CN117959398A

CN117959398A - Application of intelligence-promoting volatile oil extracted by supercritical carbon dioxide method in preparation of medicine for improving inflammatory bowel disease

Info

Publication number: CN117959398A
Application number: CN202410143947.1A
Authority: CN
Inventors: 赵辉; 杨心如; 陶永清; 杨帆
Original assignee: Tianjin University of Commerce
Current assignee: Tianjin University of Commerce
Priority date: 2024-01-31
Filing date: 2024-01-31
Publication date: 2024-05-03

Abstract

The invention provides application of intelligence-promoting volatile oil extracted by a supercritical carbon dioxide method in preparation of medicines for improving inflammatory bowel disease, and belongs to the technical field of medicines. The invention provides that the nootropic essential oil extracted by the supercritical carbon dioxide method has good biological activity, and the nootropic essential oil can play a good role in protecting the integrity of intestinal epithelial barrier at the cellular level, and can improve the physical condition of enteritis mice, protect the integrity of intestinal epithelial barrier of mice and reduce the intestinal leakage phenomenon caused by DSS. The invention provides a good theoretical basis for subsequent development of intelligence-promoting products and new drug development, and provides potential research references for future development of diets and health-care products.

Description

Application of intelligence-promoting volatile oil extracted by supercritical carbon dioxide method in preparation of medicine for improving inflammatory bowel disease

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of intelligence-promoting volatile oil extracted by a supercritical carbon dioxide method in preparation of medicines for improving inflammatory bowel diseases.

Background

Intelligence development (Alpinia oxyphylla Miquel) is an important plant with homology of medicine and food, not only can be used as edible fruit, but also is an important traditional medicine beneficial to cognition and relieving various diseases, and has the effects of warming kidney, controlling nocturnal emission, stopping excessive, warming spleen, stopping diarrhea, stopping saliva and the like in medical books. The modern pharmacological research on intelligence development mainly aims at improving the nervous system, and the intelligence development and the effective components thereof show the neuroprotection through various mechanisms, so that the intelligence development has great potential as a medicament for treating the nervous diseases. Research on active ingredients in the alpinia oxyphylla shows that the alpinia oxyphylla is rich in sesquiterpene, flavonoid substances and the like, and the ingredients have antioxidant activity; the alpinia oxyphylla and petroleum ether extract, acetoacetic acid extract and n-hexane extract have good effect on DPPH free radical scavenging and are concentration dependent.

Inflammatory bowel disease (inflammatory bowel disease, IBD), including Crohn's Disease (CD) and Ulcerative Colitis (UC), is characterized by chronic recurrent intestinal inflammation. Some researchers believe that IBD is caused by abnormal and sustained immune responses by susceptible intestinal microorganisms, but the specific cause has not been identified. Although the etiology of IBD is largely unknown, it involves complex interactions between genetic, environmental or microbiological factors and immune responses, which many researchers have experimentally explored from different perspectives. IBD is a typical case of intestinal inflammation, and many studies are therefore based on this basis.

Supercritical fluid extraction is a method of separating a substance by contacting the substance to be separated or extracted with a supercritical fluid under a specific high pressure condition, extracting a desired component by adjusting the extraction pressure and the extraction temperature of a supercritical system, and changing the pressure or the temperature. Changes in the operating parameters of supercritical fluid extraction can have a significant impact on the type and content of the separated materials. At present, research shows that the alpinia oxyphylla extract extracted by supercritical fluid carbon dioxide has certain DPPH free radical scavenging capability and reducing capability, but no report is made on whether the alpinia oxyphylla volatile oil extracted by a supercritical carbon dioxide method can be used for improving inflammatory bowel diseases.

Disclosure of Invention

In view of the above, the invention aims to provide an application of the nootropic essential oil extracted by the supercritical carbon dioxide method in preparing medicines for improving inflammatory bowel diseases.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides an application of nootropic essential oil extracted by a supercritical carbon dioxide method in preparing a medicament for improving inflammatory bowel disease, wherein the supercritical carbon dioxide method comprises the following steps of: drying fructus Alpinae Oxyphyllae, pulverizing, sieving, and extracting in extraction tank; the temperature of the extraction tank is 38-42 ℃, the separation pressure is 12-17 mpa, the flow of CO ₂ is 0.6-1.0L/min, the separation temperature is 44-46 ℃, and the extraction time is 3.5-4.5 h.

