CN114224987B - Application of flower volatile oil of Curcuma rhizome flower in preparing anti-inflammatory medicine - Google Patents
Application of flower volatile oil of Curcuma rhizome flower in preparing anti-inflammatory medicine Download PDFInfo
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- CN114224987B CN114224987B CN202210008138.0A CN202210008138A CN114224987B CN 114224987 B CN114224987 B CN 114224987B CN 202210008138 A CN202210008138 A CN 202210008138A CN 114224987 B CN114224987 B CN 114224987B
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Abstract
The invention discloses application of a flower volatile oil of turmeric flower in preparing an anti-inflammatory medicament. In the invention, the anti-inflammatory flower volatile oil of the turmeric flower is extracted from the flower of the turmeric flower by steam distillation, and the volatile oil inhibits proinflammatory mediators (NO and PGE) induced by LPS under the condition of NO cytotoxicity 2 ) And the production of pro-inflammatory cytokines (TNF- α, IL-6, and IL-1 β); the volatile oil inhibits the transcription of proinflammatory cytokine genes induced by LPS and the expression of genes and proteins for synthesizing proinflammatory mediator enzymes (iNOS and COX-2); the volatile oil can inhibit nuclear transfer of NF-kB by reducing phosphorylation and degradation of I kB alpha, and also inhibit phosphorylation of MPAKs (ERK, p38 and JNK); has obvious inhibiting effect on ear swelling induced by dimethylbenzene, and has obvious anti-inflammatory effect both in vivo and in vitro.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to application of a flower volatile oil of turmeric flower in preparation of an anti-inflammatory drug.
Background
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, chi.j.nat. Med.2017,15 (6), 0401-0416. Since natural products derived from plants have side effects lower than those of main anti-inflammatory drugs (steroids and non-steroids) on the market, natural products derived from plants have become a hot spot for the development of anti-inflammatory drugs (tradit. Med. Res.2019,4 (5), 257-268). In particular, the Essential Oils may be used as natural substitutes or supplements for the treatment of inflammation (Buchbauer, G., bohusch, R.,2010.Biological activities of Essential Oils.
There are about 93 plants of the genus zingiber (Hedychium) of the family zingiberaceae, which are mainly distributed in tropical and warm temperate regions of china, india and south-east asia (Prakash, o., chandra, m., pnentha, h., pant, a.k., rawat, d.s.,2016.Chapter 84-split girer Lily (Hedychium spp.). Oil. In: preference, v. (Ed), essential oil in Food Preservation, flavour and safety. London: academic Press, pp.737-750). Especially volatile oils from plants of the genus zingiber have great development potential and have been used in high-grade perfumes and national medicines (Procedia chem.2014, 13. Volatile oils from plants of the genus zingiber are reported to possess a variety of pharmacological activities, such as antibacterial, anti-inflammatory, antifungal, antioxidant, analgesic, cytotoxic, anticholinesterase, insect repellent and insecticidal properties (ind. Crop. Prod.2018,126,135-142, ind. Crops prod.2018,112, 353-362.
Yellow ginger flower (Hedychium flavum roxb.) is an ornamental, edible and medicinal plant that is widely cultivated in China, thailand, india and burma for its aromatic volatile oils (front. Pharmacol.2020,11,572659 wu, t.l., larsen, k.,2000.Zingiberaceae. In. Underground rhizomes of flowers of turmeric are used as food seasonings and in traditional Chinese medicine for treating bruises, rheumatism, headache, cold, cough and abdominal pain (Chinese medicinal materials, journal of Chinese materia medica resources, 1994, beijing: science publishers, p.1511; wang Yi; chinese Natural medicine color atlas, 2010, guiyang: guizhou publishing group Co., ltd. And Guizhou science publishers, p.200). Its young shoots have been used as food flavoring agents and vegetables (j.environ.sci.toxicol.food technol.2014,8 (2), 21-23). In addition, flowers of litsea cubeba are used as spices and in ethnic medicine for the treatment of dyspepsia, diarrhea and stomachache (Ai Tiemin, "journal of Chinese medicinal plant (Vol. 12)," 2013, "Beijing: beijing university medical Press, pp.381-382 Wu, T.L., larsen, K.,2000. Zingineeae. In. In particular, the flower volatile oil is used in cosmetics and as aromatic stomachic (Ai Tiemin, "Chinese medicinal plant (vol. 12)", 2013, beijing: beijing university medical publisher, pp.381-382; chinese medicinal materials Co., china Chinese medicinal materials journal of resource, 1994, beijing: scientific publisher, p.1511). However, there is no report on the chemical composition and pharmacological activity of the floral volatile oil of turmeric flower, which may hinder the development and utilization thereof.
