Application of volatile oil extracted from fructus alpiniae oxyphyllae by supercritical carbon dioxide method in preparation of drugs for improving inflammatory bowel disease

Abstract

The application provides application of supercritical carbon dioxide method extracted volatile oil of Alpiniae oxyphyllae in preparation of a medicine for improving inflammatory bowel disease, and belongs to the technical field of medicines. The application proposes that the supercritical carbon dioxide method extracted volatile oil of Alpiniae oxyphyllae has good biological activity, the volatile oil of Alpiniae oxyphyllae can play a good protection effect on the integrity of the intestinal epithelial barrier at the cell level, the volatile oil of Alpiniae oxyphyllae can improve the physical condition of intestinal inflammation mice, can protect the integrity of the intestinal epithelial barrier of the mice, and reduce the "intestinal leakage" phenomenon caused by DSS. The application provides a good theoretical basis for subsequent Alpiniae oxyphyllae product development and new drug research and development, and provides a potential research reference for future diet and health product development.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of Alpinia oxyphylla volatile oil extracted by supercritical carbon dioxide method in the preparation of drugs to improve inflammatory bowel disease. Background Technology

[0002] Alpinia oxyphylla Miquel is an important plant used for both food and medicine. It is not only a fruit but also a traditional medicine beneficial for cognitive function and alleviating various ailments. Medical texts record its effects as "warming the kidneys, strengthening essence, stopping excessive salivation, warming the spleen, stopping diarrhea, and reducing drooling." Modern pharmacological research on Alpinia oxyphylla primarily focuses on its effects on the nervous system. Alpinia oxyphylla and its active ingredients exhibit neuroprotective effects through multiple mechanisms, making it a promising treatment for neurological disorders. Studies on the active components of Alpinia oxyphylla have shown that it is rich in sesquiterpenes and flavonoids, which possess antioxidant activity. Alpinia oxyphylla and its petroleum ether extract, acetoacetic acid extract, and n-hexane extract have shown good, concentration-dependent effects in DPPH free radical scavenging.

[0003] Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is characterized by chronic, relapsing intestinal inflammation. Some researchers believe IBD is caused by abnormalities in susceptible gut microbiota and a persistent immune response, but the specific cause remains unclear. Although the etiology of IBD remains largely unknown, it involves complex interactions between genetic, environmental, or microbial factors and the immune response, and many researchers are exploring it experimentally from different perspectives. IBD is a classic case of intestinal inflammation, and therefore many studies are based on it.

[0004] Supercritical fluid extraction utilizes the properties of supercritical fluids to contact the substances to be separated or extracted under specific high-pressure conditions. By adjusting the extraction pressure and temperature of the supercritical system, the desired components are extracted, and the obtained substances are separated by changing the pressure or increasing the temperature. Changes in the operating parameters of supercritical fluid extraction can significantly affect the types and contents of the separated substances. Current research has shown that the extract of Alpinia oxyphylla extracted by supercritical carbon dioxide has certain DPPH free radical scavenging and reducing abilities, but there are no reports on whether the volatile oil of Alpinia oxyphylla extracted by supercritical carbon dioxide can be used to improve inflammatory bowel disease. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide the application of Alpinia oxyphylla volatile oil extracted by supercritical carbon dioxide in the preparation of a drug for improving inflammatory bowel disease.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of Alpinia oxyphylla volatile oil extracted by supercritical carbon dioxide in the preparation of drugs to improve inflammatory bowel disease. The supercritical carbon dioxide extraction steps include: drying, crushing, and sieving Alpinia oxyphylla fruit, and placing it in an extraction tank for extraction; the temperature of the extraction tank is 38-42℃, the separation pressure is 12-17 MPa, the CO2 flow rate is 0.6-1.0 L / min, the separation temperature is 44-46℃, and the extraction time is 3.5-4.5 h.

[0008] Preferably, the drying conditions are: 48-52℃, 23-25h.

[0009] Preferably, the pulverized material is passed through a 40-mesh sieve.

[0010] Preferably, the inflammatory bowel disease is chronic colitis induced by sodium dextran sulfate.

[0011] Preferably, the volatile oil that enhances intelligence protects the integrity of the intestinal epithelial barrier.

