Application of volatile oil from Pyrus truncatula fruit in preparation of anti-inflammatory drugs

Abstract

The invention discloses the application of volatile oil of Pyrus edulis fruit in the preparation of anti-inflammatory medicine and its preparation method. In the present invention, the volatile oil of Pyrus edulis fruit with anti-inflammatory effect is obtained by extracting the fruit of Pyrus edulis through steam distillation, and the volatile oil inhibits the production of pro-inflammatory mediators (NO and PGE2) and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) induced by LPS without cytotoxicity; the volatile oil inhibits the transcription of pro-inflammatory cytokine genes induced by LPS and the expression of genes and proteins that synthesize pro-inflammatory mediator enzymes (iNOS and COX-2); the volatile oil not only inhibits the nuclear transfer of NF-κB by reducing the phosphorylation and degradation of IκBα, but also inhibits the phosphorylation of MPAKs (ERK, p38 and JNK); it has a significant inhibitory effect on ear swelling induced by xylene, and has a significant anti-inflammatory effect both in vivo and in vitro.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and particularly relates to application of volatile oil of Pyrus truncatula fruit in preparing anti-inflammatory medicines and a preparation method thereof. Background Art

[0002] Chronic inflammation plays a crucial role in the development of many diseases, including cancer, arthritis, atherosclerosis, asthma, obesity, diabetes, and neurodegenerative diseases (Int. Immunopharmacol. 2019, 67, 98–105; Nature 2008, 454, 428–435; Chin. J. Nat. Med. 2017, 15(6), 0401–0416). Plant-derived natural products have become a hot topic in the development of anti-inflammatory drugs because their side effects are lower than those of the main anti-inflammatory drugs (steroidal and non-steroidal) on the market (Tradit. Med. Res. 2019, 4(5), 257–268). In particular, volatile oils can be used as natural alternatives or supplements for the treatment of inflammation (J. Agric. Food Chem. 2017, 65, 8586–8594).

[0003] Pyrus Mill. is a genus in the Rosaceae family. There are about 25 species in the genus worldwide, mainly distributed in Asia, Europe and North Africa. According to the Flora of China, there are 14 species of Pyrus in China. As an important economic crop in my country, Pyrus has the value of timber, ornamental, edible and medicinal. Pyrus plants are reported to have a variety of pharmacological activities, such as antibacterial, anti-inflammatory, antifungal, antioxidant, analgesic, etc. (Journal of Hebei University of Science and Technology, 2013, 27(03):37-42).

[0004] Pyrus calleryana Decne is a deciduous tree of the genus Pyrus in the Rosaceae family, and is native to my country. my country is rich in Pyrus plant resources, with a total of 14 species, and Pyrus calleryana Decne is one of the most widely distributed Pyrus plants. In China, Pyrus calleryana is naturally distributed mostly in the south, mainly in the Huaihe River and Yangtze River basins and in South China. Pyrus calleryana has been recorded in Shandong, Henan, Jiangsu, Zhejiang, Jiangxi, Anhui, Hubei, Hunan, Fujian, Guangdong, Guangxi, Yunnan, Guizhou and other provinces (Anhui Agricultural Sciences, 2020, 48(21):6-9).

[0005] Pear is a multi-purpose tree species. In addition to being traditionally used as a rootstock for grafting of Pyrus plants, it also has timber, ornamental, edible and medicinal value. Pear was first recorded in the "Famous Doctors' Records" and has a long history of medicinal use. It is cool in nature, sweet and slightly sour in taste, and enters the lung and stomach meridians. The fruit, peel, leaves, flowers and roots can all be used as medicine, with the effects of moistening dryness, promoting fluid production, clearing heat and resolving phlegm (Journal of Hebei University of Science and Technology, 2013, 27(03): 37-42). Pear has good medicinal value. The roots and leaves of Pear have the effects of moistening the lungs and relieving cough, clearing heat and detoxifying, and are mainly used to treat dry cough and acute conjunctivitis; the fruit has the effects of strengthening the stomach and digestion, astringing the intestines and stopping diarrhea, clearing heat and promoting fluid production; the branches can stop diarrhea, promote qi and soothe the stomach; the leaves and flowers can detoxify the dwarf sheep's grass and the dwarf ... However, there are currently no reports on the chemical composition and pharmacological activities of , which may hinder its development and utilization. Summary of the invention

[0006] The purpose of the present invention is to provide an application of volatile oil of Pyrus truncatula fruit in preparing anti-inflammatory drugs and a preparation method thereof, thereby exploring new uses of volatile oil of Pyrus truncatula fruit and providing a new choice for preparing anti-inflammatory drugs.