Preferably, the drying conditions are: 48-52 deg.c for 23-25 hr.

Preferably, the crushed materials are sieved by a 40-mesh sieve.

Preferably, the inflammatory bowel disease is dextran sodium sulfate induced chronic colitis.

Preferably, the nootropic essential oil protects intestinal epithelial barrier integrity.

Preferably, the nootropic essential oil reduces intestinal leakage caused by inflammatory bowel disease.

Preferably, the nootropic essential oil inhibits p65 protein phosphorylation and oxidative stress signaling NOX1-LCN-2 molecular cascade in the inflammatory NF- κB signaling pathway.

The invention also provides a medicine for improving inflammatory bowel disease, the active ingredients of the medicine comprise the nootropic essential oil extracted by a supercritical carbon dioxide method, and the supercritical carbon dioxide method comprises the following steps: drying fructus Alpinae Oxyphyllae, pulverizing, sieving, and extracting in extraction tank; the temperature of the extraction tank is 38-42 ℃, the separation pressure is 12-17 mpa, the flow of CO ₂ is 0.6-1.0L/min, the separation temperature is 44-46 ℃, and the extraction time is 3.5-4.5 h.

Preferably, the content of the nootropic essential oil in the medicine is 35-98 wt%.

Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

The invention has the beneficial effects that:

The invention provides application of the intelligence-promoting volatile oil extracted by the supercritical carbon dioxide method in preparing medicines for improving inflammatory bowel diseases. According to the invention, firstly, the safe concentration of AOE to cells is 0.5 mug/mL and 1 mug/mL on the cell level, and the TER experiment and the monolayer cell permeability experiment are carried out by adopting the two concentrations, so that the intelligence-promoting volatile oil can play a good role in protecting the integrity of intestinal epithelial barriers at the cell level. Then, animal experiments are carried out, and the survival rate, daily health detection, DAI index scoring, intestinal permeability experiment and pathological section analysis of the mice are carried out to obtain the AOE which can improve the physical condition of enteritis mice, protect the integrity of intestinal epithelial barriers of the mice and better reduce the intestinal leakage phenomenon caused by DSS. The RT-PCR and WesternBlot experiments prove that the mechanism of the AOE with good protection effect on the tight junction of intestinal epithelial barriers is to inhibit the phosphorylation of a key protein p65 in an inflammatory NF- κB signal path and inhibit the molecular cascade of oxidation stress signals NOX1-LCN-2, thereby inhibiting the release of pro-inflammatory factors of intestinal epithelium and protecting the integrity of intestinal mucosa.

Drawings

Fig. 1: MTT method detects the survival rate of AOE with different concentration gradients to Caco-2 cells;

Fig. 2: changes in TER values due to stimulation of Caco-2 by AOE antagonizing LPS;

Fig. 3: AOE antagonizes monolayer barrier permeability changes caused by LPS stimulation of Caco-2;

Fig. 4: survival curves and weight changes for mice from different treatment groups;

Fig. 5: a comprehensive evaluation chart of DAI and colon of mice in different treatment groups within seven days;

fig. 6: colon anatomical appearance of mice of different treatment groups;

fig. 7: FITC concentrations in serum of mice of different treatment groups were measured;

fig. 8: HE staining pictures of mice from different treatment groups;

Fig. 9: influence of AOE on expression level of inflammatory factor mRNA related to mice with colon inflammation;

Fig. 10: effects of AOE on expression levels of zonulin ZO-1 and occludin proteins in the colon of mice;

fig. 11: effects of AOE on IκBα and p-IκBα protein expression in NF- κB signaling pathways;

Fig. 12: effect of AOE on p65 phosphorylation in NF- κb signaling pathway;

fig. 13: effect of AOE on NOX1 and LCN-2 expression levels in the colon of inflammatory mice.

Detailed Description

The invention provides application of nootropic essential oil extracted by a supercritical carbon dioxide method in preparing medicines for improving inflammatory bowel disease. Inflammatory bowel disease as described herein includes Crohn's disease and ulcerative colitis, more preferably sodium dextran sulfate induced chronic colitis.