[0012] Preferably, the volatile oil that enhances the sense of wellness reduces leaky gut caused by inflammatory bowel disease.

[0013] Preferably, the Alpinia oxyphylla volatile oil inhibits p65 protein phosphorylation and the NOX1-LCN-2 molecular cascade of oxidative stress signaling in the inflammatory NF-κB signaling pathway.

[0014] The present invention also provides a drug for improving inflammatory bowel disease, wherein the active ingredient of the drug includes Alpinia oxyphylla volatile oil extracted by supercritical carbon dioxide method. The supercritical carbon dioxide extraction steps include: drying, crushing, and sieving Alpinia oxyphylla fruit, and placing it in an extraction tank for extraction; the temperature of the extraction tank is 38-42℃, the separation pressure is 12-17 MPa, the CO2 flow rate is 0.6-1.0 L / min, the separation temperature is 44-46℃, and the extraction time is 3.5-4.5 h.

[0015] Preferably, the content of the volatile oil in the drug is 35-98 wt%.

[0016] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0017] The beneficial effects of this invention are:

[0018] This invention proposes the application of Alpinia oxyphylla volatile oil extracted by supercritical carbon dioxide in the preparation of drugs to improve inflammatory bowel disease (IBD). Firstly, this invention determined the safe concentrations of AOE (Aloxin volatile oil) for cells to be 0.5 μg / mL and 1 μg / mL at the cellular level. TER experiments and monolayer cell permeability experiments were conducted using these two concentrations, confirming that the Alpinia oxyphylla volatile oil can effectively protect the integrity of the intestinal epithelial barrier at the cellular level. Then, animal experiments were conducted. Through mouse survival rate, daily health monitoring, DAI index scores, intestinal permeability experiments, and pathological section analysis, it was found that AOE can improve the physical condition of mice with enteritis, protect the integrity of the intestinal epithelial barrier, and better reduce the "leaky gut" phenomenon caused by DSS. RT-PCR and Western Blot experiments demonstrated that the mechanism by which AOE effectively protects the tight junctions of the intestinal epithelial barrier lies in inhibiting the phosphorylation of the key protein p65 in the inflammatory NF-κB signaling pathway and inhibiting the NOX1-LCN-2 molecular cascade of oxidative stress signals, thereby inhibiting the release of pro-inflammatory factors from the intestinal epithelium and protecting the integrity of the intestinal mucosa. Attached Figure Description

[0019] Figure 1 MTT assay was used to detect the survival rate of Caco-2 cells with different concentration gradients of AOE.

[0020] Figure 2 AOE antagonizes the TER value changes induced by LPS stimulation of Caco-2;

[0021] Figure 3 AOE antagonizes changes in monolayer barrier permeability induced by LPS stimulation of Caco-2;

[0022] Figure 4 Survival curves and weight changes of mice in different treatment groups;

[0023] Figure 5 DAI and colonic comprehensive score of mice in different treatment groups over seven days;

[0024] Figure 6 Anatomical appearance of the colon in mice from different treatment groups;

[0025] Figure 7 Detection of FITC concentration in the serum of mice in different treatment groups;

[0026] Figure 8 Images of mice stained with hematoxylin and eosin (HE) in different treatment groups;

[0027] Figure 9 Effects of AOE on the mRNA expression levels of inflammatory factors related to colitis in mice;

[0028] Figure 10 Effects of AOE on the expression levels of tight junction proteins ZO-1 and occludin in the mouse colon;

[0029] Figure 11 Effects of AOE on the expression of IκBα and p-IκBα proteins in the NF-κB signaling pathway;

[0030] Figure 12 The effect of AOE on p65 phosphorylation in the NF-κB signaling pathway;

[0031] Figure 13 Effects of AOE on the expression levels of NOX1 and LCN-2 in the colon of inflamed mice. Detailed Implementation

[0032] This invention provides the application of Alpinia oxyphylla volatile oil extracted by supercritical carbon dioxide in the preparation of drugs for improving inflammatory bowel disease. The inflammatory bowel disease described in this invention includes Crohn's disease and ulcerative colitis, more preferably chronic colitis induced by sodium dextran sulfate.