Disclosure of Invention
The purpose of the invention is as follows: provides the application of the flower volatile oil of the turmeric flower in preparing anti-inflammatory drugs, develops the new application of the flower volatile oil of the turmeric flower and provides a new choice for preparing anti-inflammatory drugs.
The invention also discovers that the flower volatile oil of the turmeric flower inhibits proinflammatory mediators (NO and PGE) induced by LPS 2 ) And the production of pro-inflammatory cytokines (TNF- α, IL-6, and IL-1 β); the volatile oil inhibits the transcription of proinflammatory cytokine genes induced by LPS and the expression of genes and proteins for synthesizing proinflammatory mediator enzymes (iNOS and COX-2); relevant mechanism researches show that the volatile oil not only inhibits nuclear transfer of NF-kB by reducing phosphorylation and degradation of I kB alpha, but also inhibits phosphorylation of MPAKs (ERK, p38 and JNK); improved ear caused by xyleneSwelling was observed. And the chemical components of the volatile oil of the turmeric flower are identified.
The technical scheme adopted by the invention is as follows:
the flower volatile oil of the turmeric flower is prepared by the following method:
crushing fresh turmeric flower pollen, mixing the crushed raw materials with distilled water according to the material-liquid ratio of 1:2-1:8, carrying out steam distillation on the mixed materials in a volatile oil extractor for 3-5 h, collecting volatile oil, and removing water by anhydrous sodium sulfate to obtain 0.31% (w/w, based on the fresh weight of the flower) volatile oil.
The invention determines the chemical components of the volatile oil of the flower of the litsea cubeba by GC-MS (see the detailed chart 1 and the table 1), wherein the main chemical components are beta-Pinene (beta-Pinene, 20.2%), alpha-Pinene (alpha-Pinene, 9.3%), alpha-Phellandrene (alpha-Phellandrene, 8.3%), 1,8-cineol (1,8-Cineole, 7.1%), E-Nerolidol (E-Nerolidol, 5.4%), limonene (limone, 4.4%), borneol (Borneol, 4.1%) and beta-Caryophyllene (beta-Caryophyllene, 3.7%).
The invention discovers that the flower volatile oil of the turmeric flower can obviously inhibit proinflammatory mediators (NO and PGE) induced by LPS (lipopolysaccharide) under the non-cytotoxic effect through experimental research 2 ) And the production of pro-inflammatory cytokines (TNF- α, IL-6, and IL-1 β); inhibits the transcription of proinflammatory cytokine genes induced by LPS and the expression of genes and proteins for synthesizing proinflammatory mediator enzymes (iNOS and COX-2); relevant mechanism researches show that the volatile oil not only inhibits nuclear transfer of NF-kB by reducing phosphorylation and degradation of I kB alpha, but also inhibits phosphorylation of MPAKs (ERK, p38 and JNK); ear swelling caused by xylene was improved.
By adopting the technical scheme, the invention discovers the new application of the flower volatile oil of the turmeric flower in treating inflammation-related diseases for the first time, provides a new choice for preparing anti-inflammatory drugs, and has important application value in the pharmaceutical industry.
