Isolation of short acylphloroglucinols from the bark of acacia nilotica and their applications

Abstract

This invention discloses a method for isolating and applying bryophyllol from sheep carcasses. The method involves using sheep carcasses as raw material, obtaining its extract, dispersing it in water, first extracting with petroleum ether, then loading the petroleum ether fraction onto a silica gel column and eluting with a chloroform-methanol gradient. The resulting crude bryophyllol is then purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic conditions are as follows: column: octadecyl-bonded silica gel; mobile phase: methanol and water in a volume ratio of 5-95:45-55. Pharmacological studies have confirmed that bryophyllol possesses good anti-inflammatory activity, with an IC50 value that inhibits LPS-induced nitric oxide release from RAW264.7 cells. 50 The value was 55.77±7.28 μM, providing an important candidate compound for the development of novel anti-inflammatory drugs.

Description

Technical Field

[0001] This invention relates to a method for extracting and separating active ingredients from plants, specifically to the separation and application of shortleaf hematoxylin from sheep scabra. Background Technology

[0002] Inflammation is the body's defensive response to injury or infection, but excessive inflammation can lead to various diseases, such as arthritis, cardiovascular disease, and neurodegenerative diseases. Currently, commonly used anti-inflammatory drugs in clinical practice mainly include nonsteroidal 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, developing novel, highly effective, and low-toxicity anti-inflammatory drugs is of significant clinical importance.

[0003] The structural formula of shortleaf hematoxylin is: Existing literature indicates that bryophyllol exists in various plants, such as pomegranate leaves and ailanthus. Patent application CN102659746A discloses a method for extracting ailanthus flowers using solvent extraction, followed by further purification of the extract to obtain the active compound bryophyllol. Bioactivity studies show that bryophyllol possesses certain antioxidant and anti-inflammatory effects.

[0004] Melastoma normale D. Don, a plant belonging to the genus Melastoma of the family Melastomaceae, is widely distributed in Guangxi, Guangdong, Yunnan, Sichuan, Taiwan, and other regions. Its dried root is the traditional Chinese medicine "Yang Kai Kou". This medicinal material is warm in nature, with a sweet, sour, and astringent taste. It has astringent, hemostatic, and detoxifying effects and is commonly used in folk medicine to treat dysentery, urinary tract infections, diphtheria, indigestion, leukorrhea, carbuncles, and ulcers. Previous studies have shown that Yang Kai Kou contains terpenoids, sterols, and ellagic acid compounds, which possess pharmacological activities such as antitumor, anti-inflammatory, and antibacterial properties. However, there are currently no publicly available reports of Yang Kai Kou containing brevifolin, nor are there any reports of brevifolin being isolated from Yang Kai Kou. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for the separation and application of short-leaved hematoxylin in sheep sacs.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] A method for isolating shortleaf hematoxylin from sheep sacs includes the following steps:

[0008] 1) Using sheep's first molt as raw material, obtain its extract;

[0009] 2) The obtained extract was dispersed in water, extracted with petroleum ether, and the petroleum ether fraction was collected;

[0010] 3) The obtained petroleum ether fraction was subjected to silica gel column chromatography and eluted with a gradient of chloroform-methanol to obtain crude hematoxylin and brevicornuate;

[0011] 4) The obtained crude bryophyllum was purified by reversed-phase high-performance liquid chromatography to obtain high-purity bryophyllum; the chromatographic conditions were as follows:

[0012] Column: packed with octadecyl bonded silica gel;

[0013] Mobile phase: Composed of methanol and water in a volume ratio of 5~95:45~55.

[0014] The sheep-head described in this application is the dried root of Melastoma normale D. Don.

[0015] In step 1) of the above separation method, a traditional solvent extraction process can be used to extract the sheep's beak to obtain an extract. Preferably, the sheep's beak is used as raw material, and water or ethanol is used as the solvent for extraction. The resulting extract is concentrated under reduced pressure to obtain the sheep's beak extract. The concentration of the ethanol is preferably 50~100 v / v%, more preferably 60~90 v / v%.

[0016] In step 2) of the above separation method, it is preferable to disperse the obtained extract in hot water at 30~50°C.