[0033] The supercritical carbon dioxide extraction method of this invention includes the following steps: drying, pulverizing, and sieving the Alpinia oxyphylla fruit, and then placing it in an extraction tank for extraction; the extraction tank temperature is 38–42℃, the separation pressure is 12–17 MPa, the CO2 flow rate is 0.6–1.0 L / min, the separation temperature is 44–46℃, and the extraction time is 3.5–4.5 h. The drying conditions are: 48–52℃ for 23–25 h, preferably 50℃ for 24 h; the pulverized fruit is sieved through a 40-mesh sieve; the extraction tank temperature is preferably 40℃, the separation pressure is preferably 15 MPa, the CO2 flow rate is preferably 0.8 L / min, the separation temperature is preferably 45℃, and the extraction time is preferably 4 h.

[0034] The Alpinia oxyphylla volatile oil extracted by this invention using a limited supercritical carbon dioxide method has high contents of naringin, Valencian mansone, gingerol, vetiverol, α-maleene, and eucalyptol, which can effectively exert the pharmacological effects of terpenoid natural products. Furthermore, the Alpinia oxyphylla volatile oil obtained by this method has a high content of naringin, resulting in better anti-inflammatory effects.

[0035] The volatile oil for improving intelligence described in this invention can prevent damage to the intestinal villi structure and crypts caused by DSS, reduce neutrophil infiltration, protect the integrity of the intestinal mucosa, protect the integrity of the intestinal epithelial barrier, alleviate colonic lesions, and reduce intestinal leakage caused by DSS.

[0036] Intestinal tight junctions are a major component of the intestinal epithelial barrier. This invention investigates the effects of the Alpinia oxyphylla volatile oil on the intestinal barrier and classical inflammatory pathways in the colon of DSS mice at the gene and molecular levels. The study found that the Alpinia oxyphylla volatile oil can increase the expression levels of ZO-1 and Occludin in the DSS-induced intestinal epithelial barrier, further demonstrating that AOE can effectively inhibit the occurrence of "leaky gut".

[0037] This invention, through the detection of IκBα and p65 phosphorylated protein content in the NF-κB signaling pathway, found that the Alpinia oxyphylla volatile oil can effectively inhibit the activation of the NF-κB signaling pathway, thereby suppressing inflammation. Simultaneously, the Alpinia oxyphylla volatile oil can inhibit NOX1 expression, thereby achieving the purpose of inhibiting LCN-2 synthesis.

[0038] The effective dose of the volatile oil for improving intelligence described in this invention is 50 mg / kg to 200 mg / kg, preferably 120 mg / kg to 180 mg / kg.

[0039] This invention also provides a drug for improving inflammatory bowel disease, the active ingredient of which includes Alpinia oxyphylla volatile oil, which is extracted by the supercritical carbon dioxide method described above. The supercritical carbon dioxide extraction steps include: drying, pulverizing, and sieving Alpinia oxyphylla fruit, and then placing it in an extraction tank for extraction; the temperature of the extraction tank is 38-42℃, the separation pressure is 12-17 MPa, the CO2 flow rate is 0.6-1.0 L / min, the separation temperature is 44-46℃, and the extraction time is 3.5-4.5 h. The drying conditions are: 48-52℃ for 23-25 ​​h, preferably 50℃ for 24 h; the pulverized fruit is sieved through a 40-mesh sieve; the temperature of the extraction tank is preferably 40℃, the separation pressure is preferably 15 MPa, the CO2 flow rate is preferably 0.8 L / min, the separation temperature is preferably 45℃, and the extraction time is preferably 4 h.

[0040] The active ingredient in the drug for improving inflammatory bowel disease described in this invention can be the Alpinia oxyphylla volatile oil extracted by supercritical carbon dioxide method as the sole active ingredient, or the Alpinia oxyphylla volatile oil extracted by supercritical carbon dioxide method can be used in combination with other active ingredients that have the effect of improving inflammatory bowel disease.

[0041] The drugs described in this invention include, but are not limited to, injectable formulations, emulsions, ointments, granules, powders, and oral liquids. The drugs also include pharmaceutically acceptable carriers. This invention does not impose any specific limitations on other excipients contained in the drugs; commonly used pharmaceutical excipients in the art may be used.

