-double bond olefinic hydrogen proton signals delta H 5.88and 6.95(d，J＝12.8Hz)，δ H 6.52and 7.63 (d, j=16.0 Hz), and four pairs of ortho-coupled aromatic protons δ H 7.74 (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) clearly shows that the acyl moiety is two trans-p-methoxycinnamoyl units, compound 2 13 C NMR confirmed two trans formsThe presence of p-methoxycinnamoyl. Compound 2 in 1 H- 1 Proton signal delta in the H COSY spectrum H 3.64 (1 h, d, j=3.8 Hz), 3.91 (1 h, d, j=8.3 Hz), 2.28 (1 h, m), 2.38 (1 h, dd, j=7.4, 2.5 Hz), 4.96 (1 h, d, j=4.6 Hz) are sequentially correlated, suggesting that they are from the same spin coupling system. Combining the related information provided by the HSQC spectrum to obtain the carbon skeleton delta C 57.4 (C-7), 81.8 (C-6), 35.6 (C-5), 41.8 (C-9), 93.1 (C-1). In HMBC spectra, proton signal delta H 2.38 (H-9) and 4.96 (H-1) are both identical to the carbon signal delta C 65.3 (C-8) is related, suggesting that C-8 is attached to C-9. In addition, delta H 2.38 Hydrogen signal and delta of (H-9) C 57.4 The carbon signals of (C-7) are related, indicating that C-7 is linked to C-8 to form a five membered ring structure. In HMBC, H-6 (delta) H 3.91)/C-8(δ C 65.3)、H-7(δ H 3.64)/C-9(δ C 41.8 The presence of the above five-membered ring is further confirmed by the correlation peak. According to the carbon signal delta C 57.4 (C-7), 65.3 (C-8), it can be inferred that the C-7, C-8 sites form an epoxy three-membered ring with oxygen; combining the related information provided by the HSQC spectrum to obtain the carbon skeleton delta C 57.4 (C-7), 65.3 (C-8), 58.7 (C-10). Alkene hydrogen signal delta H 6.44 (1H, m, H-3) and delta C 102.2 The ethylenic carbon signal of (C-4) is HMBC-related, suggesting that C-3 is linked to C-4 in the structure; combining the related information provided by the HSQC spectrum to obtain the carbon skeleton delta C 141.0 (C-3), 102.2 (C-4). Furthermore, in HMBC spectra the olefinic hydrogen protons H-3 (delta H 6.44 And C-5 (delta) C 35.6 Related to H-5 (delta) H 2.28 Respectively with C-3 (delta) C 141.0)、C-4(δ C 102.2 A) the hint that C-4 is linked to C-5. H-3 (delta) H 6.44 And C-1 (delta) C 93.1 For example, it is suggested that C-1 and C-3 are linked through a heteroatom (oxygen atom). Thereby obtaining a six-membered ring structure. Thus, the skeleton structure of iridoid was obtained. At delta H 4.89 An anomeric proton signal was observed at (d, j=5.7 hz, h-1 "), at δ H 1.06 A proton resonance signal was observed at (3 h, d, j=6.2 Hz), at δ C 66.4 A carbon signal was observed at (C-5') which was attributed to an alpha-rhamnosyl group; further, at delta H 4.59(d，J＝7.9Hz, H-1') and delta C 97.8 (C-1') has an anomeric proton and a carbon signal at delta H The region 3.21-3.85 has other signals indicating the presence of a β -glucosyl group. In HMBC spectra, according to 1 H- 1 The HCOSY spectrum can be used for deducing that the 2-bit hydrogen signal and the 4-bit hydrogen signal of rhamnose are delta respectively H 3.74 and delta H 4.94, hydrogen signal delta at position 2 of rhamnose H 3.74 carbonyl carbon delta on trans-p-methoxy cinnamoyl A group C 165.4 (C- α -c=o) has a remote correlation suggesting that the trans-p-methoxycinnamoyl a group is attached at the C-2 position of rhamnose; and rhamnose 4-position hydrogen signal delta H 4.94 carbonyl carbon delta on trans-p-methoxy cinnamoyl B group C 166.2 (C- α' -c=o) has a remote correlation suggesting that the trans-p-methoxycinnamoyl B group is attached at the C-4 position of rhamnose. The terminal hydrogen of glucose is remotely related to the carbon at the 1-position of the iridoid skeleton, which indicates that glucose is connected to the C-1 position of the iridoid skeleton; the terminal hydrogen of rhamnose is remotely related to carbon at position 6 of the iridoid skeleton, indicating that rhamnose A is linked at position C-6 of the iridoid skeleton. Each associated H, C data can be passed through 1 H-NMR、 13 C-NMR、 1 H- 1 The spectra of H COSY, HMBC, HSQC, HR-ESI-MS, etc. were assigned (see FIGS. 3-4).
