Preparation method and application of ethanol extract of fingered citron with anti-inflammatory activity

Abstract

The present invention discloses a preparation method of a bergamot ethanol extract with anti-inflammatory activity, which comprises the following operation steps: (1) Select bergamot, wash and cut into pieces; (2) Dry at 35-45 °C until constant weight, pulverize and then pass through a sieve; (3) Mix with ethanol and place in a shaker for extraction; (4) Filter, collect the filtrate, and then concentrate at 40-50 °C until the ethanol is completely removed. The present invention provides a preparation method of a bergamot ethanol extract with anti-inflammatory activity, which can fully protect the effective anti-inflammatory components in the bergamot ethanol extract, has low raw material cost, simple operation and does not affect the activity of the active ingredients, has fewer process steps and is convenient for processing and production; further, the bergamot ethanol extract prepared by the method of the present invention has a relatively significant anti-inflammatory effect, can effectively relieve colitis, can be used as a potential anti-inflammatory drug, and uses non-targeted metabolism and correlation analysis to screen out potential compounds with anti-inflammatory activity in the ethanol extract, laying a foundation for its further development and utilization.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-inflammatory compound extraction, and particularly relates to a preparation method and application of an ethanol extract of bergamot with anti-inflammatory activity. Background Art

[0002] Bergamot (Citrus medica 'Fingered'), also known as bergamot orange or bergamot fruit, belongs to the genus Citrus of the Rutaceae family. Its fruit has a golden color, a strong fragrance, and a peculiar shape similar to a hand. It is mainly produced in Fujian, Guangdong, Sichuan, Jiangsu and Zhejiang provinces. Bergamot was first recorded in "Compendium of Materia Medica" and was included in "Chinese Pharmacopoeia" in 1963. Bergamot is a fruit that can be used as both medicine and food. Its nature and flavor are pungent, bitter, sour, and warm. It contains various traditional Chinese medicine components such as flavonoids, coumarins, limettin, and active polysaccharides. It has the effects of soothing the liver and regulating qi, strengthening the stomach and stopping vomiting, and resolving phlegm and relieving cough. It can prevent and treat physical discomforts such as cough, chest tightness, and loss of appetite. The ethanol extract of bergamot contains various active components such as flavonoids and coumarins, and has various biological activities such as anti-inflammatory, antioxidant, and immune regulation, and has no toxic and side effects. It has important research value in the clinical application of traditional Chinese medicine.

[0003] Inflammatory bowel disease (IBD) is a non-specific inflammatory disease that occurs in the gastrointestinal tract, including ulcerative colitis and Crohn's disease. The incidence of inflammatory bowel disease worldwide has remained high all year round, and the affected population is gradually spreading to young people. Among them, ulcerative colitis has a long course and is prone to recurrence, and is an important inducement for rectal cancer. At present, the commonly used treatment drugs mainly include aminosalicylates, glucocorticoids, antibiotics, and immunomodulators, etc. However, most of the above drugs will produce dependence, and the treatment effect is unstable, and there is even a possibility of inducing the further deterioration of the condition. Therefore, exploring a natural drug that is safe, effective, has small toxic and side effects, and is not prone to drug resistance has become an urgent problem to be solved in the field of drug research for inflammatory bowel disease. Summary of the Invention

[0004] To solve the above technical problems and further develop the potential medicinal value of the medicine and food homologous plant, namely bergamot, the present invention provides a preparation method and application of an anti-inflammatory compound of bergamot. Using bergamot as the raw material, modern food separation technology is adopted, and 95% ethanol is used to extract and separate the alcohol-soluble components from bergamot. The ethanol extract of bergamot obtained by the method of the present invention has the effect of anti-inflammatory activity and its role in preventing colitis.

[0005] To achieve the above object, the technical solution provided by the present invention is as follows:

[0006] A preparation method of an ethanol extract of bergamot with anti-inflammatory activity, comprising the following operation steps:

[0007] (1) Select fresh fingered citron without damage, mildew or deterioration, wash and cut into pieces;

[0008] (2) Put the cut fingered citron in step (1) into an oven and dry it to constant weight at 35 - 45 °C, crush it and sieve it through a 100 - mesh sieve for standby;

[0009] (3) Mix the sieved and standby fingered citron powder in step (2) with ethanol at a solid - liquid ratio of 1:10 - 1:20 g / mL, place it in a shaker and extract at 150 r / min and 25 °C for 12 - 18 h;

[0010] (4) Filter the substance obtained after extraction in step (3), collect the filtrate, and then vacuum - concentrate it at 40 - 50 °C until the ethanol is completely removed.