[0017] In step 4) of the above separation method, the chromatographic conditions also include a detection wavelength of 254~265nm; further, the injection volume is 100~200μL, the mobile phase flow rate is 1.0~2.0mL / min, and the column temperature is 25~40℃.

[0018] Preferably, the chromatographic column mentioned in step 4) is an Agilent Zorbax Eclipse XDB-C18 column.

[0019] More preferably, the chromatographic conditions in step 4) are as follows: the column is an Agilent Zorbax Eclipse XDB-C18 column, the mobile phase consists 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℃.

[0020] The applicant discovered in experiments that bryophyllum can significantly inhibit LPS-induced NO release and has good anti-inflammatory activity. Therefore, this invention also includes the use of bryophyllum in the preparation of medicaments for treating inflammation.

[0021] The inventors of this application have for the first time discovered that bryophyllol is present in sheep orifices, and have established for the first time a method for isolating high-purity bryophyllol from sheep orifices. Through experiments, the applicants found that bryophyllol has a good anti-inflammatory effect, significantly inhibiting LPS-induced NO release. Detailed Implementation

[0022] To better explain the technical solution of the present invention, the present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto.

[0023] Example 1: Isolation of shortleaf hematoxylin

[0024] 1. Sample Source

[0025] The root (sheep-opening) sample of Melastoma pubescens used for extraction was collected from Yanshan Town, Yanshan District, Guilin City, Guangxi.

[0026] 2. Extraction and separation

[0027] 2.1) 9 kg of naturally air-dried and pulverized Melastoma pubescens root was soaked and extracted three times with 75 v / v% ethanol at room temperature for 7 days each time. The filtrates were combined and concentrated under reduced pressure to obtain 560 g of extract.

[0028] 2.2) Disperse the extract in hot water at 60°C, extract with petroleum ether, and collect the petroleum ether fraction.

[0029] 2.3) The obtained petroleum ether fraction was subjected to silica gel column chromatography and eluted with a chloroform-methanol gradient (100:0~0:100) to obtain crude hematoxylin and brevicornuate.

[0030] 2.4) The crude product was purified by reversed-phase high-performance liquid chromatography (chromatographic conditions: Agilent Zorbax Eclipse XDB-C18 column, 9.6 mm × 250 mm, 5 μm; column temperature: 25℃; 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 brevicornuol (31 mg, purity 98.6%).

[0031] 3. The obtained shortleaf hematoxylin was characterized, and its physicochemical data are as follows:

[0032] Yellow powder; ESI-MS m / z: 247.0 [MH] - .

[0033] 1H-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).

[0034] 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).

[0035] Based on the comprehensive analysis of the above mass spectrometry, one-dimensional nuclear magnetic resonance and other spectroscopic data, the compound was deduced to be breviforin.

[0036] Example 2: Isolation of shortleaf hematoxylin

[0037] Repeat Example 1, except that in step 2.1), the roots of Melastoma pubescens were extracted three times by heating and refluxing with water, each time for 6 hours.

[0038] Finally, bryophyllum hematoxylin was obtained (52 mg, purity 99.1%).

[0039] Example 3: Isolation of shortleaf hematoxylin

[0040] Repeat Example 1, except that in step 2.1), the extraction solvent is 90 v / v% ethanol.

[0041] Finally, bryophyllum hematoxylin was obtained (46 mg, purity 98.9%).

[0042] Experimental Example: In vitro anti-inflammatory activity study of bryophyllum hematoxylin

[0043] This experimental example studies the in vitro anti-inflammatory activity of bryophyllum, and the specific steps are as follows:

[0044] 1. Cytotoxicity test

[0045] (1) Weighing, dissolving and diluting the compound: Prepare a 20 mM stock solution using DMSO (biotechnology grade) as the solvent, and dilute the compound to 60, 40, 20, 10 and 5 μM using culture medium as the diluent.

[0046] (2) Cell plating: After counting the cultured cells, dilute the cells with complete culture medium and calculate the dilution factor so that each well (100 μL) contains 3~4 × 10⁶ cells. 4 Place it in a carbon dioxide incubator and incubate for 24 hours.