In combination with the above data and literature analysis, 1 h and 13 c NMR is shown in Table 1, and it can be confirmed that compound 2 (6-O- (2 ', 4' -di-trans-p-methoxycinnamonyl) -alpha-L-rhamnopyranosyl catalpol) has a structural formula shown in formula II and is an iridoid compound.
3. Structural identification of Compound 3
At the position of 1 In the H-NMR spectrum, a typical group of trans-double bond olefinic hydrogen proton signals delta H 6.50 and 7.64 (d, j=16.0 Hz), and four aromatic protons δ H 6.94 (d, j=2.2 hz, h-2 '"), 6.99 (dd, j=8.8, 2.2hz, h-4'"), 7.69 (d, j=8.8 hz, h-5 '"), 7.79 (dd, j=8.8, 2.2hz, h-6'") and one aromatic methoxy δ H 3.79 The signal of (3H, s) clearly shows that the acyl moiety is a trans-m-methoxycinnamoyl unit and, furthermore, delta in the HMBC spectra of Compound 3 H 3.80(s)/δ C 161.1 (C-3') correlation confirms the presence of trans-m-methoxycinnamoyl. Compound 3 in 1 H- 1 Proton signal delta in the H COSY spectrum H 3.64 (1 h, m), 3.93 (1 h, dd, j=8.1, 1.2 hz), 2.31 (1 h, dd, j=4.2, 1.8 hz), 2.39 (1 h, dd, j=9.7, 7.6 hz), 4.97 (1 h, s) are sequentially correlated, suggesting that they are from the same spin coupling system. Combining the related information provided by the HSQC spectrum to obtain the carbon skeleton delta C 57.4 (C-7), 81.6 (C-6), 35.5 (C-5), 41.9 (C-9), 93.1 (C-1). In HMBC spectra, proton signal delta H 2.39 (H-9) and 4.97 (H-1) are both identical to the carbon signal delta C 65.3 (C-8) is related, suggesting that C-8 is attached to C-9. In addition, delta H 2.39 Hydrogen signal and delta of (H-9) C 57.4 The carbon signals of (C-7) are related, indicating that C-7 is linked to C-8 to form a five membered ring structure. In HMBC, H-6 (delta) H 3.93)/C-8(δ C 65.3)、H-7(δ H 3.64)/C-9(δ C 41.9 The presence of the above five-membered ring is further confirmed by the correlation peak. According to the carbon signal delta C 57.4 (C-7), 65.3 (C-8), it can be inferred that the C-7, C-8 sites form an epoxy three-membered ring with oxygen; combining the related information provided by the HSQC spectrum to obtain the carbon skeleton delta C 57.4 (C-7), 65.3 (C-8), 58.7 (C-10). Alkene hydrogen signal delta H 6.44 (1H, dd, J=6.0, 1.7Hz, H-3) and delta C 102.3 The ethylenic carbon signal of (C-4) is HMBC-related, suggesting that C-3 is linked to C-4 in the structure; combining the related information provided by the HSQC spectrum to obtain the carbon skeleton delta C 140.9 (C-3), 102.3 (C-4). Furthermore, in HMBC spectra the olefinic hydrogen protons H-3 (delta H 6.44 And C-5 (delta) C 35.5 Related to H-5 (delta) H 2.31 Respectively if are notAnd C-3 (delta) C 140.9)、C-4(δ C 102.3 A) the hint that C-4 is linked to C-5. H-3 (delta) H 6.44 And C-1 (delta) C 93.1 For example, it is suggested that C-1 and C-3 are linked through a heteroatom (oxygen atom). Thereby obtaining a six-membered ring structure. Thus, the skeleton structure of iridoid was obtained. At delta H 4.85 An anomeric proton signal was observed at (d, j=1.7 hz, h-1 "), at δ H 1.20 A proton resonance signal was observed at (3 h, d, j=6.3 Hz), at δ C 68.1 A carbon signal was observed at (C-5') which was attributed to an alpha-rhamnosyl group; further, at delta H 4.60 (d, J=7.9 Hz, H-1') and delta C 97.8 (C-1') has an anomeric proton and a carbon signal at delta H The region 3.21-3.85 has other signals indicating the presence of a β -glucosyl group. In HMBC spectra, according to 1 H- 1 HCOSY spectrum can deduce that the 3-hydrogen signal of rhamnose is delta H 4.89, hydrogen Signal delta at position 3 of rhamnose H 4.89 carbonyl carbon delta on trans-Methoxycinnamoyl group C 166.2 (C- α -c=o) has a remote correlation suggesting that the trans-m-methoxycinnamoyl group is attached at the C-3 position of rhamnose. The terminal hydrogen of glucose is remotely related to the carbon at the 1-position of the iridoid skeleton, which indicates that glucose is connected to the C-1 position of the iridoid skeleton; the terminal hydrogen of rhamnose is remotely related to carbon at position 6 of the iridoid skeleton, indicating that rhamnose A is linked at position C-6 of the iridoid skeleton. Each associated H, C data can be passed through 1 H-NMR、 13 C-NMR、 1 H- 1 The spectra of H COSY, HMBC, HSQC, HR-ESI-MS, etc. were assigned (see FIGS. 5-6).