[0042] In the drug of the present invention, the content of the Alpinia oxyphylla volatile oil extracted by supercritical carbon dioxide method is 35-98 wt%, preferably 55-90%.

[0043] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Unless otherwise specified, the following embodiments are all conventional methods.

[0045] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0046] Example 1

[0047] This embodiment uses the supercritical carbon dioxide method to extract the volatile oil of Alpinia oxyphylla:

[0048] Select plump, insect-free Alpinia oxyphylla fruits and place them in a dryer at 50℃ for 24 hours. After drying, pulverize the Alpinia oxyphylla using a pharmaceutical pulverizer and pass it through a 40-mesh sieve. Weigh 200g of Alpinia oxyphylla powder and place it in an extraction tank. Set the extraction tank temperature to 40℃, the separation pressure to 15MPa, the CO2 flow rate to 0.8L / min, the separation temperature to 45℃, and the extraction time to 4 hours. Open the material tank after the instrument has cooled to room temperature.

[0049] The extracted Alpinia oxyphylla volatile oil is a greenish-blue oil with a strong, pungent odor. After standing for a period of time, the bottom of the extract becomes a viscous gel, indicating extremely high volatility. The collected volatile oil was weighed as follows: m: 52g, m0: 8g, M: 200g. The extraction rate of Alpinia oxyphylla volatile oil was calculated to be 2.2% according to the formula.

[0050] A = (m - m0) / M (*100) (Equation 1)

[0051] In the formula: A: extraction rate of Alpinia oxyphylla volatile oil, m: mass of Alpinia oxyphylla volatile oil, m0: mass of empty centrifuge tube; M: mass of Alpinia oxyphylla powder placed in the extraction tank.

[0052] 10 μL of Alpinia oxyphylla volatile oil was dissolved in 990 μL of acetonitrile and filtered through a 0.22 μm sterile filter membrane. The filtered sample solution was then placed in an analytical vial. GC-MS analysis of the Alpinia oxyphylla volatile oil sample was performed using a Trace1310, TSQ 8000 GC-MS, and Thermo Fisher TR-5MS column with a 30 m × 0.25 mm × 0.25 μm capillary column. The programmed temperature was increased from 50 °C to 200 °C at a gradient of 3 °C / min, then increased from 200 °C to 280 °C at a rate of 203 °C / min, and then held for 10 min. Helium was used as the carrier gas at a flow rate of 1 mL / min. The split ratio was 60:1, the solvent delay was 3 min, the ionization energy was 70 eV, the ion source temperature was 230 °C, the interface temperature was 250 °C, and the mass scan range was 50–300 m / z.

[0053] GC-MS analysis revealed 84 volatile components in the Alpinia oxyphylla extracted by supercritical carbon dioxide, of which 46 contained more than 0.1% and 20 contained more than 1%. These were primarily terpenoids. The GC-MS results are shown in Table 1.

[0054] Table 1. GC-MS analysis of volatile components in Alpinia oxyphylla oil

[0055]

[0056]

[0057]

[0058] Table 1 shows that 35 components with relatively high content were analyzed in the volatile oil of Alpinia oxyphylla, accounting for 48.09% of the total components. Among them, naringin (12.59%), Valencian citrus (9.8%), gingerol (7.48%), vetiverol (6.55%), α-maleene (4.43%), and eucalyptol (3.07%) accounted for the largest proportions, and most of them were terpenoid natural products.

[0059] Example 2

[0060] This embodiment uses the Alpinia oxyphylla volatile oil extracted in Example 1 for pharmacological activity study:

[0061] 1. The protective effect of volatile oils on Caco-2 cells

[0062] (1) MTT assay for cytotoxicity

[0063] A dish of Caco-2 cells was cultured, and the total number of cells was adjusted to a density of 8 × 10⁻⁶ cells using calculations. 4 Cells were seeded at a density of 80%–90% in 96-well plates. Different concentrations of Alpinia oxyphylla volatile oil (AOE) were added for further cultivation. The final AOE concentrations in the culture medium were: 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL, 12 μg / mL, 14 μg / mL, 16 μg / mL, 18 μg / mL, and 20 μg / mL. The 96-well plates were then removed, and 10 μL of a 5 mg / mL MTT solution was added to each well. The plates were cultured for another 4 hours, then DMSO was added, and the crystal violet was allowed to dissolve completely at room temperature for 1 hour. The absorbance was measured at 490 nm using a multi-energy microplate reader, and cell viability was calculated. The results are shown below. Figure 1 .