[0007] The present invention also found that the volatile oil of Pyrus edulis fruit inhibited the production of pro-inflammatory mediators (NO and PGE2) and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) induced by LPS; the volatile oil inhibited the transcription of pro-inflammatory cytokine genes induced by LPS and the expression of genes and proteins of pro-inflammatory mediator synthases (iNOS and COX-2); related mechanism studies showed that the volatile oil not only inhibited the nuclear translocation of NF-κB by reducing the phosphorylation and degradation of IκBα, but also inhibited the phosphorylation of MPAKs (ERK, p38 and JNK); and improved ear swelling caused by xylene. The chemical components of the volatile oil of Pyrus edulis fruit were identified.

[0008] The technical scheme adopted by the present invention is as follows: application of volatile oil of Pyrus truncatula fruit in preparing anti-inflammatory medicine.

[0009] The volatile oil of Pyrus truncatula fruit is used in the preparation of a drug for improving the excessive secretion of pro-inflammatory mediators and pro-inflammatory cytokines in cells induced by lipopolysaccharide.

[0010] The pro-inflammatory mediator is NO or PGE2; the pro-inflammatory cytokine is TNF-α, IL-6 or IL-1β.

[0011] The application of the volatile oil of Pyrus truncatula fruit in preparing drugs for inhibiting MAPK and NF-κB pathways activated by lipopolysaccharide.

[0012] The volatile oil from Pyrus truncatula fruit inhibited LPS-induced activation of MAPKs by suppressing the phosphorylation of ERK, p38 and JNK, and also inhibited LPS-induced activation of NF-κB by blocking the nuclear translocation of NF-κB and the phosphorylation and degradation of IκBα.

[0013] The invention discloses an application of the volatile oil of Pyrus truncatula fruit in preparing a medicine for improving ear swelling caused by xylene.

[0014] The volatile oil of Pyrus truncatula fruit and a pharmaceutically acceptable carrier are prepared into a medicine in the form of tablets, capsules, fat emulsions, suppositories, dropping pills or ointments.

[0015] The method for preparing volatile oil from Pyrus communis fruit comprises the following steps: crushing fresh Pyrus communis fruit, mixing the crushed raw material with distilled water at a material-liquid ratio of 1:3 to 1:8, and performing steam distillation in a volatile oil extractor for 3 to 5 hours, collecting volatile oil, and removing water with anhydrous sodium sulfate to obtain 0.11% (w / w, based on the fresh weight of the fruit) volatile oil from the fruit.

[0016] The present invention determines the chemical composition of the volatile oil of Pyrus truncatula fruit by GC-MS (see Figure 1 and Table 1), where the main chemical components are linalool (16.1%), α-terpineol (14.2%), E-nerolidol (11.5%), trans-2-hexenol (8.0%), palmitic acid (7.3%), geraniol (6.8%) and trans-3-hexenol (5.4%).

[0017] Through further anti-inflammatory related research, the present invention finds that the volatile oil of Pyrus communis fruit significantly inhibits the production of LPS-induced pro-inflammatory mediators (NO and PGE2) and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) without cytotoxic effects; inhibits the transcription of LPS-induced pro-inflammatory cytokine genes and the expression of genes and proteins that synthesize pro-inflammatory mediator enzymes (iNOS and COX-2); related mechanism studies show that the volatile oil not only inhibits the nuclear translocation of NF-κB by reducing the phosphorylation and degradation of IκBα, but also inhibits the phosphorylation of MPAKs (ERK, p38 and JNK); the volatile oil improves ear swelling caused by xylene.

[0018] By adopting the above technical scheme, the present invention has discovered for the first time a new use of the volatile oil of Pyrus truncatula fruit in treating inflammation-related diseases, providing a new option for preparing anti-inflammatory drugs, and has important application value in the pharmaceutical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the GC-MS chromatogram of the essential oil from Pyrus communis fruit;

[0020] Figure 2The cytotoxicity of the essential oil from Pyrus chinensis fruit on mouse macrophage RAW264.7 cells;

[0021] Figure 3 The volatile oil of Pyrus truncatula fruit inhibits the cell morphology, pro-inflammatory mediators (NO and PGE2) and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) of RAW264.7 cell line induced by LPS.

