Separation and application of brevifolin in Ophiopogon japonicus

Abstract

The invention discloses separation and application of brevifolin in Ophiopogon japonicus. The separation method comprises the following steps: taking the Ophiopogon japonicus as a raw material, obtaining an extract of the Ophiopogon japonicus, dispersing the extract in water, extracting with petroleum ether, putting the petroleum ether part on a silica gel column, carrying out gradient elution with chloroform-methanol, and purifying an obtained crude product of the Ophiopogon japonicus through reversed-phase high-performance liquid chromatography, wherein the chromatographic conditions are as follows: a chromatographic column takes octadecyl bonded silica gel as a filling agent; and the mobile phase is composed of methanol and water in a volume ratio of (5-95): (45-55). Pharmacological studies prove that the brevifolin has good anti-inflammatory activity, the half inhibitory concentration IC50 value of the brevifolin for inhibiting release of nitric oxide of RAW264.7 cells induced by LPS is 55.77 + / -7.28 mu M, and an important candidate compound is provided for developing novel anti-inflammatory drugs.

Description

Technical Field

[0001] The invention relates to a method for extracting and separating active components from plants, and in particular to the separation of brevifolia hematoxylin from ophiopogon serrata and application thereof. Background Art

[0002] Inflammation is a defensive response of the body to injury or infection, but excessive inflammatory response can lead to a variety of diseases, such as arthritis, cardiovascular disease, neurodegenerative diseases, etc. At present, the commonly used anti-inflammatory drugs in clinical practice mainly include non-steroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids, but long-term use of these drugs can cause serious side effects, such as gastrointestinal damage and immunosuppression. Therefore, the development of new, efficient and low-toxic anti-inflammatory drugs is of great clinical significance.

[0003] The structural formula of brevifoliol is Existing literature shows that brevifolia sutchuenensis exists in many plants, such as pomegranate leaves, Ailanthus altissima, etc. In the invention patent application with publication number CN102659746A, a method for extracting Ailanthus altissima flowers by solvent extraction is disclosed, and the obtained extract is further purified to obtain the active compound brevifolia sutchuenensis. Its biological activity research shows that brevifolia sutchuenensis has certain antioxidant and anti-inflammatory effects.

[0004] Melastoma normale D. Don is a plant of the genus Melastoma in the family Melastomaceae, which is widely distributed in Guangxi, Guangdong, Yunnan, Sichuan, Taiwan and other places. Its dried root is the traditional Chinese medicine "Yang Kaikou". This medicinal material is warm in nature, sweet, sour and astringent in taste, and has the effects of astringency, hemostasis and detoxification. It is commonly used in the folk to treat dysentery, gonorrhea, diphtheria, food stagnation, leukorrhea, carbuncle and ulceration. Existing studies have shown that Yang Kaikou contains compounds such as terpenes, sterols and ellagic acid, which have pharmacological activities such as anti-tumor, anti-inflammatory and antibacterial. However, there is no public report in the prior art that Yang Kaikou contains short-leaf hematoxylin, and there is no related report on the separation of short-leaf hematoxylin from Yang Kaikou. Summary of the invention

[0005] The technical problem to be solved by the invention is to provide a separation and application of breccia sutchuitol from sheep's mouth.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions: The method for separating brevifolia hematoxylin from scutellaria baicalensis comprises the following steps: 1) Using sheep's mouth as raw material to obtain its extract; 2) dispersing the obtained extract in water, extracting with petroleum ether, and collecting the petroleum ether fraction; 3) The obtained petroleum ether fraction was subjected to silica gel column chromatography and gradient eluted with chloroform-methanol to obtain crude brevifolia succinophenol; 4) The obtained crude brevifolia hematoxylin is purified by reverse phase high performance liquid chromatography to obtain high purity brevifolia hematoxylin; wherein the chromatographic conditions are: Chromatographic column: Octadecyl bonded silica gel as filler; Mobile phase: composed of methanol and water in a volume ratio of 5~95:45~55.

[0007] The sheep's mouth described in the present application is the dried root of Melastoma normale D. Don.

[0008] In step 1) of the above separation method, a conventional solvent extraction process can be used to extract the sheep's mouth opening to obtain a sheep's mouth opening extract. Preferably, the sheep's mouth opening is used as a raw material, and water or ethanol is used as a solvent for extraction, and the obtained extract is concentrated under reduced pressure to obtain a sheep's mouth opening extract. The concentration of the ethanol is preferably 50-100 v / v%, more preferably 60-90 v / v%.

[0009] In step 2) of the above separation method, the obtained extract is preferably dispersed in hot water at 30-50°C.

[0010] In step 4) of the above separation method, the chromatographic conditions also include a detection wavelength of 254-265 nm; further include: an injection volume of 100-200 μL, a mobile phase flow rate of 1.0-2.0 mL / min, and a chromatographic column temperature of 25-40°C.

[0011] Preferably, the chromatographic column in step 4) is an Agilent Zorbax Eclipse XDB-C18 chromatographic column.