[0064] Figure 1The results showed that cell viability decreased with increasing AOE concentration. No significant difference in cell viability was observed when AOE concentrations were between 0.5 and 2 μg / mL. However, cell viability decreased significantly when AOE concentrations exceeded 4 μg / mL. Therefore, subsequent experiments used low concentrations of 0.5 μg / mL and high concentrations of 2 μg / mL as the optimal dosages.

[0065] (2) Measurement of transepithelial electrical resistance (TER)

[0066] Establishing a cell tight junction model: After cell culture, cell counting was performed, and the cell density was adjusted to 8 × 10⁶ cells / year. 4 Cells per mL were seeded into 24-well plates. Cell culture medium was added to the inner chamber (AP layer) and the outer chamber (BL layer) of the plate. Cell growth was observed daily for one week after seeding, and the medium was changed as needed. After one week, as cell density increased, the medium was changed daily. From day 14, the medium was changed daily and the resistance of the chamber was measured until day 21. The cell model was considered successful when the resistance reached its peak and did not increase within two days. The successfully modeled cells were divided into four groups: control group (CON), drug-treated group 1 (0.5 AOE), drug-treated group 2 (1 AOE), and LPS stimulation group (LPS).

[0067] Drug treatment: After successful modeling, the cell culture medium was replaced with serum-free medium and cultured for 12 hours to ensure the cells were in the same growth cycle. The experiment was divided into four groups: Group 1 received AOE at a final concentration of 0.5 μg / mL; Group 2 received AOE at a final concentration of 2 μg / mL; the control group and LPS stimulation group received no AOE. After 12 hours of AOE pretreatment, Group 1, Group 2, and the LPS stimulation group were stimulated with 5 μg / mL LPS.

[0068] Resistance measurement: Resistance of cells with added LPS was measured starting at 0h, and subsequently at 2h, 4h, 8h, 12h, 24h, 36h, and 48h. The actual resistance value was calculated using the following formula:

[0069] TER=(RR 空白 )*A(Ω*cm 2 (Equation 2)

[0070] In the formula: R -- measured actual resistance; Rblank -- resistance of cell-free blank chamber; A -- membrane area.

[0071] See results Figure 2 . Figure 2In the table, “%” indicates P < 0.05, “%%” indicates P < 0.01, “%%%” indicates P < 0.001, and “%%%%” indicates P < 0.0001; “*” indicates P < 0.5AOE compared with LPS, “*” indicates P < 0.05, “**” indicates P < 0.01, “***” indicates P < 0.001, and “****” indicates P < 0.0001; “#” indicates P < 0.05, “##” indicates P < 0.01, “###” indicates P < 0.001, and “####” indicates P < 0.0001. Mean ± SD, n = 3.

[0072] Depend on Figure 2 It was found that, compared with the control group, LPS stimulation caused barrier damage. 0.5% AOE showed a better effect (P<0.0001), and 1% AOE also showed a better effect (P<0.0001). All groups adding AOE showed a good protective effect on the cell barrier. This indicates that LPS stimulation can disrupt the integrity of intestinal epithelial cells, and AOE can, to some extent, protect the cell barrier from damage and reduce the damage to cell barrier integrity caused by LPS.

[0073] (3) Cell permeability assay

[0074] Based on transepithelial electrical resistance measurement, the permeation rate of the fluorescent macromolecule FITC-glucan through cells was measured to examine the permeation rate of macromolecules such as bacteria at the cellular level and whether AOE has a protective effect. Cell culture and drug treatment in the transwell chamber were performed in the same manner as in step (2). After drug treatment, 100 μL of each of the inner and outer chambers of the Transwell chamber were placed in a 96-well plate, and the fluorescence absorbance inside and outside the chamber was measured. The excitation wavelength was set to 480 nm and the emission wavelength to 520 nm. The apparent permeability coefficient (Papp) of the transmembrane transport of the fluorescent substance FITC-glucan was calculated using the following formula.