[0022] Figure 4 The inhibitory effect of volatile oil from Pyrus communis fruit on the mRNA expressions of IL-6 (A), TNF-α (B) and IL-1β (C) in RAW264.7 cells induced by LPS;

[0023] Figure 5 The inhibitory effect of volatile oil from Pyrus communis fruit on the gene and protein expressions of iNOS (A and C) and COX-2 (B and D) in RAW264.7 cells induced by LPS;

[0024] Figure 6 The inhibitory effect of the volatile oil from Pyrus edulis fruit on the phosphorylation of MPAKs (p38, ERK and JNK) induced by LPS;

[0025] Figure 7 The inhibitory effect of the volatile oil from Pyrus edulis fruit on the activity of NF-κB induced by LPS;

[0026] Figure 8 The inhibitory effect of volatile oil from Pyrus chinensis fruit on LPS-induced nuclear translocation of NF-κB p65;

[0027] Fig. 9 Effects of essential oil from Pyrus communis fruit on xylene-induced ear swelling in mice. DETAILED DESCRIPTION

[0028] An embodiment of the present invention: fresh Pear fruit (harvested at Shansen Reservoir, Yudu County, Ganzhou City, Jiangxi Province; identified by Professor Hu Guoxiong of Guizhou University) is crushed, the crushed raw material is mixed with distilled water at a material-liquid ratio of 1:5, the mixed material is steam distilled in a volatile oil extractor for 4 hours, the volatile oil is collected, and water is removed by anhydrous sodium sulfate to obtain the fruit volatile oil, which is sealed and stored in a refrigerator at 4°C.

[0029] Quantitative analysis of the essential oil of Pyrus truncatula was performed using an Agilent 6890 gas chromatograph (GC) equipped with a flame ionization detector (FID) and an HP-5MS column (60 mMS chromatograph equipped with a flame 0.25 μm) (Agilent Technologies, CA, USA). The essential oil (1 μL) was injected in splitless mode with helium as the carrier gas (1 mL / min). The column temperature was as follows: maintained at 80 °C (2 min), increased to 230 °C (50 min) at 3 °C / min, and heated to 265 °C (7 min) at 5 °C / min. The qualitative analysis of the essential oil was performed on an Agilent 6890 / 5975C gas chromatograph-mass spectrometer (GC-MS) with the same GC parameters as those set in the GC-FID analysis. The operating parameters of MS were as follows: ion source (EI source), ion source temperature of 230°C, quadrupole temperature of 150°C, electron energy of 70 eV, emission current of 34.6 μA, multiplier voltage of 1847 V, interface temperature of 280°C, mass range of 29-500 amu. The relative abundance (%) of each chemical component was determined by peak area normalization method. The retention index (RI) was calculated by using the normalized values ​​of n-alkanes (C8-C 22 The composition of volatile oil from Pyrus edulis was determined by comparing mass spectra and retention indices in the NIST2020 and Wiley 275 databases.

[0030] Chemical components of volatile oil from Pyrus chinensis fruit Figure 1 As shown in Table 1, 48 compounds were identified, accounting for 95.7% of the total components (Table 1, Figure 1 The main components of the volatile oil of Pyrus truncatula are linalool (16.1%), α-terpineol (14.2%), E-nerolidol (11.5%), trans-2-hexenol (8.0%), palmitic acid (7.3%), geraniol (6.8%) and trans-3-hexenol (5.4%).

[0031] Table 1 Chemical composition

[0032]

[0033]

[0034] a Compounds: Arranged in order of elution from the HP-5MS column.

[0035] b The database RI (Retention Index) comes from the NIST2017 and Wiley 275 databases.

[0036] c Calculation of RI (Retention Index) was based on n-alkanes (C8-C22) on the HP-5MS column.

[0037] d Identification: MS, mass spectrum similarity matching with Wiley275 and NIST2020 databases; RI, comparison of calculated RI with that in the NIST2020 database.