[0011] Preferably, the fingered citron described in step (1) is Guangdong fingered citron, namely Guangfo fingered citron.

[0012] Preferably, the ethanol described in step (3) is an ethanol solution with a volume concentration of 95%.

[0013] Preferably, the extraction described in step (3) is to place it in a shaker and extract at 150 r / min and 25 °C for 12 - 18 h.

[0014] An anti - inflammatory drug, comprising the ethanol extract of fingered citron with anti - inflammatory activity as described above.

[0015] Application of the ethanol extract of fingered citron with anti - inflammatory activity prepared by the above method in the preparation of anti - inflammatory drugs.

[0016] Application of the ethanol extract of fingered citron with anti - inflammatory activity prepared by the above method in the preparation of drugs for treating colitis.

[0017] Compared with the prior art, the beneficial effects of the present invention:

[0018] The present invention provides a preparation method of an ethanol extract of fingered citron with anti - inflammatory activity, which can fully protect the effective anti - inflammatory components in the ethanol extract of fingered citron. Its raw material cost is low, the operation is simple and does not affect the activity of the active ingredients. The process steps are few, which is convenient for processing and production; furthermore, the ethanol extract of fingered citron prepared by the method of the present invention has a relatively significant anti - inflammatory effect, can effectively relieve colitis, can be used as a potential anti - inflammatory drug, and then uses HPLC - MS / MS technology for non - targeted metabolic analysis to screen out potential compounds with anti - inflammatory activity. Brief Description of the Drawings

[0019] Figure 1 is the inhibition rate of the ethanol extracts of Jin fingered citron, Guangfo fingered citron and Chuan fingered citron on COX - 2.

[0020] Figure 2 It is the animal experiment process.

[0021] Figure 3 is the weight change of mice during DSS induction.

[0022] Figure 4 is the DAI score of mice during DSS induction.

[0023] Figure 5 The length and macroscopic appearance of the mouse colon.

[0024] Figure 6 Representative H&E and AB-PAS staining of colon tissue.

[0025] Figure 7 is the level of inflammatory cytokines in colon tissue. DETAILED DESCRIPTION

[0026] The following is a detailed description with reference to the accompanying drawings and specific implementations, but it should be understood that the protection scope of the present invention is not limited by the specific implementations.

[0027] The DMSO solution (as solvent) and COX-2 inhibitor screening kit in the examples were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd. The raw materials and reagents used in the other examples were commercially available unless otherwise specified.

[0028] Example 1

[0029] A method for preparing a bergamot alcohol extract having anti-inflammatory activity, the operating steps are as follows:

[0030] (1) Select fresh Guangdong Buddha's Hand (Guangdong Buddha's Hand) that is not damaged, moldy or spoiled, wash and cut into pieces;

[0031] (2) placing the citron cut in step (1) in an oven at 40° C. and drying to constant weight, crushing and passing through a 100-mesh sieve for later use;

[0032] (3) mixing the sifted bergamot powder in step (2) with a 95% ethanol solution at a solid-liquid ratio of 1:15 g / mL, and placing in a shaker at 150 r / min and 25° C. for 12 h;

[0033] (4) filtering the substance obtained after extraction in step (3), collecting the filtrate, and then concentrating it under vacuum at 45° C. until the ethanol is completely removed, thereby obtaining the ethanol extract of Foeniculum villosum with anti-inflammatory activity.

[0034] Example 2

[0035] A method for preparing a bergamot alcohol extract having anti-inflammatory activity, the operating steps are as follows:

[0036] (1) Select fresh Guangdong fingered citron (Guangfo fingered citron) without damage and mildew, wash and cut into pieces;

[0037] (2) Put the cut fingered citron in step (1) into an oven and dry it at 45 °C until constant weight, then crush it and pass through a 100-mesh sieve for standby;

[0038] (3) Mix the sieved and standby fingered citron powder in step (2) with an ethanol solution with a volume concentration of 95% according to a solid-liquid ratio of 1:10 g / mL, place it in a shaker and extract at 150 r / min and 25 °C for 13 h;

[0039] (4) Filter the substance obtained after extraction in step (3), collect the filtrate, and then vacuum concentrate it at 40 °C until the ethanol is completely removed, thus obtaining the ethanol extract of fingered citron with anti-inflammatory activity.