[0012] More preferably, the chromatographic conditions in step 4) are: the chromatographic column is an Agilent Zorbax Eclipse XDB-C18 chromatographic column, the mobile phase is composed of methanol and water in a volume ratio of 5~95:45~55, the detection wavelength is 254nm, the injection volume is 100μL, the mobile phase flow rate is 2.0mL / min, and the column temperature is 25°C.

[0013] The applicant found in the experiment that brevifolia sutchuenol can significantly inhibit the release of NO induced by LPS and has good anti-inflammatory activity. Therefore, the present invention also includes the use of brevifolia sutchuenol in the preparation of drugs for treating inflammation.

[0014] The inventor team of this application first discovered that sheep open mouth contains brevipein, and first established a method for isolating high-purity brevipein from sheep open mouth. The applicant found through experiments that brevipein can significantly inhibit the release of NO induced by LPS and has a good anti-inflammatory effect. DETAILED DESCRIPTION

[0015] In order to better explain the technical solution of the present invention, the present invention is further described in detail below in conjunction with embodiments, but the embodiments of the present invention are not limited thereto.

[0016] Example 1: Separation of Brassica brevifolia 1. Sample source The root samples of Paeonia pubescens used for extraction (Yangkaikou) were collected from Yanshan Town, Yanshan District, Guilin City, Guangxi.

[0017] 2. Extraction and separation 2.1) 9 kg of naturally air-dried and crushed Paeonia pubescens roots were soaked and extracted with 75 v / v% ethanol at room temperature for 3 times, each time for 7 days. The filtrates were combined and concentrated under reduced pressure to obtain 560 g of extract.

[0018] 2.2) Disperse the extract in 60°C hot water, extract with petroleum ether, and collect the petroleum ether fraction.

[0019] 2.3) The petroleum ether fraction was subjected to silica gel column chromatography and gradient eluted with chloroform-methanol (100:0-0:100) to obtain crude brevifolia succinophenol.

[0020] 2.4) The crude product was purified by reverse phase high performance liquid chromatography (chromatographic conditions: chromatographic column: Agilent ZorbaxEclipse XDB-C18 column, 9.6 mm×250 mm, 5 μm; column temperature: 25°C; mobile phase: CH3OH-H2O (5:95~45:55), 0 min~40 min, retention time: 30.5 min; injection volume: 100 μL, flow rate: 2.0 mL / min; detection wavelength: 254 nm) to obtain high-purity brevifolia hematoxylin (31 mg, purity 98.6%).

[0021] 3. The obtained shortleaf hematoxylin was characterized, and its physicochemical data are as follows: Yellow powder; ESI-MS m / z: 247.0 [MH] - .

[0022] 1 H-NMR (Pyridine-d5, 500 MHz) d : 7.99 (1H, s, H-7), 3.45 (2H, d,J = 3.6Hz, H-9), 2.58 (2H, d, J = 3.6 Hz, H-10).

[0023] 13 C-NMR (Pyridine-d5, 125 MHz) d : 195.5 (C-11), 161.7 (C-8), 150.9 (C-6), 146.2 (C-2), 145.9 (C-4), 142.2 (C-5), 142.1 (C-3), 116.7 (C-3a), 114.5 (C-7a), 108.8 (C-7), 33.5(C-10), 24.4 (C-9).

[0024] Based on the comprehensive analysis of the above mass spectrometry, one-dimensional nuclear magnetic resonance and other spectral data, the compound was deduced to be Brevifoliol.

[0025] Example 2: Separation of Brassica brevifolia Example 1 was repeated, except that in step 2.1), the root of Melastoma pubescens was extracted with water by heating under reflux for 3 times, each time for 6 h.

[0026] Finally, shortleaf hematoxylin (52 mg, purity 99.1%) was obtained.

[0027] Example 3: Separation of Brassica brevifolia Example 1 was repeated, except that in step 2.1), the extraction solvent was 90 v / v% ethanol.

[0028] Finally, shortleaf hematoxylin (46 mg, purity 98.9%) was obtained.

[0029] Experimental example: Study on the anti-inflammatory activity of brevifolia phenol in vitro This experimental example is a study on the in vitro anti-inflammatory activity of brevifolia sutchuenol. The specific steps are as follows: 1. Cytotoxicity Test (1) Compound weighing, dissolving and diluting: DMSO (biotechnology grade) was used as the solvent to prepare a 20 mM stock solution. The compound was diluted to 60, 40, 20, 10, or 5 μM using culture medium as the diluent for later use.

[0030] (2) Cell plating: After the cultured cells are counted, dilute the cells with complete medium and calculate the dilution multiple so that each well (100 μL) contains 3 to 4 × 10 4 , placed in a carbon dioxide incubator for 24 hours.

[0031] (3) Drug administration: a drug-added group, a blank group, and a background group were set up. The drug-added group was added with 5 μL of drug; no cell solution was added to the background group, and 100 μL of culture medium was added to the empty wells; the blank group: nothing was added (three replicate wells were set up for each group). After drug addition, the cells were placed in an incubator and cultured for 24 h.