The supercritical carbon dioxide method comprises the following extraction steps: drying fructus Alpinae Oxyphyllae, pulverizing, sieving, and extracting in extraction tank; the temperature of the extraction tank is 38-42 ℃, the separation pressure is 12-17 mpa, the flow of CO ₂ is 0.6-1.0L/min, the separation temperature is 44-46 ℃, and the extraction time is 3.5-4.5 h. The drying conditions are as follows: 48-52 ℃, 23-25 hours, preferably 50 ℃,24 hours; sieving the crushed materials with a 40-mesh sieve; the temperature of the extraction tank is preferably 40 ℃, the separation pressure is preferably 15Mpa, the flow rate of CO ₂ is preferably 0.8L/min, the separation temperature is preferably 45 ℃, and the extraction time is preferably 4h.

The nootkatone, the Valencia, the gingerol, the vetiverol, the alpha-malene and the eudesmol in the nootkatone, the Valencia, the gingerol and the eudesmol are extracted by the limited supercritical carbon dioxide method, so that the pharmacological effects of terpene natural products can be effectively exerted. The nootkatone content in the nootkatone volatile oil obtained by the method is high, and the nootkatone volatile oil has better anti-inflammatory effect.

The intelligence-promoting volatile oil can avoid damage of DSS to intestinal villus structure and crypt, reduce neutrophil infiltration, protect intestinal mucosa integrity, protect intestinal epithelial barrier integrity, alleviate colon lesions and reduce intestinal leakage caused by DSS.

The intestinal tight junctions are the major components of the intestinal epithelial barrier. According to the invention, the influence of the nootropic essential oil on intestinal barriers and classical inflammatory pathways in the colon of the DSS mice is researched from the gene and molecular level, and the nootropic essential oil is found to be capable of improving the expression quantity of ZO-1 and Occludin tightly connected in intestinal epithelial barriers induced by DSS, so that the AOE is further proved to be capable of effectively inhibiting the occurrence of intestinal leakage.

The invention discovers that the nootropic essential oil can effectively inhibit the activation of NF- κB signal path through detecting the content of IκBα and p65 phosphorylated proteins in the NF- κB signal path, thereby inhibiting the occurrence of inflammation. Meanwhile, the nootropic essential oil can inhibit the expression of NOX1, so that the aim of inhibiting the synthesis of LCN-2 is fulfilled.

The effective dose of the nootropic essential oil is 50 mg/kg-200 mg/kg, preferably 120 mg/kg-180 mg/kg.

The invention also provides a medicine for improving inflammatory bowel disease, the active ingredients of the medicine comprise nootropic essential oil, the nootropic essential oil is extracted by the supercritical carbon dioxide method, and the supercritical carbon dioxide method comprises the following steps: drying fructus Alpinae Oxyphyllae, pulverizing, sieving, and extracting in extraction tank; the temperature of the extraction tank is 38-42 ℃, the separation pressure is 12-17 mpa, the flow of CO ₂ is 0.6-1.0L/min, the separation temperature is 44-46 ℃, and the extraction time is 3.5-4.5 h. The drying conditions are as follows: 48-52 ℃, 23-25 hours, preferably 50 ℃,24 hours; sieving the crushed materials with a 40-mesh sieve; the temperature of the extraction tank is preferably 40 ℃, the separation pressure is preferably 15Mpa, the flow rate of CO ₂ is preferably 0.8L/min, the separation temperature is preferably 45 ℃, and the extraction time is preferably 4h.

The active ingredient in the medicine for improving inflammatory bowel disease can take the nootropic essential oil extracted by the supercritical carbon dioxide method as the only active ingredient, and can also be combined with other active ingredients with the effect of improving inflammatory bowel disease.

The medicine of the invention comprises but is not limited to injection preparations, emulsion, ointment, granules, powder and oral liquid. The medicament of the invention also comprises a pharmaceutically acceptable carrier. The invention has no special limitation on other auxiliary materials contained in the medicine, and the auxiliary materials commonly used in the field of medicine can be adopted.

