Compound essential oil for intervening obesity and application

Abstract

The invention relates to the technical field of traditional Chinese medicine, in particular to compound essential oil for intervening obesity and application. The compound essential oil for intervening obesity comprises the following components in parts by weight: 8-30 parts of agilawood essential oil; the first component comprises rhizoma cyperi essential oil and ginger essential oil; the second component comprises pericarpium citri reticulatae essential oil, pericarpium citri reticulatae viride essential oil, pericarpium zanthoxyli essential oil, rhizoma atractylodis essential oil and fructus aurantii essential oil; the mass ratio of the agilawood essential oil to the first component to the second component is 3: 3: 10. Under the guidance of the traditional Chinese medicine aromatherapy theory, the compatibility of the traditional Chinese medicine essential oil in the related research is adjusted and improved, and through the synergistic interaction between the agilawood and the common formula components, the obvious treatment effect on obesity is achieved.

Description

[Technical field]

[0001] The invention relates to the technical field of traditional Chinese medicine, and in particular to a compound essential oil for intervening obesity and an application thereof. [Background technology]

[0002] Obesity is a major risk factor for health problems such as metabolic syndrome, cardiovascular disease and cancer. The prevention and treatment of obesity is crucial to improving the overall health of individuals and society. Related treatment methods such as Western medicine, dieting, and surgery are not only difficult to maintain long-term effects, but also expensive and may cause side effects. For example, oral medications currently have certain gastrointestinal reactions and liver and kidney toxicity, and the invasive administration of subcutaneous injections of drugs brings inconvenience, gastrointestinal reactions or nervous system side effects.

[0003] Chinese herbal essential oils are a complementary and alternative treatment for obesity that combines the principles of Chinese aromatherapy with the mechanisms of Western medicine. They are also a potential effective way to treat obesity. In related studies, compound essential oils generally have complicated raw materials, are not targeted enough for the treatment of obesity, and have little efficacy.

[0004] Therefore, it is necessary to provide a compound essential oil for intervening obesity and its application to solve the above problems. [Summary of the invention]

[0005] In view of the above problems, the purpose of the present invention is to provide a compound essential oil and its application for intervening in obesity. The present invention adjusts and improves the compatibility of Chinese medicine essential oils in related research, and achieves significant therapeutic effects on obesity through the synergistic enhancement between agarwood and common formula components.

[0006] To this end, the present invention adopts the following technical solution:

[0007] A compound essential oil for intervening obesity, characterized in that it comprises, by weight:

[0008] 8-30 parts of agarwood essential oil;

[0009] A first component, the first component comprising cyperus rotundus essential oil and ginger essential oil;

[0010] A second component, wherein the second component includes tangerine peel essential oil, green peel essential oil, Zanthoxylum essential oil, atractylodes essential oil and fructus aurantii essential oil;

[0011] The mass ratio of the agarwood essential oil, the first component and the second component is 3:3:10.

[0012] In some embodiments, a third component is further included in parts by weight, wherein the third component includes Magnolia bark essential oil and Curcuma zedoaria essential oil, and the mass ratio between the third component and the other components as described above is 2:3:3:10.

[0013] In some embodiments, the following are included by weight:

[0014] 8-30 parts of the agarwood essential oil, 6-20 parts of the tangerine peel essential oil, 6-20 parts of the green peel essential oil, 6-20 parts of the Zanthoxylum essential oil, 6-20 parts of the Atractylodes lancea essential oil, 6-20 parts of the Citrus aurantium essential oil, 4-15 parts of the Cyperus rotundus essential oil, 4-15 parts of the ginger essential oil, 2-10 parts of the Magnolia bark essential oil and 2-10 parts of the Curcuma zedoaria essential oil.

[0015] In some embodiments, the following are included by weight:

[0016] 9-25 parts of the agarwood essential oil, 8-15 parts of the tangerine peel essential oil, 8-15 parts of the green peel essential oil, 8-15 parts of the Zanthoxylum essential oil, 8-15 parts of the atractylodes essential oil, 8-15 parts of the fructus aurantii essential oil, 5-12 parts of the cyperus rotundus essential oil, 5-12 parts of the ginger essential oil, 3-9 parts of the magnolia bark essential oil and 3-9 parts of the zedoaria essential oil.