[0038] Pharmacological Example 1: Toxicity of Pyrus communis Fruit Volatile Oil to RAW264.7 Cells

[0039] RAW264.7 cells were cultured in DMEM medium containing penicillin (100 U / mL), streptomycin (100 μg / mL), fetal bovine serum (10%) and glutamine (2 mM). The cytotoxic effect of essential oils on RAW264.7 cell line was evaluated by MTT assay. Essential oils dissolved in DMSO were serially diluted half-fold with culture medium (maximum final concentration of DMSO was 0.05%). Cells (2×10 4 After culturing for 24 h, diluted volatile oil solution was added to a final concentration of 0, 15.63, 31.25, 62.5, 125, 250 and 500 μg / mL and cultured for 24 h. MTT solution (10 μL, 5 mg / mL in PBS) was added and incubated for 4 h, the supernatant was removed, and 150 μL DMSO was added to dissolve the formazan crystals. The optical density at 490 nm was measured by Varioskan Lux microplate reader (ThermoFisher Scientific, USA).

[0040] like Figure 2 As shown, compared with the control, the essential oil (EO) in the range of 15.63-250 μg / mL of EO from Pyrus edulis fruit did not show significant cytotoxicity to RAW264.7 cells (p>0.05). Therefore, the EO concentrations (31.25, 62.5, and 125 μg / mL) that were not cytotoxic to RAW264.7 cells were selected for subsequent testing.

[0041] Pharmacological Example 2: Pyrus truncatula fruit volatile oil inhibits LPS-induced morphological changes and the release of pro-inflammatory mediators and cytokines in RAW264.7 cells

[0042] RAW264.7 cells were seeded in 96-well plates (2×10 4 / per well) and cultured for 24h. After pretreatment with fresh culture medium containing different doses (31.25, 62.5 and 125μg / mL) of volatile oil for 2h, lipopolysaccharide (LPS, 1μg / mL) was added and incubated for another 24h. Changes in RAW264.7 cell morphology were recorded using a LeicaDMi8 inverted microscope (Leica Microsystems, Germany). Dexamethasone (DXM, 20μg / mL) was used as a positive control drug. After collecting the cell supernatant, NO secreted by RAW264.7 cells was measured according to the instructions in the NO detection kit (Beyotime, Shanghai, China). In addition, the levels of PGE2, IL-1β, IL-6 and TNF-α were assessed using their respective ELISA kits according to the manufacturer's instructions.

[0043] like Figure 3 As shown in A, the cells in the control group were round and smooth. The RAW264.7 cells treated with LPS became larger and irregular in shape, while the cell morphology of the volatile oil treatment group changed little. Figure 3 As shown in BF, LPS induction alone significantly increased the levels of NO, PGE2, TNF-α, IL-1β, and IL-6 compared with the control group. When the cells were pretreated with volatile oil (31.25, 62.5, 125 μg / mL), the release of NO, PGE2, TNF-α, IL-1β, and IL-6 was significantly inhibited compared with the LPS group (p<0.05). In particular, the inhibitory effect of volatile oil at a dose of 125 μg / mL on PGE2 and TNF-α exceeded or was equivalent to DXM (20 μg / mL). The above results indicate that the volatile oil of Pyrus truncatula fruit inhibited the release of proinflammatory mediators and cytokines in LPS-induced RAW264.7 cells in a dose-dependent manner.

[0044] Pharmacological Example 3: Pyrus serrata fruit volatile oil inhibits LPS-induced TNF-α, IL-6 and IL-1β gene expression