In the medicine, the content of the nootropic essential oil extracted by the supercritical carbon dioxide method is 35-98wt%, preferably 55-90%.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

In the following examples, conventional methods are used unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1

In the embodiment, the intelligent volatile oil is extracted by a supercritical carbon dioxide method:

Selecting fructus Alpinae Oxyphyllae with full grain and no worm hole, placing into dryer, drying at 50deg.C for 24 hr, pulverizing with pulverizer, and sieving with 40 mesh sieve. 200g of the intelligent powder is weighed and placed in an extraction tank, the temperature of the extraction tank is set to be 40 ℃, the separation pressure is set to be 15mpa, the flow of CO ₂ is set to be 0.8L/min, the separation temperature is set to be 45 ℃, the extraction time is set to be 4 hours, and the material tank is opened after the instrument is cooled to normal temperature.

The obtained volatile oil is in the form of green oil, has strong pungent smell, and has strong volatility, and the bottom of the extract is in viscous colloid state after standing for a period of time. Weighing the collected volatile oil: m:52g, m ₀: 8g, M:200g. The extraction rate of the volatile oil is 2.2 percent according to the formula calculation.

A= (M-M ₀)/M (×100) (formula 1)

Wherein: a: intelligence-promoting volatile oil extraction rate, m: mass of volatile oil for promoting intelligence, m ₀: empty centrifuge tube mass; m: putting into an extraction tank to promote intelligence powder quality.

10 Mu L of the nootropic essential oil was dissolved in 990 mu L of acetonitrile and then filtered through a 0.22 mu m sterile filter, and the filtered sample solution was placed in an analytical flask. GC-MS analysis was performed on samples of the nootropic volatile oils using Trace1310, TSQ 8000GC-MS and Siemens flight TR-5MS,30 mX0.25 mm X0.25 μm capillary columns. The program temperature was increased from 50 ℃ to 200 ℃, gradient 3 ℃/min, from 200 ℃ to 280 ℃ at a rate of 203 ℃/min, followed by a 10min hold. Helium is the carrier gas at a flow rate of 1mL/min. Split ratio 60:1, delaying the solvent for 3min, and having ionization energy of 70eV, ion source temperature of 230 ℃, interface temperature of 250 ℃ and mass scanning range of 50-300m/z.

The GC-MS analysis results show that 84 types of the volatile oil extracted by supercritical carbon dioxide have 46 types of volatile oil with the content exceeding 0.1 percent and 20 types of volatile oil with the content exceeding 1 percent, and the volatile oil is mainly terpenoid. The GC-MS results are analyzed as shown in Table 1.

TABLE 1GC-MS analysis of the volatile oil composition for developing Intelligence

As can be seen from Table 1, 35 ingredients with higher content were analyzed in the nootropic essential oil, which accounted for 48.09% of the total ingredients. Wherein, the components of the natural product comprise the components of the natural product such as the naringin (12.59%), the Valencia orange alkene (9.8%), the gingerol (7.48%), the vetiverol (6.55%), the alpha-maleene (4.43%), the eudesmol (3.07%) and the like, and the natural product is mainly terpene natural products.

Example 2

In this example, pharmacological activity studies were performed using the nootropic essential oil extracted in example 1:

1. protection effect of volatile oil for improving intelligence on Caco-2 cells

(1) MTT method for detecting cytotoxicity

One dish of Caco-2 cells was cultured, and the total number of cells was seeded on a 96-well plate by counting the number of cells adjusted to 8X 10 ⁴/mL. Culturing until the density reaches 80% -90%, adding the essential oil (AOE) with different concentrations for culturing, wherein the final concentrations of the AOE in the culture medium are respectively as follows: 0.5. Mu.g/mL, 1. Mu.g/mL, 2. Mu.g/mL, 4. Mu.g/mL, 6. Mu.g/mL, 8. Mu.g/mL, 10. Mu.g/mL, 12. Mu.g/mL, 14. Mu.g/mL, 16. Mu.g/mL, 18. Mu.g/mL, 20. Mu.g/mL. The 96-well plate was taken out, 10. Mu.L of prepared MTT solution at a concentration of 5mg/mL was added to the well, the culture was continued for 4 hours, DMSO was added, and the plate was left at room temperature for 1 hour to sufficiently dissolve crystal violet. The absorbance was measured at 490nm wavelength using a kinetic energy microplate reader to calculate the cell viability, the results are shown in FIG. 1.