[0040] Example 3

[0041] Replace the Guangdong fingered citron in step (1) with Jinhua fingered citron (Jinfuo fingered citron) and Sichuan fingered citron (Chuanfo fingered citron) respectively, and the remaining operations are the same as in Example 1, obtaining the ethanol extract of Jinhua fingered citron and the ethanol extract of Sichuan fingered citron respectively.

[0042] Example 4

[0043] Determination of the in vitro anti-inflammatory activities of the ethanol extract of fingered citron with anti-inflammatory activity prepared in Example 1 and the ethanol extracts prepared in Example 3

[0044] COX-2 enzyme anti-inflammatory test:

[0045] 4.1. Test materials:

[0046] The ethanol extract of fingered citron with anti-inflammatory activity prepared in Example 1, the ethanol extract of Jinhua fingered citron and the ethanol extract of Sichuan fingered citron prepared in Example 3; DMSO solution (as a solvent) was purchased from Sinopharm Chemical Reagent Co., Ltd.; COX-2 inhibitor screening kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.;

[0047] 4.2. Kit preparation:

[0048] 1) Dissolve other reagents in the kit except rhCOX-2 to room temperature, centrifuge to precipitate the solution to the bottom of the tube and then mix well for standby; COX-2 Probe, COX-2 Cofactor (50×), COX-2 Substrate (50×) are prepared in DMSO, water bath at 37 °C for 0.5 - 2 min to room temperature, and store in the dark for standby (store in the dark at -20 °C for long term);

[0049] 2) Cofactor working solution: According to the requirement of 5 μl of Cofactor working solution for each sample, take an appropriate amount of Cofactor (50×) and dilute it 50 times with assay buffer at a ratio of 1:49, and store it at 4°C for use on the same day;

[0050] 3) COX-2 working solution: According to the requirement of 5 μl of COX-2 working solution for each sample, take an appropriate amount of rhCOX-2 (25×) and dilute it 25 times with assay buffer at a ratio of 1:24, and store it in an ice bath for use within 1 h;

[0051] 4) Substrate working solution: According to the requirement of 5 μl of Substrate working solution for each sample, take an appropriate amount of Substrate (50×), add an equal volume of Substrate buffer and dilute it, vortex and mix well, then dilute the mixture 125 times with ultrapure water, and store it in an ice bath for use within 1 h;

[0052] 5) Preparation of positive drug solution: Take an appropriate amount of COX-2 positive inhibitor Celecoxib provided in the kit, dissolve it with DMSO as the solvent to prepare a solution with a concentration of 0.3429 μg / ml, and the final concentration in the reaction system is 0.019 μg / ml (50 nM);

[0053] 6) Preparation of sample solution: Take appropriate amounts of the ethanol extracts of Fructus Citri Sarcodactylis with anti-inflammatory activity prepared in Example 1, the ethanol extracts of Fructus Citri Sarcodactylis 'Jin' prepared in Example 3, and the ethanol extract of Fructus Citri Sarcodactylis 'Chuan' respectively, dissolve them with an appropriate amount of DMSO to obtain an ethanol extract working solution with a concentration of 200 μg / ml.

[0054] 4.3. Test method:

[0055] 1) Use a 96-well enzyme-labeled plate. According to the arrangement of each well, add assay buffer (COX-2 reagent buffer solution), Cofactor working solution (COX-2 cofactor working solution) (vibrate) (5 μl for each well), COX-2 working solution (vibrate) (5 μl), sample solution (5 μl) (add 5 μl of sample solvent to the blank group), mix well with a multi-channel pipette, and incubate at 25°C for 5 min; then place it on ice (4°C refrigerator) and incubate for 10 min to cool down; add 5 μl of Probe working solution to each well, mix well with a multi-channel pipette; add 5 μl of low-temperature Substrate working solution to each well, and operate as much as possible in a low-temperature environment, mix well with a multi-channel pipette;

[0056] 2) Immediately measure the fluorescence at 37°C after adding the working solution, with an excitation wavelength of 560 nm and an emission wavelength of 590 nm (measure once every 30 s for 15 consecutive min);

[0057] 3) The “dose-response-inhibition” model of GraphPad Prism was used and the following formula was used to calculate the inhibition rate of the drug on COX-2 enzyme activity, draw the dose-effect curve, and predict the IC50;

[0058] 4) COX-2 inhibition rate = (RFU initial enzyme activity - RFU sample) / (RFU initial enzyme activity - RFU blank control) × 100%