[0032] (4) Result test: After taking out the incubator, add 10 μL of CCK8 solution to each well of the original 96-well plate, place it in a 37°C incubator and incubate it for 30 min to 60 min. Take it out and test it on an ELISA reader (absorbance: 450 nm). Calculate the cytotoxic activity according to the following formula: Cytotoxicity = (A 样品 -A 背景 ) / (A 空白 -A 背景 )×100% 2. Anti-inflammatory activity of compounds (1) Compound weighing, dissolving and diluting: DMSO (biotechnology grade) was used as the solvent to prepare a 20 mM stock solution. The compound was diluted to 60, 40, 20, 10, or 5 μM using culture medium as the diluent for later use.

[0033] (2) Cell plating: After the cultured cells are counted, dilute the cells with complete medium and add cells at a calculated dilution factor so that each well (100 μL) contains 3 to 4 × 10 4 , placed in a carbon dioxide incubator for 24 hours.

[0034] (3) Drug administration: Lps was diluted with PBS solution to a final concentration of 1 μg / mL. A drug-treated group, a model group, and a blank group were set up. The drug-treated group was added with 5 μL of drug, followed by 5 μL of 1 μg / mL Lps solution; the model group was only added with 5 μL of 1 μg / mL Lps solution; the blank group was not treated (three replicates were set for each group) and was cultured in an incubator for 24 h after drug addition.

[0035] (4) Result test: After taking the 96-well plate out of the incubator, take another 96-well plate, take 50 μL of the experimental solution (50 μL of supernatant needs to be aspirated from the drug-added group, model group, and blank group), add 50 μL of G1 and 50 μL of G2 respectively, and directly test with an enzyme-labeled instrument (absorbance 540 nm).

[0036] (5) Calculation formula Inhibition rate = [1-(A 样品 -A 空白 ) / (A LPS -A 空白 )]×100% 3. Experimental results The survival rate of RAW264.7 cells was 109.7±8.4% after 24 hours of treatment with brevifolia hematoxylin at a concentration of 60 μM. The half inhibitory concentration of drug treatment for inhibiting LPS-induced nitric oxide release in RAW264.7 cells was 55.77±7.28 μM after 24 hours of treatment, and the half inhibitory concentration of positive control dexamethasone for inhibiting LPS-induced nitric oxide release in RAW264.7 cells was 109.8±13.62 μM.

[0037] In summary, the brevifolia hematoxylin isolated from sheep larvae in the present invention has been confirmed by in vitro pharmacological experiments to have the activity of inhibiting the release of nitric oxide from RAW264.7 cells induced by LPS, wherein IC 50 The value was 55.77±7.28 μM (the IC 50 value was 109.8±13.62 μM), providing important candidate compounds for the development of new anti-inflammatory drugs.

Claims

1. A method for separating brevifolia hematoxylin from ophiopogon serrata, comprising the following steps: 1) Using sheep's mouth as raw material to obtain its extract; 2) dispersing the obtained extract in water, extracting with petroleum ether, and collecting the petroleum ether fraction; 3) The obtained petroleum ether fraction was subjected to silica gel column chromatography and gradient eluted with chloroform-methanol to obtain crude brevifolia succinophenol; 4) The obtained crude brevifolia hematoxylin is purified by reverse phase high performance liquid chromatography to obtain high purity brevifolia hematoxylin; wherein the chromatographic conditions are: Chromatographic column: Octadecyl bonded silica gel as filler; Mobile phase: composed of methanol and water in a volume ratio of 5~95:45~55.

2. The separation method according to claim 1, characterized in that: In step 4), the chromatographic conditions also include: a detection wavelength of 254-265 nm.

3. The method according to claim 2, characterized in that: In step 4), the chromatographic conditions also include: an injection volume of 100-200 μL, a mobile phase flow rate of 1.0-2.0 mL / min, and a chromatographic column temperature of 25-40°C.

4. The separation method according to any one of claims 1 to 3, characterized in that: In step 4), the chromatographic column is an Agilent Zorbax Eclipse XDB-C18 chromatographic column.

5. The separation method according to claim 4, characterized in that: In step 4), the chromatographic conditions are as follows: the chromatographic column is an Agilent Zorbax Eclipse XDB-C18 chromatographic column, the mobile phase is composed of methanol and water in a volume ratio of 5~95:45~55, the detection wavelength is 254nm, the injection volume is 100μL, the mobile phase flow rate is 2.0mL / min, and the column temperature is 25°C.

6. The separation method according to any one of claims 1 to 3, characterized in that: In step 1), sheep scallops are used as raw materials, and water or ethanol is used as solvent for extraction, and the obtained extract is concentrated under reduced pressure to obtain sheep scallops extract.

7. The separation method according to claim 6, characterized in that: The concentration of the ethanol is 50-100 v / v%.

8. Application of shortleaf hematoxylin in the preparation of drugs for treating inflammation.