Drawings
FIG. 1 is a GC-MS chromatogram of a flower volatile oil of Curcuma rhizome;
FIG. 2 shows cytotoxicity of the flower volatile oil of Curcuma rhizome flower on mouse macrophage RAW 264.7;
FIG. 3 shows the cell morphology of LPS-induced RAW264.7 cell line and pro-inflammatory mediators (NO and PGE) by floral volatile oil of Curcuma rhizome 2 ) And the inhibitory effects of proinflammatory cytokines (TNF-alpha, IL-6 and IL-1 beta);
FIG. 4 shows the inhibitory effect of floral volatile oil of Curcuma longa on mRNA expression of TNF- α (A), IL-6 (B) and IL-1 β (C) in RAW264.7 cells induced by LPS;
FIG. 5 shows the inhibitory effect of the flower volatile oil of Curcuma xanthorrhiza on the expression of iNOS (A and C) and COX-2 (B and D) genes and proteins in RAW264.7 cells induced by LPS;
FIG. 6 shows inhibition of LPS-induced phosphorylation of MPAKs (p 38, ERK and JNK) by floral volatile oil of Curcuma longa;
FIG. 7 shows the inhibitory effect of floral volatile oil of Curcuma longa on LPS-induced NF- κ B activity;
FIG. 8 shows the inhibitory effect of the floral volatile oil of Curcuma xanthorrhiza on nuclear transfer NF- κ B p induced by LPS;
FIG. 9 shows the effect of floral volatile oil from Curcuma xanthorrhiza on the degree of swelling of mouse ears induced by xylene.
Detailed Description
The embodiment of the invention comprises the following steps: pulverizing fresh Curcuma rhizome flower (collected in Nayong county of Bijie city, guizhou province; identified by professor Hu Guoxiong of Guizhou university), mixing the pulverized raw materials with distilled water according to the material-liquid ratio of 1:5, subjecting the mixed material to steam distillation in a volatile oil extractor for 4h, collecting volatile oil, removing water with anhydrous sodium sulfate to obtain the volatile oil of the flower, and sealing and storing in a refrigerator at 4 ℃.
The chemical components of the flower volatile oil of the turmeric flower of the embodiment are as follows: chemical composition was quantified by Agilent 6890 gas chromatograph and flame ionization detector (GC-FID). GC-FID analysis conditions: the sample introduction amount is 1 μ L, the chromatographic column is HP-5MS (60 m × 0.25mm × 0.25 μm) elastic quartz capillary column, the initial temperature is 70 deg.C (reserved for 2 min), the temperature is increased to 180 deg.C (55 min) at 2 deg.C/min, and then the temperature is increased to 260 deg.C (8 min) at 10 deg.C/min; the temperature of the vaporization chamber is 250 ℃; the carrier gas is high-purity He (99.999%); the column front pressure is 18.53psi, the carrier gas flow is 1.0mL/min, the split ratio is 20:1, solvent delay time: 6.0min. The chemical composition of the flower volatile oil was identified by Agilent 6890/5975C gas chromatography-mass spectrometer (GC-MS). The GC-MS gas chromatography conditions were the same as for GC-FID. The setting of the MS is as follows: the ion source is an EI source; the ion source temperature is 230 ℃; the temperature of the quadrupole rods is 150 ℃; electron energy 70eV; emission current 34.6 μ A; multiplier voltage 1847V; the interface temperature is 280 ℃; the mass range is 29 to 500amu. Calculating a retention index (RI value) by normal alkane (C8-C30); the chemical components of the volatile oils were determined by comparing the RI and mass spectra in the NIST2020 and Wiley275 databases, and the relative mass fractions of each chemical component were determined by peak area normalization.
The chemical components of the flower volatile oil of the litsea cubeba are shown in figure 1 and table 1, 55 chemical components are identified in total, and account for 97.9 percent of the total peak area, wherein the main chemical components are beta-Pinene (beta-Pinene, 20.2 percent), alpha-Pinene (alpha-Pinene, 9.3 percent), alpha-Phellandrene (alpha-Phellandrene, 8.3 percent), 1,8-cineol (1,8-Cineole, 7.1 percent), E-Nerolidol (E-Nerolidol, 5.4 percent), limonene (limone, 4.4 percent), borneol (Borneol, 4.1 percent) and beta-caryophylene (beta-Caryophyllene, 3.7 percent).