[0075] Papp=ΔQ / (Δt*A*C o (cm*s) -1 (Equation 3)

[0076] Where: ΔQ -- transport amount Δt; Δt -- incubation time; A -- membrane area; C o --Initial concentration of Caco-2 cells in the intracellular compartment.

[0077] See results Figure 3 . Figure 3 In the text, "*" indicates a comparison with the LPS group, and "***" indicates P < 0.001, Mean ± SD, n = 3.

[0078] Depend on Figure 3 It can be seen that the cell permeability after LPS stimulation increased by more than double compared with the Con group, but the cells pretreated with AOE showed a significant decrease compared with the LPS group, indicating that AOE can protect the cell barrier from damage and prevent large molecules from entering the blood.

[0079] 2. Protective effect of Alpinia-enhancing volatile oil on intestinal inflammation in mice.

[0080] Animal experiments:

[0081] Healthy male C57 BL / 6 mice were randomly divided into four groups: a blank control group, a dextran sulfate sodium salt (DSS, molecular weight: 36000-50000 Da) treatment group, an AOEL group, and an AOEH group, with 10 mice in each group. After one week of acclimatization, the blank control group was given a normal diet, while the DSS, AOEL, and AOEH groups were fed normal feed with 3% DSS dissolved in their drinking water. The AOEL and AOEH groups were administered 50 mg / kg and 200 mg / kg of AOE by gavage daily, respectively, using olive oil for dissolution, for 7 consecutive days. Blood was collected from the ocular venous plexus after the feeding period. Mice were euthanized by cervical dislocation, and tissue samples were collected and stored in sampling tubes at -80°C for subsequent molecular experiments.

[0082] After collecting blood and keeping it at room temperature for about 2 hours in the dark, place it in a centrifuge and centrifuge at 3000 r / min and 4℃ for 10 minutes. After centrifugation, use a pipette to gently aspirate the clear yellowish-white supernatant into a new EP tube and store it in an ultra-low temperature freezer at -80℃ in the dark.

[0083] (1) Health check

[0084] After a week of acclimatization, starting from day 1, the weight and mortality of mice in each group were recorded, and a disease activity index score was calculated. The scoring criteria are shown in Tables 2 and 3.

[0085] Table 2 Colon Scoring Criteria

[0086]

[0087] Table 3. Clinical Disease Activity Scoring Criteria

[0088]

[0089]

[0090] See results Figures 4-5 . Figure 4 In the graph, A represents the mouse survival curve, and B represents the change in body weight. Figure 4It was observed that on the seventh day of modeling, mice in the DSS group experienced mortality (10%), while no mice in either the AOE group showed mortality. Starting on day 6, the DSS group mice experienced a sharp decline in body weight, while the AOE group showed a more gradual decline. As shown in Figure B, the DSS group mice experienced severe weight loss, while the AOE groups, although also showing weight loss, exhibited a better overall trend than the DSS group. These findings, based on survival rate and body weight, demonstrate that both AOE groups can protect against the weight loss induced by DSS stimulation in mice. Figure 5 The images show the DAI and colonic comprehensive score charts for mice. A represents the DSS group score, B represents the AOEL group score, and C represents the AOEH group score. Figure 5 It can be seen that severe rectal bleeding began to occur in the DSS group on day 5, while the situation improved in both AOE groups, with rectal bleeding and hemorrhage occurring on day 6, and fewer mice experiencing severe rectal bleeding.

[0091] The mice were dissected after the experiment, and the results are shown below. Figure 6 . Figure 6 In the diagram, A shows a comparison of anatomical images of the large intestine, and B shows statistical data on colon length. Figure 6 The results showed that in the Con group, the stool was formed and granular, the cecum was brownish-yellow, and the colon was longer. In contrast, the DSS group exhibited congestion and edema, thickened intestines, unformed stools, bloody stools in the cecum, and a shorter colon. Compared to the DSS group, both AOE groups had longer colons, less bloody stools, thinner intestinal walls, and improved congestion. These results indicate that AOE can improve DSS-induced enteritis in mice, and higher concentrations of AOE are more effective than lower concentrations.