In combination with the above data and literature analysis, 1 h and 13 c NMR is shown in Table 1, and can prove that the structural formula of the compound 3 (6-O- (3' -O-trans-m-methoxycinnamonyl) -alpha-L-rhamnopyranosyl catalpol) is shown in a formula III, and is an iridoid compound.
TABLE 1 Compounds 1-3 (DMSO-d 6 ) A kind of electronic device 1 H spectrum 13 C spectral data
Example 3 anti-inflammatory Activity experiment of iridoid Compounds
This example is conducted with the compound 6-O- (2 '- (3' -O-trans-p-methoxycinnamonyl-. Alpha. -L-rhamnopyranosyl) -3 '-O-trans-p-methoxycinnamonyl) -alpha. -L-rhamnopyranosyl catalpol (1), 6-O- (2', in vitro anti-inflammatory Activity study experiments of 4 '-di-trans-p-methoxycinnamonyl) -alpha-L-rhamnopyranosyl catalpol (2), and 6-O- (3' -O-trans-m-methoxycinnamonyl) -alpha-L-rhamnopyranosyl catalpol (3) on LPS-induced RAW264.7 cells.
1. Experimental materials and instruments
Clean bench (air technologies, inc. Of safe, su zhou); constant temperature CO 2 Cell incubator (Thermo company, usa); an electric heating thermostatic water bath (Shanghai BoXie Co., ltd.); inverted microscope (LEICA company, germany); multifunctional microplate reader (TECAN company, switzerland); refrigerated centrifuge (Pink black horse Co.); electronic balance (Shanghai Tianmei balance instruments Co., ltd.); vortex oscillators (Haimen Korla); PBS phosphate buffer (beijing solibao); nitric oxide kit (bi yun tian biotechnology); mouse TNF-. Alpha.EL ISA kit, mouse IL-6ELISA kit (Wuhan Irey Co.); cell Counting Kit-8 (Med Chem Express Co., USA); LPS (Sigma Co., USA); DMEM (Sigma, usa); DMSO (Shanghai microphone Biochemical technologies Co., ltd.); indomethacin (Sigma, usa).
2. Test method
1. Cytotoxicity and anti-inflammatory Activity assays
Inoculating RAW264.7 cells in logarithmic phase into 96-well cell culture plate, adding 100 μl of culture medium per well to obtain cell density of 2×10 5 Culturing in incubator24h. The experiments were divided into blank and dosing groups. No drug was added to the blank, and each dosing group was incubated with compound 1,2,3 diluted to different concentrations with DMEM medium for 24h, 3 wells repeated for each group. After 24h, the 96-well plates were removed from the incubator, 10. Mu.L of CCK-8 was added to each well, and after incubation at 37℃for L h, the absorbance (A) was measured at a wavelength of 450nm and the cell viability was calculated and 3 experiments were repeated.
Inoculating RAW264.7 cells in logarithmic phase into 96-well cell culture plate, adding 100 μl of culture medium per well to obtain cell density of 2×10 5 The cells are cultured in an incubator for 24 hours. The experiment groups are blank group, model group, positive medicine group and medicine adding group. The drug groups were incubated for 24h with LPS (1. Mu.g/mL) stimulated cells after each addition of different concentrations of compound 1,2,3, l h, and 3 wells were repeated for each group with indomethacin as positive control. After 24h, 50. Mu.L of supernatant was aspirated into 96-well cell culture plates, then 50. Mu.L of Griess reagent I and Griess reagent II were sequentially added, gently shaken several times to mix them well, absorbance A was measured at 540nm wavelength with a microplate reader, NO inhibition was calculated from the standard curve, and fitting curves were obtained using GraphPad Prism 8 software to determine the IC for inhibition of NO release by Compound 1,2,3, respectively 50 Values.