TABLE 1 chemical composition of floral volatile oil of turmeric flower
a A compound: in order of elution from the HP-5MS column.
b Database RI (retention index) is from NIST 2017 and Wiley275 of the database.
c RI (retention index) was calculated based on n-alkanes (C8-C22) on an HP-5MS column.
d And (3) identification: MS (Mass Spectrometry)Matching mass spectrum similarity to Wiley275 and NIST2020 databases; RI, calculated RI, was compared to Wiley275 and NIST2020 databases.
e tr：trace(trace<0.1％)。
Pharmacological example 1: toxicity of flower volatile oil of Curcuma rhizome flower on RAW264.7 cell
RAW264.7 cells were cultured in DMEM medium containing penicillin (100U/mL), streptomycin (100. Mu.g/mL), fetal bovine serum (10%) and glutamine (2 mM). The cytotoxic effect of the essential oils on the RAW264.7 cell line was assessed by MTT assay. Essential oils dissolved in DMSO were diluted half-fold serially with medium (maximum final DMSO concentration 0.05%). Cells in 96-well plates (2X 10) 4 One cell/well) for 24h, adding the diluted volatile oil solution to final concentrations of 0, 3.91, 7.81, 15.63, 31.25, 62.5 and 125 μ g/mL, and culturing for 24h. MTT solution (10. Mu.L, 5mg/mL in PBS) was added, after incubation for 4h, the supernatant was removed and 150. Mu.L of DMSO was added to dissolve the formazan crystals. The optical density at 490nm was measured by a Varioskan Lux microplate reader (Thermo Fisher Scientific, USA).
As shown in FIG. 2, the volatile oil (EO) at a concentration ranging from 3.91 to 31.25. Mu.g/mL did not show significant cytotoxicity (p > 0.05) to RAW264.7 cells as compared to the control. Therefore, volatile oil concentrations that were not cytotoxic to RAW264.7 cells (7.81, 15.63, and 31.25 μ g/mL) were selected for subsequent testing.
Pharmacological example 2: the flower volatile oil of Curcuma rhizome flower inhibits LPS-induced morphological change of RAW264.7 cell and release of proinflammatory mediators and cytokines
RAW264.7 cells were seeded in 96-well plates (2X 10) 4 One/each well) for 24h. After 2h of pre-treatment with fresh medium containing different doses (0, 7.81, 15.63 and 31.25. Mu.g/mL) of the volatile oil, lipopolysaccharide (LPS, 1. Mu.g/mL) was added and incubation continued for 24h. Changes in the morphology of RAW264.7 cells were recorded using a Leica DMi8 inverted microscope (Leica Microsystems, germany). Dexamethasone (DXM, 20. Mu.g/mL) was used as a positive control. After collection of cell supernatants, NO secreted by RAW264.7 cells was measured according to the instructions in the NO detection kit (Beyotime, shanghai, china). In addition, rootPGE was assessed using the respective ELISA kits according to the manufacturer's instructions 2 IL-1 beta, IL-6 and TNF-alpha.
As shown in FIG. 3A, the cells of the control group were rounded and smooth in surface. RAW264.7 cells treated with LPS were large in size and irregular in shape, while the volatile oil treated group showed less morphological changes. As shown in FIGS. 3B-F, LPS induction alone significantly increased NO, PGE, compared to the control group 2 TNF-alpha, IL-1 beta and IL-6 levels. When cells were pretreated with volatile oil (7.81, 15.63, 31.25. Mu.g/mL), NO, PGE compared to LPS group 2 TNF-alpha, IL-1 beta and IL-6 release were significantly inhibited (p)<0.05). Particularly, the inhibition effect of the volatile oil with the dose of 15.63 mu g/mL on NO, TNF-alpha and IL-1 beta is more than or equal to that of DXM (20 mu g/mL), which indicates that the inhibition effect of the volatile oil on NO, TNF-alpha and IL-1 beta is stronger than that of DXM. The above results indicate that the volatile oil inhibits LPS-induced release of pro-inflammatory mediators and cytokines in RAW264.7 cells in a dose-dependent manner.