[0017] In some embodiments, the following are included by weight:

[0018] 10-21 parts of the agarwood essential oil, 9-13 parts of the tangerine peel essential oil, 9-13 parts of the green peel essential oil, 9-13 parts of the Zanthoxylum essential oil, 9-13 parts of the atractylodes essential oil, 9-13 parts of the fructus aurantii essential oil, 6-10 parts of the cyperus rotundus essential oil, 6-10 parts of the ginger essential oil, 5-8 parts of the magnolia bark essential oil and 5-8 parts of the zedoaria essential oil.

[0019] In some embodiments, the preparation methods of the agarwood essential oil, the tangerine peel essential oil, the green peel essential oil, the Zanthoxylum bungeanum essential oil, the Atractylodes lancea essential oil, the Citrus aurantium essential oil, the Cyperus rotundus essential oil, the ginger essential oil, the Magnolia bark essential oil and the Curcuma zedoaria essential oil include any one or more of steam distillation, pressing, supercritical extraction and oil separation.

[0020] At the same time, the present invention also provides an application of a compound essential oil in the preparation of a drug for intervening in obesity, characterized in that the drug type is an inhalation preparation or a topical ointment, and the compound essential oil is any compound essential oil for intervening in obesity as described above.

[0021] Compared with related means for preventing or treating obesity, the present invention has obvious beneficial effects. Specifically, it has been verified that the technical scheme described above can be used to intervene in obesity by sniffing or transdermal administration for 6 or 8 weeks, which can significantly reduce the body weight, feeding efficiency and fasting blood glucose of db / db mice or HFD mice, and significantly improve the glucose tolerance and insulin sensitivity of HFD mice; by adopting the present invention, the weight of the liver and peritesticular white fat (eWAT) of db / db mice and HFD mice is significantly reduced, and the weight of brown fat (BAT) is significantly increased; by adopting the present invention, fatty degeneration of the liver of db / db mice or HFD mice is significantly improved, the accumulation of white fat droplets is reduced, and the accumulation of brown fat droplets is increased, and there is no skin irritation in the transdermal administration method; by adopting the present invention, the expression levels of pharmacodynamic mechanism indicators related to the glucose and lipid metabolism pathway, liver function, "brain-gut axis" appetite pathway, bile acid metabolism pathway and fat heat production pathway of db / db mice and HFD mice are significantly improved. Under the guidance of the theory of aromatherapy in traditional Chinese medicine, the present invention adjusts and improves the compatibility of traditional Chinese medicine essential oils in related research, and achieves a significant therapeutic effect on obesity through the synergistic enhancement between agarwood and common formula components.

Brief Description of the Drawings

[0022] In order to more clearly illustrate the beneficial effects of the embodiments of the present invention, the following briefly introduces the drawings obtained after experimental verification of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 This is a schematic diagram of the results of body weight and feeding efficiency experiments on two types of mice after using two compound essential oils and western medicine respectively. Figures A and C are the research results of db / db mice, and Figures B and D are the research results of HFD mice.

[0024] Figure 2 This is a schematic diagram of the results of fasting blood glucose, glucose tolerance test and insulin sensitivity test on experimental mice after using two kinds of compound essential oils and western medicine respectively. Figures A, C, and E are the research results of db / db mice, and Figures B, D, and F are the research results of HFD mice.

[0025] Figure 3 This is a schematic diagram of the results of the liver and peritesticular white fat / brown fat weight analysis experiment on experimental mice after using two compound essential oils and western medicine respectively. Figures A, C, and E are the research results of db / db mice, and Figures B, D, and F are the research results of HFD mice.

[0026] Figure 4This is a schematic diagram of the results of the HE staining analysis experiment on the liver, peritesticular white fat / brown fat of the two types of mice, and the abdominal skin of db / db mice after using two compound essential oils and western medicine in experimental mice. Figure A is the research result of db / db mice, and Figure B is the research result of HFD mice.

[0027] Figure 5 This is a schematic diagram of the results of the experiment in which two kinds of compound essential oils and western medicine were used in experimental mice, and then the serum efficacy index analysis experiments were performed on db / db mice using a fully automatic biochemical analyzer or ELISA. Figure A reflects the GLU level, Figure B reflects the INS level, Figure C reflects the TC level, Figure D reflects the TG level, Figure E reflects the LDL-C level, Figure F reflects the HDL-C level, Figure G reflects the AST level, and Figure H reflects the ALT level.

[0028] Figure 6 This is a schematic diagram of the results of the experiment in which two kinds of compound essential oils and western medicine were used in experimental mice, and the serum efficacy index analysis experiments were performed on HFD mice using a fully automatic biochemical analyzer or ELISA. Figure A reflects the GLU level, Figure B reflects the INS level, Figure C reflects the TC level, Figure D reflects the TG level, Figure E reflects the LDL-C level, Figure F reflects the HDL-C level, Figure G reflects the AST level, and Figure H reflects the ALT level.