[0059] 4.4、Test results Figure 1 As shown in the figure: the higher the inhibition rate of the sample on COX-2, the better its anti-inflammatory effect. Figure 1 As shown, at 200ug / mL, among the three kinds of bergamot, the inhibition rate of Guang Fo Shou on COX-2 enzyme is the highest, with an inhibition rate of 37.522%, followed by Jin Fo Shou, with an inhibition rate of 29.699% on COX-2, and Sichuan Fo Shou has the lowest inhibition rate on COX-2, with an inhibition rate of 17.233%. The results show that among the three kinds of bergamot, Guang Fo Shou has the best anti-inflammatory effect, followed by Jin Fo Shou, and Sichuan Fo Shou has the worst anti-inflammatory effect. The present invention finds that Guang Fo Shou ethanol extract has good anti-inflammatory effect through in vitro COX-2 enzyme inhibitor screening test, and can be used as a potential anti-inflammatory drug.

[0060] Example 5

[0061] Determination of anti-inflammatory activity of bergamot alcohol extract in vivo

[0062] The present invention uses dextran sulfate sodium (DSS) to induce a colitis mouse model, and then selects Bergamot ethanol extract (BEE) for in vivo experiments based on the above in vitro anti-inflammatory test results. It is found that the Bergamot ethanol extract can effectively alleviate colitis and can be used as a potential drug for preventing or treating colitis.

[0063] Colitis Animal Test

[0064] 5.1 Experimental Materials:

[0065] 5.1.1 Experimental Animals:

[0066] SPF male C57BL6 / J mice, 7 weeks old, weighing 18-22 g, were purchased from Spav (Beijing) Biotechnology Co., Ltd. and kept under standard conditions; all mice were acclimated for 7 days before the experiment.

[0067] 5.1.2 Other reagents:

[0068] Dextran sulfate sodium (DSS, molecular weight 36 - 50 kDa) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); Salicylazosulfapyridine (SASP) was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China), and the ethanol extract of Fructus Citri Sarcodactylis was the ethanol extract of Fructus Citri Sarcodactylis with anti-inflammatory activity prepared in Example 1.

[0069] 5.2 Experimental design:

[0070] Forty mice were randomly divided into 5 groups, namely: control group, DSS group, DSS + Salicylazosulfapyridine group (SASP), DSS + BEE low-dose group (BEE-L), and DSS + BEE high-dose group (BEE-H). From day 8 to day 28, the control group and DSS group were only gavaged with normal saline, and the SASP group, BEE-L group, and BEE-H group were respectively gavaged with 100 mg / kg body weight of salicylazosulfapyridine, 50 mg / kg BW BEE, and 100 mg / kg BW BEE. In the fourth week, only the mice in the control group drank distilled water, and the remaining mice drank distilled water containing 3% DSS to establish a colitis model for 7 days ( Figure 2 ).

[0071] 5.3 Detection indicators

[0072] 1) Disease Activity Index (DAI): During the DSS induction period, the fecal morphology (normal, 0 points; soft feces, 2 points; diarrhea, 4 points), blood in the stool condition (normal, 0 points; slight blood in the stool, 2 points; severe blood in the stool, 4 points), and percentage of weight loss (0, 0 points; 1% - 5%, 1 point; 5% - 10%, 2 points; 10% - 20%, 3 points; > 20%, 4 points) of the mice in each group were recorded daily and scored. The DAI score was the average of the scores of these three indicators;

[0073] 2) Colon length: After dissecting the mice, a ruler was used to measure the colon length of the mice;

[0074] 3) H&E and AB-PAS staining: The colon tissues were fixed in paraformaldehyde and Carnoy's fixative, dehydrated, and embedded in paraffin. Then, 4-μm thick sections were cut according to the standard method, stained with H&E and AB-PAS, and the inflammatory infiltration and the number of goblet cells in the colon were observed under an optical microscope;

[0075] 4) Determination of inflammatory factors TNF-α and IL-10: The colon tissues (10 mg) were homogenized with pre-cooled PBS at a ratio of 1:18, centrifuged to collect the supernatant, and the levels of TNF-α and IL-10 were detected according to the instructions of the enzyme-linked immunosorbent assay kit;

[0076] 5) Statistical analysis: GraphPad Prism was used to perform one-way analysis of variance (ANOVA) on the data and draw graphs. The mean and standard deviation of all reported data were from at least three independent biological replicates, and a P value < 0.05 was considered statistically significant compared to the DSS group.