FIG. 1 shows that cell viability decreases with increasing AOE concentration, and that cell viability does not show significant differences when AOE concentration is 0.5-2 μg/mL. Cell viability was significantly reduced at AOE concentrations above 4 μg/mL. The subsequent experiments were therefore dosed at low concentrations of 0.5. Mu.g/mL and at high concentrations of 2. Mu.g/mL, respectively.

(2) Cell transepithelial resistance (TER) assay

Establishing a cell tight junction model: cell counting is carried out after cell culture, the cell density is adjusted to be 8 multiplied by 10 ⁴/mL, a cell is placed in a 24-hole plate, cell liquid is taken and inoculated into the cell chamber (AP layer), and cell culture liquid is added into the cell chamber (BL layer). Cell growth was observed daily for one week after inoculation, cell density was increased after one week, medium was changed daily, culture was started to change every day until 14d and cell resistance was measured to 21d, and modeling was considered successful when resistance reached peak and no growth occurred within two days. The modeled cells were divided into four groups: control group (CON), dosing group 1 (0.5 AOE), dosing group 2 (1 AOE), LPS stimulated group (LPS).

And (3) adding medicines: after modeling is successful, the cell culture solution is changed into serum-free culture solution to be cultured for 12 hours, so that the cells are in the same growth cycle. The experiments were divided into 4 groups: drug addition group 1 was added with AOE at a final concentration of 0.5 μg/mL; drug addition group 2 was added with AOE at a final concentration of 2 μg/mL; the control and LPS stimulated groups did not add AOE. After pretreatment with AOE for 12h, 5. Mu.g/mL LPS was added to each of the drug-added group 1, drug-added group 2 and LPS-stimulated group to stimulate cells.

Resistance measurement: the cells to which LPS was added were measured for resistance values from 0h, followed by resistance values detection for 2h,4h,8h,12h,24h,36h,48 h. The actual resistance value is calculated according to the following formula:

ter= (R-R _{Blank space})*A(Ω*cm²) (formula 2)

Wherein: r- -measuring the actual resistance; r blank-cell free blank cell resistance; a- -membrane area.

The results are shown in FIG. 2. In fig. 2, "%": in the Control group, P <0.05, "%" represents P <0.01, "%" represents P <0.001, "%%" represents P <0.0001, compared with the LPS group; "*": comparing 0.5AOE with LPS group, "" means P <0.05, "" means P <0.01, "" means P <0.001, "" means P <0.0001; "#":1AOE compared to LPS group, "#" indicates P <0.05, "#" indicates P <0.01, "#" indicates P <0.001, "# #" indicates P <0.0001, "# SD, n=3.

As can be seen from fig. 2, 0.5AOE showed better effect (P < 0.0001) and 1AOE also showed better effect (P < 0.0001) when compared to control group, and groups added with AOE all showed good effect of protecting cell barrier. The damage of the integrity of the cell barrier caused by LPS can be reduced by demonstrating that the integrity of the intestinal epithelial cells can be damaged under the stimulation of LPS and the damage of the cell barrier can be protected to a certain extent by AOE.

(3) Cell permeability assay

On the basis of transepithelial resistance measurement, it was examined whether or not a fluorescent macromolecular substance FITC-dextran has a protective effect on the cellular level and the passing rate of a macromolecular substance such as bacteria and the like was simulated by detecting the passing rate of the fluorescent macromolecular substance FITC-dextran through cells. And (2) culturing cells in a cell and adding medicines, wherein after the medicines are added, 100 mu L of each of the inner chamber and the outer chamber of the Transwell cell is placed in a 96-well plate, the fluorescence absorbance inside and outside the cell is measured, the excitation wavelength is 480nm, and the emission wavelength is 520nm. The apparent permeability coefficient of the fluorescent substance FITC-dextran for transmembrane transport (Papp) was calculated by the following formula.

Papp=Δq/(Δt×a×c _o)(cm*s^-1) (formula 3)

Wherein: the transport quantity of delta Q-delta t; Δt- -incubation time; a- -membrane area; c _o - -initial concentration in the inner chamber of Caco-2 cells.