[0045] RAW264.7 cells were seeded in 6-well plates (6×10 5 The cells were then pretreated with different doses (31.25, 62.5 and 125 μg / mL) of volatile oil for 2 h and then treated with LPS (1 μg / mL) for 24 h. Total RNA was extracted from cells using Total RNA KitI (Omega Bio-Tek, Norcross, GA, USA), and then analyzed by RT Easy TMII kit (FOREGENE, Chengdu, China) was used to reverse transcribe into single-stranded cDNA. Real Time PCReasyTM-SYBR Green I kit (FOREGENE, Chengdu, China) was used to reverse transcribe into single-stranded cDNA. TM The qRT-PCR reaction was performed using a Real-Time System (Bio-Rad, CA, USA). The primer sequences are as follows: TNF-α (forward, 5′-GCA AAG GGGAG TGG TCA-3′; reverse, 5′-CTG GCT CTG TGA GGA AGG-3′), IL-1β (forward, 5′-CCTGTG TCT TTC CCG TGG AC-3′; reverse, 5′-CAT CTC GGA GCC TGT AGT GC-3′), IL-6 (forward, 5′-ATT TCC TCT GGT CTT CTG G-3′; reverse, 5′-GAC TCT GGC TTT GTC TTTC-3′), iNOS (forward, 5′-TCG GGT TGA AGT GGT ATG C-3′; reverse, 5′-GAG GCC AGT GTGTGG GTC T-3′), COX-2 (forward, 5′-TGA CTG CCC AAC TCC CAT-3′;reverse,5′-GAA CCCAGG TCC TCG CTT-3′), and GAPDH (forward, 5′-GGT GAA GGT CGG TGT GAA CG-3′; reverse, 5′-CTC GCT CCT GGA AGA TGG TG-3′). qRT-PCR results were analyzed using Bio-Rad CFXMaestro1.0 software, and GAPDH was used as a reference gene.

[0046] To determine whether volatile oils attenuate cytokine production by modulating the transcriptional levels of genes, the mRNA levels of IL-6, TNF-α, and IL-1β were analyzed using qRT-PCR. Figure 4 As shown in AC, after induction with LPS alone, the mRNA expression levels of IL-6, TNF-α, and IL-1β were significantly increased compared with the control group. However, the volatile oil significantly inhibited the mRNA expression levels of TNF-α, IL-6, and IL-1β induced by LPS in a dose-dependent manner. The above data indicate that the volatile oil of Pyrus sutchuenensis fruit reduces the secretion of TNF-α, IL-6, and IL-1β by inhibiting the expression of LPS-induced genes.

[0047] Pharmacological Example 4: Pyrus chinensis fruit volatile oil inhibits LPS-induced iNOS and COX-2 protein and gene expression

[0048] RAW264.7 cells were seeded in 6-well plates (6×10 5 After culturing for 24 hours with 5% skim milk, the cells were incubated with a primary antibody at 4 °C overnight, washed 3 times with TBST, and then exposed to a HRP-labeled secondary antibody at room temperature for 1 hour. Imaged by ChemiDoctouch imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Imaged by Image Lab 6.0 (Bio-Rad, CA, USA) for analyzing the intensity of protein bands.

[0049] Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are key enzymes for the synthesis of NO and PGE2, respectively (J. Ethnopharmacol. 2013, 147, 208–214). In order to investigate whether volatile oils attenuate the release of proinflammatory mediators by regulating the expression of enzymes that catalyze their synthesis, the protein and mRNA expression levels of iNOS and COX-2 were detected by Western blotting and qRT-PCR, respectively. Figure 5 As shown in A and C, LPS stimulation alone significantly promoted the protein and mRNA expressions of iNOS compared with the control group. However, after pretreatment with volatile oil, the protein and mRNA levels decreased significantly in a dose-dependent manner compared with LPS treatment alone. Figure 5 As shown in B and D, the expression levels of COX-2 protein and mRNA increased significantly after LPS induction, and the volatile oil dose-dependently inhibited the levels of COX-2 protein and mRNA in RAW264.7 cells induced by LPS. The results showed that the volatile oil of Pyrus truncatula fruit significantly inhibited the release of NO and PGE2 by inhibiting the expression of iNOS and COX-2 at the transcriptional and translational levels.

[0050] Pharmacological Example 5: Pyrus communis fruit volatile oil inhibits the phosphorylation level of MAPKs induced by LPS

[0051] LPS stimulates phosphorylation and activation of mitogen-activated protein kinases (MAPKs), including c-Jun N-terminalkinase (JNK), p38, and extracellular signal-regulated kinase (ERK), which subsequently regulates the expression of pro-inflammatory genes (TNF-α, IL-6, and IL-1β) by affecting the activation of AP-1 transcription factors (Food Chem. Toxicol. 2021, 147, 111915). Therefore, the protein levels of p38, p-p38, ERK, p-ERK, JNK, and p-JNK were measured using Western blotting ( Figure 6 ). After stimulation with LPS alone, the phosphorylation levels of p38, ERK and JNK increased significantly compared with the control group. The volatile oil significantly inhibited the phosphorylation levels of p38, ERK and JNK induced by LPS in a dose-dependent manner. The above results indicate that the volatile oil of Pyrus truncatula fruit can effectively inhibit the phosphorylation of MAPKs (p38, ERK and JNK) induced by LPS, thereby inhibiting the activation of the MAPK pathway.