2. ELISA (enzyme-Linked immuno sorbent assay) for detecting content of inflammatory factors
Inoculating RAW264.7 cells in logarithmic phase into 96-well cell culture plate, adding 100 μl of culture medium per well to obtain cell density of 2×10 5 The cells are cultured in an incubator for 24 hours. The experiment groups are blank group, model group, positive medicine group and medicine adding group. The drug groups were incubated for 24h with LPS (1. Mu.g/mL) stimulated cells after each addition of different concentrations of compound 1,2,3, l h, and 3 wells were repeated for each group with indomethacin as positive control. After 24h, the cell supernatant was collected and transferred to an EP tube, labeled, centrifuged at 1000 Xg for 20min at 4℃to remove particles and polymer, and the supernatant was aspirated into a fresh EP tube and dispensed. Each sample was provided with 3 biological replicate wells. The operation steps are as follows: 1. standard wells, blank wells and sample wells were set separately. Standard wells were filled with 100 μl of diluted standard, blank wells100 mu L of standard substance&Sample dilutions and the remaining wells were filled with 100 μl of sample to be tested (all samples to be tested and standards were recommended to set up multiple wells in the test). The ELISA plate was covered and incubated at 37℃for 90 minutes. Prompting: during sample adding, the sample is added to the bottom of the ELISA plate, the hole wall is not touched as much as possible, and the sample is gently shaken and uniformly mixed, so that bubbles are avoided. The sample application time is preferably controlled within 10 minutes. 2. The liquid in the hole is thrown out without washing. 100. Mu.L of biotinylated antibody working solution was added to each well, and the ELISA plate was covered with a membrane and incubated at 37℃for 1 hour. 3. And (5) throwing out the liquid in the hole, and drying the hole by beating on clean absorbent paper. Adding 350 mu L of washing liquid into each hole, soaking for 1 minute, sucking or throwing away the liquid in the ELISA plate, and drying. This plate washing step was repeated 3 times. Immediately after the plate washing is completed, the next step operation is performed, and the micro-porous plate is not required to be dried. 4. Each well was added with 100. Mu.L of the enzyme conjugate working solution, and the ELISA plate was covered with a membrane and incubated at 37℃for 30 minutes. 5. And (3) throwing out the liquid in the holes, washing the plate for 5 times, and carrying out the same method as the step (3). 6. Each well was filled with 90. Mu.L of primer solution (TMB), and the ELISA plate was covered with a film and incubated at 37℃for about 15 minutes in the absence of light. Prompting: shortening or lengthening as appropriate according to the actual color development, but not exceeding 30 minutes. When a clear gradient occurs in the standard well (a clear blue gradient occurs in the first 4 wells), it can be terminated. The microplate reader was turned on 15 minutes in advance for preheating. 7. The reaction was terminated by adding 50. Mu.L of a stop solution to each well. Prompting: the order of addition of the stop solution should be as similar as possible to the order of addition of the substrate solution. 8. The optical density (OD value) of each well was measured immediately with an enzyme-labeled instrument at a wavelength of 450 nm.
3. Experimental results
1. Effect of Compounds on cytotoxicity and anti-inflammatory Activity
RAW264.7 cells were incubated for 24h with 40, 80, 160. Mu.M of Compound 1,2,3, respectively, and the results showed that Compound 1,2,3 had no toxic effect on the cells at 40, 80, 160. Mu.M.
The results of inhibition of NO release at various concentrations are shown in FIG. 7. From the graph, the release amount of NO in normal RAW264.7 cells is lower, and after LPS induction, the content of NO in cell supernatant is obviously increased to 14.09 mu M, which indicates that the model construction is successful; when dry with different concentrations of compound 1,2,3, giveReduced NO content in supernatant of medicinal group cells, compound 1,2,3 and positive drug indomethacin IC for inhibiting NO release 50 45.80, 51.17, 68.42 and 100.33 μm respectively, the compounds 1,2,3 showed better anti-inflammatory effect, and the anti-inflammatory effect of the compounds 1,2,3 was better than that of the positive drug.