[0029] Figure 7 This is a schematic diagram of the results of the experiment in which two kinds of compound essential oils and western medicine were used in experimental mice, and then the serum mechanism index analysis experiments were performed on db / db mice using a fully automatic biochemical analyzer or ELISA. Figure A reflects the LEP level, Figure B reflects the GLP-1 level, Figure C reflects the GIP level, Figure D reflects the GCG level, Figure E reflects the GDF15 level, Figure F reflects the FXR level, and Figure G reflects the UCP1 level.

[0030] Figure 8 This is a schematic diagram of the results of immunofluorescence staining analysis experiments on experimental mice after using two compound essential oils and western medicine respectively. Figure A is an immunofluorescence staining diagram, Figure B reflects the expression level of LEPR-positive cells, Figure C reflects the expression level of GLP-1R-positive cells, Figure D reflects the expression level of GIPR-positive cells, Figure E reflects the expression level of GCGR-positive cells, Figure F reflects the expression level of GDF15-positive cells, Figure G reflects the expression level of FXR-positive cells, and Figure H reflects the expression level of UCP1-positive cells.

[0031] Fig. 9This is a schematic diagram of the results of RT-qPCR analysis experiments on experimental mice after using two compound essential oils and western medicine respectively. Figure A reflects the LEPR mRNA expression level, Figure B reflects the GLP-1R mRNA expression level, Figure C reflects the GIPR mRNA expression level, Figure D reflects the GCGR mRNA expression level, Figure E reflects the DF15 mRNA expression level, Figure F reflects the FXR mRNA expression level, and Figure G reflects the UCP1 mRNA expression level. [Specific implementation method]

[0032] Embodiment 1 of the present invention

[0033] A compound essential oil for intervening obesity, comprising: by weight: 8-30 parts of agarwood essential oil, 6-20 parts of tangerine peel essential oil, 6-20 parts of green tangerine peel essential oil, 6-20 parts of prickly ash essential oil, 6-20 parts of atractylodes essential oil, 6-20 parts of fructus aurantii essential oil, 4-15 parts of cyperus rotundus essential oil, 4-15 parts of ginger essential oil, 2-10 parts of magnolia bark essential oil and 2-10 parts of zedoaria essential oil.

[0034] It should be noted that, in the present embodiment, the preparation method of the agarwood essential oil, the tangerine peel essential oil, the green peel essential oil, the Zanthoxylum bungeanum essential oil, the Atractylodes lancea essential oil, the Citrus aurantium essential oil, the Cyperus rotundus essential oil, the ginger essential oil and the Curcuma zedoaria essential oil can be a steam distillation method; in other embodiments, it can also be obtained by any one or more of a pressing method, a supercritical extraction method and an oil separation method.

[0035] It can be understood that the agarwood essential oil, the tangerine peel essential oil, the green peel essential oil, the Zanthoxylum bungeanum essential oil, the Atractylodes lancea essential oil, the Citrus aurantium essential oil, the Cyperus rotundus essential oil, the ginger essential oil and the Curcuma zedoaria essential oil can be either finished essential oils purchased through the above-mentioned preparation method or homemade essential oils obtained through the above-mentioned preparation method.

[0036] Embodiment 2 of the present invention

[0037] A compound essential oil for intervening obesity, comprising: by weight: 9-25 parts of agarwood essential oil, 8-15 parts of tangerine peel essential oil, 8-15 parts of green tangerine peel essential oil, 8-15 parts of prickly ash essential oil, 8-15 parts of atractylodes essential oil, 8-15 parts of fructus aurantii essential oil, 5-12 parts of cyperus rotundus essential oil, 5-12 parts of ginger essential oil, 3-9 parts of magnolia bark essential oil and 3-9 parts of zedoaria essential oil.

[0038] It should be noted that, in the present embodiment, the preparation method of the agarwood essential oil, the tangerine peel essential oil, the green peel essential oil, the Zanthoxylum bungeanum essential oil, the Atractylodes lancea essential oil, the Citrus aurantium essential oil, the Cyperus rotundus essential oil, the ginger essential oil and the Curcuma zedoaria essential oil can be a steam distillation method; in other embodiments, it can also be obtained by any one or more of a pressing method, a supercritical extraction method and an oil separation method.