Pharmacological example 3: the flower volatile oil of Curcuma rhizome flower inhibits the expression of TNF-alpha, IL-6 and IL-1 beta genes induced by LPS
RAW264.7 cells were seeded in 6-well plates (6X 10) 5 One/each well) for 24h. Subsequently, cells were pretreated with different doses (0, 7.81, 15.63 and 31.25. Mu.g/mL) of the essential oil for 2h, followed by LPS (1. Mu.g/mL) for 24h. E.z.n.a was used.
RNA Kit I (Omega Bio-Tek, norcross, GA, USA) Total RNA from cells was extracted and then according to RT Easy TM II kit (FOREGENE, chengdu, china) was reverse-transcribed into single-stranded cDNA. Real Time PCR easy (TM) -SYBR Green I kit (FOREGENE, chengdu, china) was used in CFX Connect TM The Real-Time System (Bio-Rad, CA, USA) instrument performs qRT-PCR reactions. The primer sequences are as follows: TNF-alpha (forward, 5'-GCA AAG GGA GAG TGG TCA-3'; reverse,5'-CTG GCT CTG TGA GGA AGG-3'), IL-1 beta (forward, 5'-CCT GTG TCT TTC CCG TGG AC-3'; reverse,5'-CAT CTC GGA GCC TGT AGT GC-3'), IL-6 (forward, 5'-ACA ACC ACG GCC TTC CCT ACT T-3'; reverse,5' -TTT)CTC ATT TCC ACG ATT TCC C-3 '), iNOS (forward, 5'-TCG GGT TGA AGT GGT ATG C-3'; reverse,5'-GAG GCC AGT GTG TGG GTC T-3'), COX-2 (forward, 5'-TGA CTG CCC AAC TCC CAT-3'; reverse,5'-GAA CCC AGG TCC TCG CTT-3'), and GAPDH (forward, 5'-AGC CTC GTC CCG TAG ACA AAA-3'; reverse,5'-GAG GCA ACA ATC TCC ACT TT-3'). The qRT-PCR results were analyzed using Bio-Rad CFX Maestro 1.0 software, GAPDH as reference gene.
To determine whether the essential oil attenuated cytokine production by modulating the transcriptional level of the gene, mRNA levels of TNF- α, IL-6 and IL-1 β were analyzed using qRT-PCR. As shown in FIGS. 4A-C, the mRNA expression levels of TNF-. Alpha.IL-6 and IL-1. Beta. Were significantly increased compared to the control group after LPS alone induction. However, the volatile oils significantly inhibited LPS-induced mRNA expression levels of TNF- α, IL-6 and IL-1 β in a dose-dependent manner. The data indicate that the essential oils reduce their secretion by inhibiting the expression of TNF-alpha, IL-6 and IL-1 beta genes induced by LPS.
Pharmacological example 4: the flower volatile oil of Curcuma rhizome flower inhibits the LPS-induced iNOS and COX-2 protein and gene expression
RAW264.7 cells were seeded in 6-well plates (6X 10) 5 Cells/well) for 24h, pre-treated with different concentrations (0, 7.81, 15.63 and 31.25 μ g/mL) of essential oil for 2h and treated with 1 μ g/mL LPS for an additional 24h. Subsequently, total protein was extracted using RIPA lysis buffer, and nuclear and cytoplasmic proteins were prepared by a nuclear and cytoplasmic protein extraction kit (Beyotime, shanghai, china). Protein concentration was quantified using the enhanced BCA protein assay kit. The proteins (20-40. Mu.g) were separated by 8% SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% skim milk for 1h, incubation with primary antibody at 4 ℃ overnight, TBST washing 3 times, then exposure to HRP-labeled secondary antibody for 1h at room temperature. Imaging was performed by ChemiDoc touch imaging system (Bio-Rad Laboratories, inc., hercules, calif., USA). The intensity of the protein band was analyzed by Image Lab 6.0 (Bio-Rad, CA, USA).