The results are shown in FIG. 3. In fig. 3, "": in comparison to the LPS group, "+" means P <0.001, mean±sd, n=3.

As can be seen from fig. 3, the cell permeability after LPS stimulation was doubled compared with that of the Con group, but cells pretreated with AOE added in advance all showed significant decrease compared with that of the LPS group, indicating that AOE can protect the cell barrier from damage and the tissue macromolecular substances from entering the blood.

2. Protection effect of volatile oil for improving intelligence on intestinal inflammation of mice

Animal experiment:

Healthy male C57 BL/6 mice are selected, and the mice are randomly divided into 4 groups, namely a blank control group, a dextran sodium sulfate salt (DSS, molecular weight: 36000-50000 Da) treatment group, AOEL groups and AOEH groups, wherein 10 mice are used in each group. After one week of split-cage adaptation, the blank group was given a normal diet, and the DSS group, AOEL group, AOEH group were given a full dissolution of 3% DSS in the drinking water, giving a normal feed. Groups AOEL and AOEH were subjected to 50mg/kg and 200mg/kg AOE per day, respectively, and the AOE was dissolved in olive oil and fed continuously for 7 days. Blood was collected from the ocular venous plexus after the completion of feeding. The mice are killed by neck removal, the mice are obtained, tissues are placed in a material obtaining pipe and stored in an ultralow temperature refrigerator at the temperature of minus 80 ℃ to be used for the subsequent molecular experiment.

Standing the obtained blood in dark place at normal temperature for about 2h, centrifuging at 3000r/min and 4deg.C for 10min, slightly sucking clear yellow-white supernatant with a pipette, placing in a new EP tube, and storing in dark place at-80deg.C in an ultralow temperature refrigerator.

(1) Health detection

One week after the split cage adaptation, the weight and death of each group of mice was recorded starting at 1d and scored for disease activity index. The scoring criteria are shown in tables 2-3.

TABLE 2 colon scoring criteria

TABLE 3 clinical disease Activity scoring criteria

The results are shown in FIGS. 4 to 5. In fig. 4, a is a survival curve of mice, and B is a change in body weight. As can be seen from fig. 4, mice in DSS group died at day seven of molding, with a mortality rate of 10%, while mice in AOE group had no mortality. Starting on day 6, mice in the DSS group had a steep weight loss, while AOE group had a slower trend toward a lower body weight, and in figure B, mice in the DSS group had a severe weight loss, while AOE group had a lower body weight but overall trend better than DSS group. From the survival and weight cases above, it was demonstrated that both groups of AOEs can protect mice from weight loss caused by DSS stimulation. FIG. 5 is a graph of the combined score of mouse DAI and colon, A scored for DSS mice, B scored for AOEL mice, and C scored for AOEH mice. As can be seen from FIG. 5, the DSS group started to develop severe hematochezia rectal bleeding on day 5, while the AOE group improved in both cases, with hematochezia and bleeding on day 6, and fewer mice with severe hematochezia.

The mice were dissected after the end of the experiment and the results are shown in fig. 6. In fig. 6, a is a large intestine anatomical map contrast and B is colon length statistics. As can be seen from fig. 6, the stool in the intestine of the Con group is shaped into a granule, the cecum is brown yellow, the colon length is long, while the DSS group is in a condition of engorged water and thick intestine, the stool is not shaped, the stool color in the cecum is blood, and the colon length is short. The AOE groups have longer colon length than the DSS groups, have little bloody stool, have thick intestinal walls and improve congestion. The results demonstrate that AOE can improve DSS-induced enteritis in mice, and that high-concentration AOE has better effect at lower concentration.

(2) Mouse intestinal mucosa barrier integrity detection

Fluorescein isothiocyanate-dextran (FITC-dextran) permeability assay: the mice were fed with water and water on the day before they were harvested, and on the next morning, the mice were perfused with FITC at a dose of 0.5mg/g by gavage according to their body weight. 200 mu L of each of the gradient FITC and the serum sample is taken and added into a black 96-well plate, fluorescence absorbance is detected at the wavelength of 485nm of excitation light and 528nm of emitted light, the absorbance of the gradient FITC is drawn into a standard curve, and the concentration of FITC-dextran in serum is calculated according to the standard curve by the mouse serum sample, namely FITC penetrating into the serum from the intestinal tract of the mouse.