2. Effect of Compounds on secretion of inflammatory factors TNF-alpha, IL-6 in cells
Inoculating RAW264.7 cells in logarithmic phase into 96-well cell culture plate, adding 100 μl of culture medium per well to obtain cell density of 2×10 5 The cells are cultured in an incubator for 24 hours. The experiment groups are blank group, model group, positive medicine group and medicine adding group. The drug groups were incubated for 24h with LPS (1. Mu.g/mL) stimulated cells after each addition of different concentrations of compound 1,2,3, l h, and 3 wells were repeated for each group with indomethacin as positive control. After 24h, the cell supernatant was collected and transferred to an EP tube, labeled, centrifuged at 1000 Xg for 20min at 4℃to remove particles and polymer, and the supernatant was aspirated into a fresh EP tube and dispensed. Each sample was provided with 3 biological replicate wells. The operation steps are as follows: 1. standard wells, blank wells and sample wells were set separately. Standard wells were filled with 100 μl of standard diluted at a double ratio, and blank wells were filled with 100 μl of standard&Sample dilutions and the remaining wells were filled with 100 μl of sample to be tested (all samples to be tested and standards were recommended to set up multiple wells in the test). The ELISA plate was covered and incubated at 37℃for 90 minutes. Prompting: during sample adding, the sample is added to the bottom of the ELISA plate, the hole wall is not touched as much as possible, and the sample is gently shaken and uniformly mixed, so that bubbles are avoided. The sample application time is preferably controlled within 10 minutes. 2. The liquid in the hole is thrown out without washing. 100. Mu.L of biotinylated antibody working solution was added to each well, and the ELISA plate was covered with a membrane and incubated at 37℃for 1 hour. 3. And (5) throwing out the liquid in the hole, and drying the hole by beating on clean absorbent paper. Each well was filled with 350. Mu.L of washing solutionSoaking for 1 min, sucking or throwing away the liquid in the ELISA plate, and drying. This plate washing step was repeated 3 times. Immediately after the plate washing is completed, the next step operation is performed, and the micro-porous plate is not required to be dried. 4. Each well was added with 100. Mu.L of the enzyme conjugate working solution, and the ELISA plate was covered with a membrane and incubated at 37℃for 30 minutes. 5. And (3) throwing out the liquid in the holes, washing the plate for 5 times, and carrying out the same method as the step (3). 6. Each well was filled with 90. Mu.L of primer solution (TMB), and the ELISA plate was covered with a film and incubated at 37℃for about 15 minutes in the absence of light. Prompting: shortening or lengthening as appropriate according to the actual color development, but not exceeding 30 minutes. When a clear gradient occurs in the standard well (a clear blue gradient occurs in the first 4 wells), it can be terminated. The microplate reader was turned on 15 minutes in advance for preheating. 7. The reaction was terminated by adding 50. Mu.L of a stop solution to each well. Prompting: the order of addition of the stop solution should be as similar as possible to the order of addition of the substrate solution. 8. The optical density (OD value) of each well was measured immediately with an enzyme-labeled instrument at a wavelength of 450 nm.
As shown in FIGS. 8-9, the concentrations of the inflammatory factors TNF-alpha and IL-6 released by macrophages in the model group after 24h of stimulation with LPS, which were obtained by pre-treating cells l h with compounds 1,2,3 at concentrations of 10, 40 and 160. Mu.M, were significantly higher than those in the blank group, indicating that the inflammatory cell model was successfully constructed. After the compound is treated, the corresponding production amount of the compounds 1, 2and 3 is lower than that of a model group, and the expression levels of the two proteins of TNF-alpha and IL-6 can be down-regulated, so that the compound can have good anti-inflammatory activity.