[0052] Pharmacological Example 6: Volatile oil from Pyrus edulis fruit inhibits LPS-induced NF-κB activity

[0053] NF-κB is a transcription factor that regulates the transcription of genes for proinflammatory cytokines (TNF-α, IL-6, and IL-1β) and proinflammatory enzymes (iNOS and COX-2) (Oncogene, 1999, 18, 6853–6866). Under normal circumstances, NF-κB is a heterodimer composed of p50 and p65 subunits, which exists in the cytoplasm in an inactive form in combination with the κB inhibitor (IκB). After LPS stimulation, IκB is phosphorylated and rapidly degraded, and then the subunits of NF-κB can freely translocate to the nucleus and activate the transcription of proinflammatory genes (Nat. Immunol. 2002, 3(1), 20–26). Therefore, Western blotting was used to evaluate the effects of volatile oils on LPS-induced nuclear translocation of NF-κB and phosphorylation and degradation of IκBα. Figure 7 As shown in A and B, after LPS induction, the phosphorylation and degradation levels of IκBα were significantly increased; however, volatile oil significantly inhibited LPS-induced IκBα phosphorylation and degradation in a concentration-dependent manner. Figure 7As shown in C and D, after induction with LPS alone, the level of NF-κB p65 subunit in the nucleus increased significantly, while its level in the cytoplasm decreased significantly, indicating that after LPS induction, NF-κB p65 translocated to the nucleus. However, the volatile oil at high doses of 62.5 μg / mL and 125 μg / mL significantly upregulated the level of NF-κB p65 in the cytoplasm, while dose-dependently downregulated the level of NF-κB p65 in the nucleus, indicating that the volatile oil inhibited the nuclear translocation of p65 induced by LPS. Pharmacological Example 7: Volatile oil from Pyrus chinensis fruit inhibits the nuclear translocation of NF-κB p65 induced by LPS

[0054] RAW264.7 cells (6×10 5 Cells were seeded into 6-well plates with coverslips (100 μg / mL) and incubated for 24 h. Subsequently, volatile oil (125 μg / mL) was pretreated for 2 h, and then LPS (1 μg / mL) was treated for 24 h. After washing 3 times with PBS, the cells were fixed with 4% paraformaldehyde solution for 15 min and permeabilized with 0.3% Triton X-100 for 5 min. Then, the cells were blocked in 5% BSA for 1 h, washed 3 times with PBS, and incubated with the primary antibody NF-κB p65 at 4 °C overnight. After washing 3 times with PBS, AlexaFluor 488-labeled secondary antibody was added and incubated for 1 h. After cells were stained with DAPI for 5 min, they were observed by Leica TCS SP8 laser confocal scanning microscope (Leica Microsystems, Germany).

[0055] like Figure 8 As shown, NF-κB p65 (green) in untreated RAW264.7 cells appeared in the cytoplasm; however, after LPS induction, the green fluorescence was mainly concentrated in the nucleus. After treatment with volatile oil (125 μg / mL), the green fluorescence was mainly distributed in the cytoplasm, indicating that the volatile oil of Pyrus truncatula fruit inhibited the nuclear translocation of NF-κB p65 induced by LPS.

[0056] Pharmacological Example 8: Effect of Pyrus truncatula Fruit Volatile Oil on Xylene-Induced Ear Swelling (In Vivo Anti-Inflammatory Experiment)