Inducible Nitric Oxide Synthase (iNOS) and cyclooxygenase-2 (COX-2) are the synthesis of NO and PGE, respectively 2 Key enzyme of (2) ((J.Ethnopharmacol.2013,147, 208-214). To investigate whether the volatile oils attenuate the release of pro-inflammatory mediators by modulating the expression of enzymes that catalyze their synthesis, the expression levels of proteins and mRNAs for iNOS and COX-2 were examined by Western blotting and qRT-PCR, respectively. As shown in FIGS. 5A and C, LPS stimulation alone significantly promoted the expression of iNOS protein and mRNA compared to the control group. However, after pretreatment with the essential oil, protein and mRNA levels were significantly reduced in a dose-dependent manner compared to treatment with LPS alone. In addition, as shown in FIGS. 5B and D, the expression levels of COX-2 protein and mRNA were hardly detected in the control group. However, after LPS induction, the expression levels of COX-2 protein and mRNA increased significantly, and the volatile oil dose-dependently inhibited COX-2 protein and mRNA levels in LPS-induced RAW264.7 cells. The results show that the volatile oil can obviously inhibit NO and PGE by inhibiting the expression of iNOS and COX-2 on the level of transcription and translation 2 Is released.
Pharmacological example 5: floral volatile oil of Curcuma longa inhibits phosphorylation levels of LPS-induced MAPKs upon stimulation by LPS, phosphorylates and activates mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK), and subsequently regulates the expression of pro-inflammatory genes (TNF-. Alpha., IL-6 and IL-1. Beta.) by affecting the activation of AP-1 transcription factors (Food chem. Toxicol.2021,147, 111915). Thus, protein levels of p38, p-p38, ERK, p-ERK, JNK, and p-JNK were measured using Western blotting (FIG. 6). The phosphorylation levels of p38, ERK and JNK were significantly increased relative to the control group after LPS stimulation alone. Interestingly, the volatile oil significantly inhibited LPS-induced phosphorylation levels of p38, ERK and JNK in a dose-dependent manner. The results show that the volatile oil can effectively inhibit the phosphorylation of MAPKs (p 38, ERK and JNK) induced by LPS, thereby inhibiting the activation of MAPK pathway.
Pharmacological example 6: the flower volatile oil of Curcuma rhizome flower inhibits NF-kB activity induced by LPS
NF-. Kappa.B is a transcription factor that regulates the transcription of genes for proinflammatory cytokines (TNF-. Alpha., IL-6 and IL-1. Beta.) and proinflammatory enzymes (iNOS and COX-2) (Oncogene, 1999,18,6853-6866). Normally, NF-. Kappa.B is a heterodimer composed of p50 and p65 subunits, and exists in the cytoplasm in an inactive form in combination with a kappa B inhibitor (I.kappa.B). Following stimulation with LPS, I κ B is phosphorylated and rapidly degraded, and then subunits of NF- κ B can freely translocate to the nucleus and activate transcription of pro-inflammatory genes (nat. Immunol.2002,3 (1), 20-26). Thus, western blot was used to assess the effect of the essential oils on LPS-induced nuclear transfer of NF- κ B and phosphorylation and degradation of I κ B α. As shown in FIGS. 7A and B, phosphorylation and degradation levels of I.kappa.Balpha.were significantly increased after LPS induction; however, the essential oils significantly inhibited LPS-induced phosphorylation and degradation of I κ B α in a concentration-dependent manner. Furthermore, as shown in FIGS. 7C and D, the levels of NF- κ B p subunit in the nucleus were significantly elevated and their levels in the cytoplasm were significantly reduced after LPS induction alone, indicating that NF- κ B p translocates to the nucleus after LPS induction. However, volatile oil 15.63 μ g/mL and a high dose of 31.25 μ g/mL significantly up-regulated the level of NF-. Kappa. B p65 in the cytoplasm, while dose-dependently down-regulated the level of NF-. Kappa. B p65 in the nucleus, indicating that volatile oil inhibits LPS-induced nuclear translocation of p 65. Pharmacological example 7: the volatile oil of Curcuma rhizome flower inhibits nuclear translocation of NF-kappa B p65 induced by LPS