The results are shown in FIG. 7. In the figure, "+": in comparison with DSS, "+," means P <0.0001, mean±sd, n=9. As can be seen from fig. 7, the fluorescence value in serum of mice in DSS group is 7 times higher than that of control group, and the fluorescence value in serum is significantly reduced (P < 0.0001) in AOE two groups, which indicates that AOE has good anti-inflammatory effect, has good protection effect on intestinal mucosa barrier caused by enteritis of mice, and improves occurrence of "intestinal leakage" phenomenon.

(3) Pathology detection

Making colon tissue slice of mouse, dewatering, transparent, wax-immersing, slicing, baking, and collecting box. HE staining was performed by deparaffinization-hematoxylin staining-eosin staining-dehydration transparency of tissue sections and then taking photographs at appropriate positions.

The results are shown in FIG. 8. As can be seen from fig. 8, the intestinal villi of the mice in the CON group are complete and clear, and the structure is complete. However, the pathological results of the DSS group show that the intestinal villi of the mice cannot see the complete structure, and with the occurrence of villus shedding, the crypt disappears, neutrophils are infiltrated, while the intestinal tract of the mice treated by AOE can retain part of the villi structure, and the crypt is clearly visible, and the situation of neutrophil infiltration is also shown but improved compared with the mice of the DSS group.

3. Research on protection mechanism of essential oil for improving intelligence on intestinal inflammation

Extraction of RNA from colon tissue of mice: RNA from the colon tissue of the mouse was extracted by Trizol method and reverse transcribed according to the instructions of the full-gold Trans one-step reverse transcription kit factory, and the concentration of cDNA was measured using an ultra-micro spectrophotometer. Diluting with RNase-free water to a specific concentration, and storing in a refrigerator at-20deg.C.

Primer design was performed using NCBI website, the primer sequences are as shown in table 4:

TABLE 4qRT-PCR primers

Timing quantitative PCR (RT-PCR) reaction: the resulting cDNA was diluted. RT-PCR reactions were performed according to the system required by the instructions of Roche FASTSTART ESSENTIAL DNA GREEN MASTER kit. When RT-PCR experiments are carried out, GAPDH and beta-actin are used as standard reference genes, and three repeated holes are simultaneously arranged on each gene sample. The reaction procedure was set as follows: pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 15s, and extension at 60 ℃ for 60s; the process was run for 40 cycles. After the PCR experiment reaction is finished, the analysis of a dissolution curve is carried out by using Roche LC96 general analysis software, and the specificity of the product is confirmed. After the data are determined to be valid, the Ct value of the data is calculated, the average delta Ct is taken, and the value of 2 ^-ΔΔ Ctd is calculated as the relative quantitative analysis data. The results are shown in FIG. 9, wherein A is the mRNA expression level of IL-1β in each group, B is the mRNA expression level of IL-6 in each group, and C is the mRNA expression level of IL-10 in each group.

As can be seen from fig. 9, the levels of pro-inflammatory factors were relatively low in mice under normal conditions, and the pro-inflammatory factors IL-1β (P < 0.01) and IL-6 (P < 0.05) were significantly elevated under stimulation by DSS. However, it can be seen in the AOE group that both pro-inflammatory factors showed some reduction (P < 0.01) (P < 0.05) and a clear concentration dependence upon AOE addition. Whereas the anti-inflammatory factor IL-10 shows a completely opposite trend to the former two. The AOE is shown to inhibit the expression of pro-inflammatory factor mRNA and promote the expression of anti-inflammatory factor mRNA.

Western blot experiments: PIPA and PMSF solutions are added according to the ratio of 1:100, and the crushed beads are put into a cell tissue crusher which is pre-cooled in a refrigerator in advance to be crushed until the total protein of the tissue is extracted in a homogenate state, and then BCA protein quantification is carried out. Adding loading buffer into the obtained protein sample according to the ratio of 1:4, and boiling at 100deg.C for 10min to denature the protein. And then, performing gel running, membrane transferring, sealing, primary antibody incubation and secondary antibody development to obtain an experimental result. The results are shown in FIGS. 10 to 13.