[0039] It can be understood that the agarwood essential oil, the tangerine peel essential oil, the green peel essential oil, the Zanthoxylum bungeanum essential oil, the Atractylodes lancea essential oil, the Citrus aurantium essential oil, the Cyperus rotundus essential oil, the ginger essential oil and the Curcuma zedoaria essential oil can be either finished essential oils purchased through the above-mentioned preparation method or homemade essential oils obtained through the above-mentioned preparation method.

[0040] Embodiment 3 of the present invention

[0041] A compound essential oil for intervening obesity, comprising: by weight: 10-21 parts of agarwood essential oil, 9-13 parts of tangerine peel essential oil, 9-13 parts of green tangerine peel essential oil, 9-13 parts of prickly ash essential oil, 9-13 parts of atractylodes essential oil, 9-13 parts of fructus aurantii essential oil, 6-10 parts of cyperus rotundus essential oil, 6-10 parts of ginger essential oil, 5-8 parts of magnolia bark essential oil and 5-8 parts of zedoaria essential oil.

[0042] It should be noted that, in the present embodiment, the preparation method of the agarwood essential oil, the tangerine peel essential oil, the green peel essential oil, the Zanthoxylum bungeanum essential oil, the Atractylodes lancea essential oil, the Citrus aurantium essential oil, the Cyperus rotundus essential oil, the ginger essential oil and the Curcuma zedoaria essential oil can be a steam distillation method; in other embodiments, it can also be obtained by any one or more of a pressing method, a supercritical extraction method and an oil separation method.

[0043] It can be understood that the agarwood essential oil, the tangerine peel essential oil, the green peel essential oil, the Zanthoxylum bungeanum essential oil, the Atractylodes lancea essential oil, the Citrus aurantium essential oil, the Cyperus rotundus essential oil, the ginger essential oil and the Curcuma zedoaria essential oil can be either finished essential oils purchased through the above-mentioned preparation method or homemade essential oils obtained through the above-mentioned preparation method.

[0044] The pathogenesis of obesity is multifactorial and multi-mechanistic, involving genetics, environment, lifestyle, endocrine regulation, inflammatory response, intestinal flora, etc. Studies have shown that obesity increases the risk of type 2 diabetes by affecting β-cell function, adipose tissue biology, and multi-organ insulin resistance, and these changes can usually be improved or even normalized by appropriate weight loss.

[0045] Obesity is a heterogeneous disease, and common related factors include genes, epigenetics, neurobehavior, endocrine and metabolism. At present, clinical candidate drugs are concentrated in four target areas: leptin (LEP), growth hormone releasing peptide, mitochondrial uncoupler and growth differentiation factor. Studies have found that the GIPR / GLP-1R co-agonist tesiparatide can reduce the weight of obese patients by more than 20%; GIPR / GLP-1R / GCGR agonists (triple agonists) acting on appetite homeostasis regulatory molecules in the gut-brain axis and intestinal insulinotropic molecules can further enhance the efficacy of dual GIPR and GLP-1R agonists, improve energy expenditure, weight loss and blood sugar control. GDF15 targets and regulates the ineffective circulation of calcium ions in muscle tissue through the adrenergic receptor axis, resists the reduction of energy expenditure caused by diet control, and thus solves the bottleneck problem of weight rebound in obesity treatment. FXR forms a systemic regulatory mechanism of whole body metabolism by activating the signaling pathway network of the intestine, liver and neuronal hormones. UCP1 dissipates energy as heat by uncoupling oxidative phosphorylation from ATP synthesis.

[0046] In order to fully reflect the beneficial effects of the technical solution of the present invention, the present invention also uses the compound essential oil provided in Example 3 and the comparative essential oil for experimental comparison and verification. The components of the comparative essential oil are used in many related studies and are drug compatibility that can be easily anticipated by professionals in the field, including tangerine peel essential oil, green peel essential oil, prickly ash essential oil, atractylodes essential oil and fructus aurantii essential oil by weight, each essential oil 9-13 parts.

[0047] After experimental verification, the experimental example obtained is as follows:

[0048] 1 Experimental process

[0049] 1.1 Experimental animals

[0050] db / db mouse study: db / db mice, 5 weeks old, male, weighing 20-30 g.

[0051] HFD mouse study: C57BL / 6J mice, 13 weeks old, male, weighing 30g-35g, fed a high-fat diet for 8 weeks.

[0052] Blank study: C57BL / 6J mice, 5 weeks old, male, weighing 15-20 g;

[0053] C57BL / 6J mice, 13 weeks old, male, weighing 25-30 g.