RAW264.7 cells (6X 10) 5 Cells per well) were seeded into 6-well plates with coverslips and incubated for 24h. Subsequently, pre-treatment with the volatile oil (31.25. Mu.g/mL) was performed for 2h, followed by LPS (1. Mu.g/mL) for 24h. After 3 times of PBS washing, the column was fixed with 4% paraformaldehyde solution for 15min, and then permeabilized with 0.3% Triton X-100 for 5min. Then, the cells were blocked in 5-vol BSA for 1h, washed 3 times with PBS and incubated overnight at 4 ℃ with primary anti-NF-. Kappa. B p 65. After 3 PBS washes, alexa Fluor 488-labeled secondary antibody was added and incubated for 1h. Cells were stained with DAPI for 5min and observed by Leica TCS SP8 confocal laser scanning microscopy (Leica Microsystems, germany).
As shown in FIG. 8, NF- κ B p (green) in untreated RAW264.7 cells appeared in the cytoplasm; however, after LPS induction, green fluorescence is mainly concentrated in the nucleus. After treatment with volatile oil (31.25. Mu.g/mL), green fluorescence was mainly distributed in the cytoplasm, indicating that volatile oil inhibited LPS-induced nuclear translocation of NF- κ B p.
Pharmacological example 8: influence of floral volatile oil of Curcuma longa on xylene-induced ear swelling (in vivo anti-inflammatory assay)
The inflammatory mice are modeled by adopting a mouse auricle swelling method, 60 Kunming mice are randomly divided into 6 groups, each group is male and female, and 10 mice are in each group; a blank control group, a model group, a dexamethasone group (7.5 mg/kg) and low (3.75 g/kg), medium (7.5 mg/kg) and high (15 mg/kg) dose groups of the flower volatile oil (EO) of the turmeric flower are respectively established. The medicine is administrated once a day, the volume of the medicine liquid is 20mL/kg, and the medicine liquid is administrated by gastric gavage for 7 days continuously. The grouping is specifically as follows:
blank control group: the same volume of physiological saline containing DMSO was administered daily;
model group: the same volume of physiological saline containing DMSO was administered daily;
positive drug group (Dexamethasone, DXM): 7.5mg/kg is administered daily;
low dose group: 3.75mg/kg is administered daily;
the medium dose group: 7.5mg/kg is administered daily;
high dose group: 15mg/kg was administered daily.
The experiment is started after the mice are adapted for 3d, each administration group is continuously administered with the gastric lavage for 7 days and is administered once a day, wherein the normal group is administered with the same volume of physiological saline containing DMSO for the gastric lavage, and the experiment is carried out 1h after the last administration. Except for the blank control group, 30 microliter of dimethylbenzene is uniformly coated on the front and back sides of the left ear of the mouse, and the right ear is not treated and is used for comparison; after the mice were inflamed for 30min, the mice were sacrificed by dislocation, both ears of the mice were cut off, and the ears were punched out at the same positions of the left and right ears of the mice using an electric ear swelling punch with a diameter of 8mm, and weighed. Recording the weights of the left and right ear sheets, and calculating swelling degree and ear swelling inhibition rate.