In conclusion, the iridoid compounds 1, 2and 3 separated from the war bone leaves can inhibit the generation of NO, have the effect of inhibiting the expression of inflammatory factors TNF-alpha and IL-6, and have good anti-inflammatory activity.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (9)
1. The novel iridoid compound with anti-inflammatory activity is characterized by having the following structural formulas:
2. a process for the preparation of novel iridoid compounds with anti-inflammatory activity according to claim 1, characterized in that it comprises the following steps:
s1, taking the war bone leaves, soaking the war bone leaves in an alcohol solvent, filtering the solution, repeating the filtering at least once, combining the solution to obtain an extracting solution, and removing the solvent in the extracting solution to obtain a total extract;
s2, subjecting the total extract obtained in the step S1 to macroporous adsorption resin column chromatography, performing gradient elution by using mixed eluent of alcohol and water, collecting gradient eluent of each gradient, detecting by using a TLC (thin layer chromatography) plate, combining similar elution fractions, concentrating, and collecting 6 fractions Fr.1-6;
s3, performing gradient elution on the fourth fraction Fr.4 obtained in the step S2 by using a mixed eluent of alcohol and water through a column chromatography method, collecting gradient eluents of each gradient, detecting through a TLC (thin layer chromatography) plate, combining similar elution fractions, concentrating, and collecting 8 fractions Fr.4- (1-8);
s4, sequentially performing gradient elution on the first fraction Fr.4-1 obtained in the step S3 by using a mixed eluent of alcohol and water through a column chromatography, detecting through a thin layer chromatography, combining similar fractions and concentrating, collecting to obtain a subfraction Fr.4-1- (1-10), performing gradient elution on the subfraction Fr.4-1-5 by using a mixed eluent of alcohol and water through a gel column chromatography, detecting through a thin layer chromatography, combining similar elution fractions and concentrating, and collecting to obtain 9 fractions Fr.4-1-5- (1-9);
s5, eluting the 4 th fraction Fr.4-1-5-4 obtained in the step S4 through HSCCC chromatography, detecting through a TLC (thin-layer chromatography) plate, combining similar elution fractions, concentrating, and collecting to obtain 6 subfractions Fr. I-VI;
s6, separating and purifying the 4 th fraction Fr. IV obtained in the step S5 through semi-preparative HPLC to obtain compounds 1 and 2 shown in the structure in the claim 1; separating and purifying the 5 th fraction Fr. V obtained in the step S5 by semi-preparative HPLC to obtain the compound 3 shown in the structure of claim 1.
3. The method according to claim 2, wherein in step S1:
the alcohol solvent is methanol or ethanol;
the dosage ratio of the war bone leaf to the alcohol solvent is 15kg (30-90) L;
soaking at room temperature; the soaking time is (5-9) d;
the repetition number is (2-4).
4. The method according to claim 2, wherein in step S2:
the macroporous adsorption resin column is a D101 column, HPD-100 and HP20;
the alcohol is methanol or ethanol;
the volume ratio gradient of the alcohol to the water is 10:90-100:0.
5. The method according to claim 2, wherein in step S3:
column chromatography using C18 column, C8, MCI, HP20SS;
the alcohol is methanol or ethanol;
the volume ratio gradient of alcohol and water is 40:60-100:0 in sequence.
6. The method according to claim 2, wherein in step S4:
the column chromatography adopts HP20SS column, C18 and MCI;
gel column chromatography adopts Sephadex LH-20 and Sephadex G;
the alcohol is methanol or ethanol;
the volume ratio gradient of alcohol to water in the column chromatography is 20:80-100:0;
the volume ratio gradient of alcohol and water in gel column chromatography is 20:80-100:0.
7. The method according to claim 2, wherein the elution conditions in step S5 are: the solvent system is a two-phase solvent system of ethyl acetate, n-butanol and water, the rotating speed is 880rpm, the flow rate is 3.0mL/min, the detection wavelength is 230nm, the column temperature is 25 ℃, the elution time is 280min, and the sample injection amount is 320mg; the solvent concentration volume ratio of ethyl acetate, n-butanol and water is 5:5:10, 7:3:10, 7.5:2.5:10, 8:2:10, preferably 7.5:2.5:10.
8. The method according to claim 2, wherein in step S6:
separating and purifying the subfraction Fr. IV by semi-preparative high performance liquid chromatography; the elution conditions were: methanol and water are used as mobile phases for gradient elution, and the gradient elution program is as follows: 0-35min,40:60-68:32 (methanol: water, v/v); the flow rate is 3.0mL/min, the detection wavelength is 230nm, and compound 1 (24 min) and compound 2 (30 min) are respectively obtained;
separating and purifying the subfraction Fr. V by semi-preparative high performance liquid chromatography; the elution conditions were: methanol and water are used as mobile phases for gradient elution, and the gradient elution program is as follows: 0-20min,40:60-50:50 (methanol: water, v/v), flow rate of 3.0mL/min, detection wavelength of 230nm, to obtain compound 3 (13 min).
9. Use of novel iridoid compounds with anti-inflammatory activity according to claim 1, characterized in that: is used for preparing anti-inflammatory drugs or health care products.
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