[0077] 5.4. Experimental results:

[0078] As Figures 3 - 5 shown, the DSS-induced colitis model mice showed obvious diarrhea, bloody stools, and weight loss, with a significant increase in the DAI score and a significant shortening of the colon length. In addition, H&E and AB-PAS staining showed that obvious inflammatory lesions occurred in the colon tissues of the model group, including obvious damage to the epithelial surface, reduction of goblet cells, crypt deformation, extensive infiltration of inflammatory cells, etc. ( Figure 6 ), and at the same time, the level of the pro-inflammatory factor TNF-α was significantly increased, and the level of the anti-inflammatory factor IL-10 was significantly decreased ( Figure 7 ). On the contrary, the ethanol extract of Citrus medica var. sarcodactylis treatment significantly alleviated DSS-induced colon inflammation. Compared with the model group, the DAI score of the experimental group was significantly decreased, the shortening of the colon length was significantly alleviated, and the damage of colon tissues was significantly reduced and the levels of inflammatory factors were restored, showing a dose-dependent manner.

[0079] 5.5. Component analysis of the ethanol extract of Citrus medica var. sarcodactylis

[0080] The present invention uses HPLC-MS / MS technology to perform non-targeted metabolite analysis on the ethanol extracts of Guangfo Fingers, Jinfo Fingers, and Chuanfo Fingers. Eventually, 2007 and 1920 small molecule compounds are detected in the ethanol extracts of the three kinds of Fingers under positive and negative ion modes respectively: Differential metabolites with significantly different peak areas between Guangfo Fingers and Jinfo Fingers, Chuanfo Fingers are selected in the positive and negative spectra, and substances that may affect the anti-inflammatory activity of Fingers are preliminarily screened. Eventually, 22 and 32 target compounds are screened under positive and negative ion modes respectively, including 9 organic acids, 6 glycosides, 5 esters, 6 ketones, 2 phenols, 2 ethers, 2 amines, 3 alcohols, 1 quinone, 4 heterocyclics, and 14 other compounds including short peptides, nitriles, and hydrocarbons. Based on the 54 compounds screened, according to the ratio of the peak areas of Guangfo Fingers to Jinfo Fingers and Chuanfo Fingers, 10 compounds with the most significant peak area differences are selected as potential anti-inflammatory active ingredients under positive and negative ion modes respectively. The component contents of resveratrol-4'-methyl ether, aspartic acid-glutamic acid-histidine-proline, cysteine-arginine-threonine-tryptophan, (6RS)-6,19-cyclodioxo-24,24-difluoro-25-hydroxy-6,19-dihydroxyvitamin D3 / (6RS)-6,19-cyclodioxo-24,24-difluoro-25-hydroxy-6,19-dihydroergocalciferol, genipin 1-β-gentiobioside, 4-oxymethyl gallate, cinnamyl alcohol D2 glucoside, tocopherol, butylated hydroxyanisole and other substances in the ethanol extract of Fingers are significantly correlated with the COX-2 inhibition rate (p<0.05), and the component content of tritriacontanoic acid is extremely significantly correlated with the COX-2 inhibition rate (p<0.01), indicating that the above 10 components have a certain impact on the anti-inflammatory activity and may be potential anti-inflammatory active substances

[0081] The foregoing description of the specific exemplary embodiments of the present invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many changes and variations are possible in light of the above teaching. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the invention and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the invention, as well as various different selections and changes. The scope of the present invention is intended to be defined by the claims and their equivalents.

Claims

1. Use of an ethanol extract of fingered citron with anti-inflammatory activity in the preparation of a medicament for treating colitis, characterized in that, Preparation method of bergamot ethanol extract with anti-inflammatory activity, comprising the following operation steps: (1) Select bergamot without damage and mildew, wash and cut into pieces; the bergamot is Guangdong bergamot; (2) Dry the bergamot cut in step (1) at 35-45 °C until constant weight, crush and then pass through a sieve; (3) Mix the sieved bergamot powder in step (2) with ethanol at a solid-liquid ratio of 1:10-1:20 g / mL and extract; the ethanol is an ethanol solution with a volume concentration of 95%; (4) Filter the substance obtained after extraction in step (3), collect the filtrate, and then concentrate it at 40-50 °C until the ethanol is completely removed.

2. Use of the ethanol extract of Citrus medica L. var. sarcodactylis (Noot.) Swingle with anti-inflammatory activity according to claim 1 in the preparation of a medicament for treating colitis, characterized in that: The extraction described in step (3) is to place it in a shaker at 150 r / min and 25 °C for extraction for 12-18 h.

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