Swelling degree = left ear mass-right ear mass
Ear swelling inhibition (%) = (degree of swelling in model group-degree of swelling in administered group)/degree of swelling in model group × 100%
As shown in table 2 and fig. 9, the floral volatile oil of turmeric flower has a dose-dependent inhibitory effect on the xylene-induced swelling of mouse pinna. Compared with the model group (swelling degree: 16.58 +/-1.57 mg), the dexamethasone group (DXM, swelling degree: 6.99 +/-1.21) has obvious inhibition (p is less than 0.001), and the high (swelling degree: 5.05 +/-0.67 mg), medium (swelling degree: 7.06 +/-0.22 mg) and low (swelling degree: 8.33 +/-0.59 mg) dose groups of the flower volatile oil of the turmeric flower have obvious inhibition (p is less than 0.001). In conclusion, the floral volatile oil of the turmeric flower has a remarkable inhibiting effect on the ear swelling induced by the dimethylbenzene, and has a remarkable anti-inflammatory effect in vivo.
TABLE 3 inhibitory Effect of floral volatile oil of Curcuma longa on xylene-induced ear swelling (Mean + -SD, n = 10)
Note: model versus blank group ( ### p is less than 0.001); drug group comparison model group ( *** p＜0.001)；(-)
Indicating none.
In combination with the above pharmacological examples, the floral volatile oil of Curcuma longa inhibits LPS-induced pro-inflammatory mediators (NO and PGE) at non-toxic concentrations 2 ) And the production of pro-inflammatory cytokines (TNF- α, IL-6, and IL-1 β); the volatile oil inhibits the transcription of proinflammatory cytokine genes induced by LPS and the expression of genes and proteins for synthesizing proinflammatory mediator enzymes (iNOS and COX-2); relevant mechanism researches show that the volatile oil not only inhibits nuclear transfer of NF-kB by reducing phosphorylation and degradation of I kB alpha, but also inhibits phosphorylation of MPAKs (ERK, p38 and JNK); improved ear swelling due to xylene; has significant anti-inflammatory effect in vivo and in vitro.
Claims (6)
1. The application of the volatile oil of the turmeric flower in preparing anti-inflammatory drugs is characterized in that: the flower volatile oil of the turmeric flower is prepared by the following method:
crushing fresh turmeric flower pollen, mixing the crushed raw materials with distilled water according to a feed-liquid ratio of 1 to 3 to 1, wherein the unit g/mL is obtained, performing steam distillation in a volatile oil extractor for 3 to 5 hours, collecting volatile oil, and removing water by using anhydrous sodium sulfate to obtain the flower volatile oil.
2. Use according to claim 1, characterized in that: application of flower volatile oil of Curcuma rhizome flower in preparing medicine for improving excessive secretion of proinflammatory mediators and proinflammatory cytokines in RAW264.7 cells induced by Lipopolysaccharide (LPS), wherein the proinflammatory mediators are NO and PGE 2 The proinflammatory cytokines are TNF-alpha, IL-6 and IL-1 beta.
3. Use according to claim 1, characterized in that: application of flower volatile oil of Curcuma rhizome flower in preparing medicine for inhibiting LPS activated MAPK and NF-kB pathway is provided.
4. Use according to claim 3, characterized in that: the volatile oil of Curcuma xanthorrhiza flower inhibits the activation of MAPKs induced by LPS through inhibiting the phosphorylation of ERK, p38 and JNK, and simultaneously inhibits the activation of NF-kappa B induced by LPS through blocking the nuclear transfer of NF-kappa B and the phosphorylation and degradation of I kappa B alpha.
5. Use according to claim 1, characterized in that: application of volatile oil of Curcuma rhizome flower in preparing medicine for improving ear swelling caused by xylene is provided.
6. The use as claimed in claim 1, wherein the flower volatile oil of Curcuma rhizome flower and pharmaceutically acceptable carrier are formulated into tablet, capsule, fat emulsion, suppository, dripping pill, and ointment.
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| hamidou.Chemical Composition,Antifungal and Insecticidal Activities of Hedychium Essential oils.molecules.2013,4308-4327. * |
| s.mollenbeck.Chemical Composition and Analyses of Enantiomers of Essential Oils from Madagascar.FLAUOUR AND FRAGRANCE JOURNAL.第12卷63-69. * |
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