As can be seen from fig. 10, the content of the mouse tight junction related proteins ZO-1 and Occludin in the DSS group is significantly reduced, the disruption of the tight junction causes the cell side barrier to be disrupted, the protein content controlling the barrier is reduced, and thus the function of controlling the entry and exit of substances is disrupted, and some bacteria, endotoxin and the like may enter the blood to cause inflammation and other related diseases. And the AOE groups can up-regulate the expression level of the tight junction ZO-1, and simultaneously can improve the expression level of Occludin. It is demonstrated that AOE can inhibit the occurrence of inflammation from the standpoint of controlling the integrity of the intestinal barrier, and can effectively inhibit the occurrence of "intestinal leakage".

The NF-. Kappa.B signaling pathway is a classical signaling pathway for inflammatory responses. As can be seen from fig. 11, DSS can cause NF- κb to be activated in colon tissue of mice, and expression of phosphorylated ikbα (P < 0.001) under DSS stimulation is increased, contributing to colitis in mice. The expression level of phosphorylated IκBα (P < 0.001) in AOE group is inhibited, and the decrease of the expression level of phosphorylated IκBα can prevent the phosphorylation of P65 to enter the nucleus, and can prevent the activation of NF- κB signal path to a certain extent.

The key to NF-. Kappa.B signaling pathway activation is the transfer into the nucleus following p65 phosphorylation, so whether p65 phosphorylates into the nucleus is a key signal considering the extent of activation of this signaling pathway. As can be seen from fig. 12, DSS was able to induce p65 protein expression in the colon tissue of mice, whereas p65 protein expression in the colon tissue of mice after AOE treatment was significantly reduced, demonstrating that AOE was able to down-regulate p65 protein phosphorylated expression. During the onset of IBD, the expression level of LCN-2 in the colonic epithelium was significantly up-regulated. As can be seen from fig. 13, DSS was able to induce expression of NOX1 and LCN-2 proteins in colon tissue of mice, whereas expression of NOX1 and LCN-2 proteins in colon tissue of mice after treatment by AOE was significantly reduced. It was demonstrated that AOE could prevent NF- κB signaling through down-regulating p65 phosphorylation and nuclear entry, thereby inhibiting inflammation.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The application of the volatile oil extracted by the supercritical carbon dioxide method in preparing the medicine for improving inflammatory bowel disease is characterized in that the supercritical carbon dioxide method comprises the following steps: drying fructus Alpinae Oxyphyllae, pulverizing, sieving, and extracting in extraction tank; the temperature of the extraction tank is 38-42 ℃, the separation pressure is 12-17 mpa, the flow of CO ₂ is 0.6-1.0L/min, the separation temperature is 44-46 ℃, and the extraction time is 3.5-4.5 h.

2. The use according to claim 1, wherein the drying conditions are: 48-52 deg.c for 23-25 hr.

3. The use according to claim 1, wherein the crushed material is sieved through a 40 mesh sieve.

4. The use according to claim 1, wherein the inflammatory bowel disease is dextran sodium sulfate induced chronic colitis.

5. The use according to claim 1, wherein the nootropic volatile oil protects intestinal epithelial barrier integrity.

6. The use according to claim 1, wherein the nootropic volatile oil reduces intestinal leakage caused by inflammatory bowel disease.

7. The use according to claim 1, wherein the nootropic essential oil inhibits p65 protein phosphorylation and oxidative stress signaling NOX1-LCN-2 molecular cascades in the inflammatory NF- κb signaling pathway.

8. A medicament for improving inflammatory bowel disease, wherein an active ingredient of the medicament comprises a nootropic essential oil extracted by a supercritical carbon dioxide method, and the supercritical carbon dioxide method comprises the following steps of: drying fructus Alpinae Oxyphyllae, pulverizing, sieving, and extracting in extraction tank; the temperature of the extraction tank is 38-42 ℃, the separation pressure is 12-17 mpa, the flow of CO ₂ is 0.6-1.0L/min, the separation temperature is 44-46 ℃, and the extraction time is 3.5-4.5 h.

9. The medicament according to claim 8, wherein the content of the nootropic essential oil in the medicament is 35-98 wt%.

10. The medicament of claim 8, further comprising a pharmaceutically acceptable carrier.