[0047] (3) Drug administration: Set up a drug-added group, a blank group, and a background group. The drug-added group was given 5 μL of drug; the background group was given no cell slurry and 100 μL of culture medium was added to the empty well; the blank group was given nothing (three replicates were set up for each group). After drug administration, the cells were placed in an incubator and incubated for 24 h.

[0048] (4) Result testing: After removing the incubator, add 10 μL of CCK8 solution to each well of the original 96-well plate, incubate at 37℃ for 30-60 min, and then test using a microplate reader (absorbance: 450 nm). Calculate the cytotoxic activity using the following formula:

[0049] Cytotoxicity rate = (A 样品 -A 背景 ) / (A 空白 -A 背景 )×100%

[0050] 2. Anti-inflammatory activity of the compound

[0051] (1) Weighing, dissolving and diluting the compound: Prepare a 20 mM stock solution using DMSO (biotechnology grade) as the solvent, and dilute the compound to 60, 40, 20, 10 and 5 μM using culture medium as the diluent.

[0052] (2) Cell plating: After counting the cultured cells, dilute the cells with complete culture medium, calculate the dilution factor, and add cells to each well (100 μL) to achieve a concentration of 3~4 × 10⁶ cells / well. 4 Place it in a carbon dioxide incubator and incubate for 24 hours.

[0053] (3) Drug administration: Lps were diluted with PBS solution to a final concentration of 1 μg / mL. Three groups were set up: drug group, model group and blank group. 5 μL of drug was added to the drug group, followed by 5 μL of 1 μg / mL Lps solution. 5 μL of 1 μg / mL Lps solution was added to the model group. No treatment was given to the blank group (three replicates were set up for each group). After drug administration, the wells were incubated in an incubator for 24 h.

[0054] (4) Results test: After removing the 96-well plate from the incubator, take another 96-well plate, take 50 μL of experimental solution (50 μL of supernatant needs to be aspirated from the drug group, model group and blank group), add 50 μL of G1 and 50 μL of G2 respectively, and test directly with an ELISA reader (absorbance 540 nm).

[0055] (5) Calculation formula

[0056] Inhibition rate = [1-(A)] 样品 -A 空白 ) / (A LPS -A 空白 )]×100%

[0057] 3. Experimental Results

[0058] After 24 hours of treatment with 60 μM hematoxylin and eosin (HPO), the survival rate of RAW264.7 cells was 109.7 ± 8.4%. The half-maximal inhibitory concentration (IC50) of the drug in inhibiting LPS-induced nitric oxide release from RAW264.7 cells after 24 hours of treatment was 55.77 ± 7.28 μM, while the positive control dexamethasone had an IC50 of 109.8 ± 13.62 μM.

[0059] In summary, the bryophyllol isolated from sheep orifices by this invention has been confirmed by in vitro pharmacological experiments to have inhibitory activity against LPS-induced nitric oxide release from RAW264.7 cells, with an IC50 concentration of [missing information]. 50 The value was 55.77 ± 7.28 μM (IC50 of the positive control dexamethasone). 50 The value was 109.8 ± 13.62 μM, providing an important candidate compound for the development of novel anti-inflammatory drugs.

Claims

1. A method for isolating shortleaf hematoxylin from sheep molts, comprising the following steps: 1) Using sheep's first molt as raw material, obtain its extract; 2) The obtained extract was dispersed in water, extracted with petroleum ether, and the petroleum ether fraction was collected; 3) The obtained petroleum ether fraction was subjected to silica gel column chromatography and eluted with a gradient of chloroform-methanol to obtain crude hematoxylin and brevicornuate; 4) The obtained crude bryophyllum was purified by reversed-phase high-performance liquid chromatography to obtain high-purity bryophyllum; the chromatographic conditions were as follows: Column: packed with octadecyl bonded silica gel; 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: the detection wavelength is 254~265nm.

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

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 column.

5. The separation method according to claim 4, characterized in that, In step 4), the chromatographic conditions are as follows: the column is an Agilent Zorbax Eclipse XDB-C18 column, the mobile phase consists 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℃.

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

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