[0057] Inflammatory mice were modeled by the ear swelling method. A total of 60 Kunming mice were randomly divided into 6 groups, half male and half female, with 10 mice in each group; a blank control group, a model group, a dexamethasone group (1g / kg), and a low-dose group (1%, v / v), a medium-dose group (5%, v / v), and a high-dose group (10%, v / v) of the volatile oil (Essential oil, EO) of Pear fruit were established respectively. The samples were diluted with olive oil and prepared as needed. The left ears of mice in the low-dose group, the medium-dose group, and the high-dose group were smeared with each concentration group once a day (50μL) for three consecutive days, and the right ears were smeared with the same amount of olive oil as a normal control. The blank group was not treated as a negative control. The dexamethasone group was smeared with compound dexamethasone acetate cream, which was evenly applied to the left ear until it was completely absorbed. On the third day, 20 minutes after the corresponding administration of each dose group and dexamethasone group, 50 μL of xylene was evenly applied to the left ears of mice in the model group and each dose group, and the time was 15 minutes. Then, a 6mm ear piece was punched in the same position of the mouse ear with a 6mm puncher, and its weight was immediately weighed with an analytical balance. The difference in weight between the left and right ear pieces of each mouse was used as the swelling degree, and the swelling rate and inhibition rate were calculated by the weight of the ear piece.

[0058] Swelling degree (mg) = left ear weight - right ear weight

[0059] Ear swelling inhibition rate (%) = (left ear weight - right ear weight) / right ear weight × 100%

[0060] As shown in Table 2 and Fig. 9 As shown, the volatile oil of Pyrus edulis fruit has a dose-dependent inhibitory effect on xylene-induced ear swelling in mice. Compared with the model group (swelling degree: 17.85±1.07mg), the inhibitory effect of the dexamethasone group (DXM, swelling degree: 6.93±1.11mg) was significant (p<0.001), and the high (swelling degree: 5.35±0.87mg), medium (swelling degree: 7.46±0.42mg), and low (swelling degree: 8.53±0.69mg) dose groups were all significant (p<0.001). In summary, the volatile oil of Pyrus edulis fruit has a significant inhibitory effect on xylene-induced ear swelling and has a significant anti-inflammatory effect in vivo.

[0061] Table 2 Inhibitory effect of volatile oil from Pyrus communis fruit on xylene-induced ear swelling (Mean±SD, n=10)

[0062]

[0063]

[0064] Note: Model group vs blank group ( ### p<0.001); drug group vs model group ( *** p<0.001).

[0065] Based on the above pharmacological examples, the volatile oil of Pyrus truncatula fruit inhibited the production of LPS-induced proinflammatory mediators (NO and PGE2) and proinflammatory cytokines (TNF-α, IL-6 and IL-1β) at non-toxic concentrations; the volatile oil inhibited the transcription of LPS-induced proinflammatory cytokine genes and the expression of genes and proteins that synthesized proinflammatory mediator enzymes (iNOS and COX-2); related mechanism studies showed that the volatile oil not only inhibited the nuclear translocation of NF-κB by reducing the phosphorylation and degradation of IκBα, but also inhibited the phosphorylation of MPAKs (ERK, p38 and JNK); it improved the ear swelling caused by xylene; and it had significant anti-inflammatory effects both in vivo and in vitro.

Claims

1. Application of volatile oil from Pyrus truncatula fruit in the preparation of anti-inflammatory drugs.

2. The use according to claim 1, characterized in that: The volatile oil of Pyrus truncatula fruit is used in the preparation of a drug for improving the excessive secretion of pro-inflammatory mediators and pro-inflammatory cytokines in cells induced by lipopolysaccharide.

3. The use according to claim 2, characterized in that: The pro-inflammatory mediator is NO or PGE2; the pro-inflammatory cytokine is TNF-α, IL-6 or IL-1β.

4. The use according to claim 1, characterized in that: The application of the volatile oil of Pyrus truncatula fruit in preparing drugs for inhibiting MAPK and NF-κB pathways activated by lipopolysaccharide.

5. The use according to claim 4, characterized in that: The volatile oil from Pyrus chinensis fruit inhibited LPS-induced activation of MAPKs by suppressing the phosphorylation of ERK, p38 and JNK, and also inhibited LPS-induced activation of NF-κB by blocking the nuclear translocation of NF-κB and the phosphorylation and degradation of IκBα.

6. The use according to claim 1, characterized in that: The invention discloses an application of the volatile oil of Pyrus truncatula fruit in preparing a medicine for improving ear swelling caused by xylene.

7. The use according to any one of claims 1 to 6, characterized in that: The volatile oil of Pyrus truncatula fruit and a pharmaceutically acceptable carrier are prepared into a medicine in the form of tablets, capsules, fat emulsions, suppositories, dropping pills or ointments.

Images