[0092] (2) Detection of intestinal mucosal barrier integrity in mice

[0093] FITC-dextran permeability assay: Mice were fasted and dehydrated the day before sampling. The following morning, mice were administered 0.5 mg / g FITC via gavage, based on their body weight. 200 μL of gradient FITC and serum samples were added to black 96-well plates. Fluorescence absorbance was measured at excitation wavelengths of 485 nm and emission wavelengths of 528 nm. A standard curve was plotted using the absorbance of the gradient FITC. The concentration of FITC-dextran in the mouse serum samples was calculated according to the standard curve, representing the FITC permeated from the mouse intestine into the serum.

[0094] See results Figure 7 In the figure, "*" indicates a comparison with DSS, and "****" indicates P < 0.0001, Mean ± SD, n = 9. (From...) Figure 7It was found that the fluorescence value in the serum of mice in the DSS group was 7 times that of the control group, which was much higher than that of the blank control group. In contrast, the fluorescence value in the serum of both AOE groups was significantly reduced (P<0.0001), indicating that AOE has a good anti-inflammatory effect and a good protective effect on the intestinal mucosal barrier caused by enteritis in mice, thus improving the occurrence of "leaky gut".

[0095] (3) Pathological examination

[0096] To prepare mouse colon tissue sections, the process involves several steps: dehydration, clearing, paraffin embedding, sectioning, and baking. The sections are then ready for use. HE staining involves dewaxing and rehydrating the tissue sections, hematoxylin staining, eosin staining, dehydration and clearing, mounting, and then photographing at appropriate locations.

[0097] See results Figure 8 .Depend on Figure 8 It can be seen that the intestinal villi of the CON group mice were intact and the crypts were clear, with a complete structure. However, the pathological results of the DSS group mice showed that the intestinal villi of the mice no longer had a complete structure, and there was villi loss, crypt disappearance, and neutrophil infiltration. The intestinal tract of mice treated with AOE was able to retain some villi structure, and the crypts were also clearly visible. Although neutrophil infiltration was also present, it was improved compared to the DSS group mice.

[0098] 3. Research on the protective mechanism of Alpinia oxyphylla volatile oil against intestinal inflammation

[0099] RNA extraction from mouse colon tissue: RNA was extracted from mouse colon tissue using the Trizol method and reverse transcribed according to the instructions of the Trans One-Step Reverse Transcription Kit manufacturer. The concentration of cDNA was measured using an ultra-micro spectrophotometer. The cDNA was diluted to a specific concentration with RNase-free water and stored at -20°C for later use.

[0100] Primers were designed using the NCBI website, and the primer sequences are shown in Table 4:

[0101] Table 4 Primers used for qRT-PCR

[0102]

[0103]

[0104] RT-PCR reaction: The obtained cDNA was diluted. RT-PCR was performed according to the specifications outlined in the Roche Faststart Essential DNA Green Master kit instructions. GAPDH and β-actin were used as standard internal control genes, with three replicate wells for each gene sample. The reaction program was: 95℃ pre-denaturation for 10 min → 95℃ denaturation for 15 s → 60℃ extension for 60 s; this process was repeated for 40 cycles. After the PCR reaction, melting curves were analyzed using Roche LC96 general analysis software to confirm product specificity. After confirming the data validity, the Ct value was calculated, and the average value ΔCt was used to calculate the 22. -ΔΔ The Ctd values ​​were used as relative quantitative analysis data. Results are shown below. Figure 9 In the figure, A represents the mRNA expression level of IL-1β in each group, B represents the mRNA expression level of IL-6 in each group, and C represents the mRNA expression level of IL-10 in each group.

[0105] Depend on Figure 9 It was found that under normal conditions, the levels of pro-inflammatory factors in mice are relatively low. Under DSS stimulation, the levels of pro-inflammatory factors IL-1β (P<0.01) and IL-6 (P<0.05) were significantly increased. However, in the AOE group, the addition of AOE resulted in a certain degree of decrease in both pro-inflammatory factors (P<0.01)(P<0.05), exhibiting a clear concentration-dependent effect. The anti-inflammatory factor IL-10 showed a completely opposite trend. This indicates that AOE inhibits the expression of pro-inflammatory factor mRNA and promotes the expression of anti-inflammatory factor mRNA.

[0106] Western blot protein immunoblotting: PIPA and PMSF solutions were added at a ratio of 1:100. The fragmentation beads were placed in a pre-chilled cell homogenizer and homogenized until a slurry was obtained. Total protein was extracted and quantified using BCA. Loading buffer was added to the protein sample at a ratio of 1:4, and the sample was boiled at 100°C for 10 min to denature the protein. The process included gel electrophoresis, transfer to a membrane, blocking, incubation with primary and secondary antibodies, and development. Results are shown below. Figures 10-13 .

[0107] Depend on Figure 10It can be seen that the contents of tight junction-related proteins ZO-1 and Occludin in the mice of the DSS group decreased significantly. The disruption of tight junctions triggered the destruction of the paracellular barrier, and the protein content controlling this barrier decreased, resulting in the disruption of the function of controlling the entry and exit of substances. The entry of some bacteria, endotoxins, etc. into the blood may trigger inflammation and other related diseases. Both AOE groups were able to up-regulate the expression level of tight junction ZO-1 and simultaneously increase the expression level of Occludin. This shows that AOE can inhibit the occurrence of inflammation from the perspective of controlling the integrity of the intestinal barrier and can effectively inhibit the occurrence of "intestinal leakage".

[0108] The NF-κB signaling pathway is a classical signal transduction pathway for inflammatory responses. Figure 11 It can be seen that DSS can cause the activation of NF-κB in the colon tissues of mice, and the expression level of phosphorylated IκBα (P<0.001) will increase under DSS stimulation, prompting the occurrence of colitis in mice. The expression level of phosphorylated IκBα (P<0.001) in the AOE group was inhibited. The decrease in the expression level of phosphorylated IκBα can prevent the phosphorylation of P65 and thus its entry into the nucleus, which can, to a certain extent, prevent the activation of the NF-κB signaling pathway.

[0109] The key to the activation of the NF-κB signaling pathway lies in the transfer of phosphorylated p65 to the nucleus after phosphorylation. Therefore, whether p65 is phosphorylated and enters the nucleus has become the key signal for considering the activation degree of this signaling pathway. Figure 12 It can be seen that DSS can induce the expression of p65 protein in the colon tissues of mice, while the expression of p65 protein in the colon tissues of mice after treatment with AOE decreased significantly, indicating that AOE can down-regulate the expression of phosphorylated p65 protein. During the onset of IBD, the expression level of LCN-2 in the colonic epithelium increased significantly. Figure 13 It can be seen that DSS can induce the expression of NOX1 and LCN-2 proteins in the colon tissues of mice, while the expression of NOX1 and LCN-2 proteins in the colon tissues of mice after treatment with AOE decreased significantly. This shows that AOE can prevent the transduction of the NF-κB signaling pathway by down-regulating the phosphorylation and nuclear entry of p65, thereby inhibiting the occurrence of inflammation.

[0110] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. The application of Alpinia oxyphylla volatile oil extracted by supercritical carbon dioxide method in the preparation of drugs to improve inflammatory bowel disease, characterized in that, The supercritical carbon dioxide extraction steps include: drying, crushing, and sieving the Alpinia oxyphylla fruit, and then placing it in an extraction tank for extraction; the temperature of the extraction tank is 38-42℃, the separation pressure is 12-17 MPa, the CO2 flow rate is 0.6-1.0 L / min, the separation temperature is 44-46℃, and the extraction time is 3.5-4.5 h. The volatile oil containing the brain-boosting compounds includes naringin, Valencia mansene, gingerol, vetiverol, α-maleene, and eucalyptol. The inflammatory bowel disease mentioned is chronic colitis induced by sodium dextran sulfate.

2. The application according to claim 1, characterized in that, The drying conditions are: 48–52℃, 23–25h.

3. The application according to claim 1, characterized in that, The powder is then passed through a 40-mesh sieve.

4. The application according to claim 1, characterized in that, The volatile oil containing the eucalyptus oil protects the integrity of the intestinal epithelial barrier.

5. The application according to claim 1, characterized in that, The volatile oil containing the nutrient-rich substance reduces leaky gut caused by inflammatory bowel disease.

6. The application according to claim 1, characterized in that, The volatile oil from Alpinia oxyphylla inhibits p65 protein phosphorylation and the NOX1-LCN-2 molecular cascade of oxidative stress signaling in the inflammatory NF-κB signaling pathway.

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