[0054] Experimental Animal Production License Number: SCXK(Su)2020-0009, Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.

[0055] 1.2 Animal grouping and establishment of pathological model

[0056] The db / db mouse model is a genetic obesity model used to simulate human type 2 diabetes. In this experimental example, 5-week-old male db / db mice were fed with ordinary feed to establish the db / db mouse model.

[0057] The HFD mouse model is an obesity model induced by a high-fat diet, which is used to simulate obesity and metabolic syndrome caused by unhealthy eating habits. In this experiment, 5-week-old male C57BL / 6J mice were fed a high-fat diet until 13 weeks of age to establish the HFD mouse model.

[0058] The db / db mice and HFD mice described above were used, and each type of mice was fed with normal feed or high-fat feed according to the group, with unlimited food and water, and the light and dark periods were 12 hours, respectively, and the temperature was constant at 22° C. They were randomly divided into the following groups, n=7-9.

[0059] The db / db mice were divided into the following groups: blank group (CON), model group (MOD), western medicine group (SEMA), control essential oil inhalation administration group (CXEO_HD_I), compound essential oil inhalation administration group provided in Example 3 of the present invention (CEO_HD_I), control essential oil transdermal administration group (CXEO_HD_T), and compound essential oil transdermal administration group (CEO_HD_T) provided in Example 3 of the present invention.

[0060] HFD mice were divided into the following groups: blank group (CON), model group (MOD), western medicine group (SEMA), control essential oil inhalation administration group (CXEO_HD_I), compound essential oil inhalation administration group (CEO_HD_I) provided in Example 3 of the present invention, control essential oil transdermal administration group (CXEO_HD_T), and compound essential oil transdermal administration group (CEO_HD_T) provided in Example 3 of the present invention.

[0061] 1.3 Administration and dosage

[0062] A drug efficacy experiment is conducted first to screen the optimal drug efficacy dose from low and high doses as the compound essential oil research dosage, i.e., the dosage of the compound essential oil and the comparison essential oil provided in Example 3 of the present invention.

[0063] 1.3.1 Drug administration by inhalation

[0064] The main body of the aromatherapy smelling device is a transparent organic glass box with a specification of 20x20x20cm. The atomizer is placed in the middle of the box. The atomizer is used to hold essential oil, and the essential oil is the compound essential oil or the comparison essential oil for intervention of obesity provided in Example 3 of the present invention. The essential oil is added to the spray bottle of the atomizer, and distilled water is added for dilution. Then the mice are evenly placed in the box. The atomizer continuously atomizes the essential oil. When the spray bottle of the atomizer no longer produces mist, the box is opened and the mice are taken out. At this time, the sniffing is completed, and the sniffing time for each group is 1h.

[0065] Among them, for db / db mice and HFD mice, after dilution with distilled water, the administration concentration of the control essential oil inhalation administration group and the compound essential oil inhalation administration group provided in Example 3 of the present invention was 260uL, once a day, for 8 consecutive weeks.

[0066] 1.3.2 Transdermal administration

[0067] 24 hours before the experiment, the mouse abdomen was depilated to complete the pre-drug treatment. The depilated area of ​​the mouse was used as the administration site, and essential oil was applied to the depilated area, wherein the essential oil was the compound essential oil provided in Example 3 of the present invention and the comparative essential oil.

[0068] Among them, for the HFD mouse grouping, the jojoba oil was diluted, and the dosing concentrations of the control essential oil transdermal administration group and the compound essential oil transdermal administration group provided in Example 3 of the present invention were 24%, the dosage for the first 4 weeks was 0.4 mL, and the dosage for the next 4 weeks was 0.8 mL, once a day, for 8 consecutive weeks.

[0069] For the db / db mouse grouping, the administration concentrations of the control essential oil transdermal administration group and the compound essential oil transdermal administration group provided in Example 3 of the present invention were 12% and the administration dose was 0.8 mL, once a day, for 6 consecutive weeks.

[0070] 1.3.3 Western medicine injection

[0071] For db / db mice, semaglutide was injected subcutaneously at a dose of 0.08 mg / kg, calculated based on the adult dose using body surface area, once every 3 days for 6 weeks.

[0072] For HFD mice, semaglutide was injected subcutaneously at a dose of 0.08 mg / kg in the first 4 weeks and 0.16 mg / kg in the second 4 weeks, once every 3 days for 8 weeks, calculated based on the adult dose using body surface area.

[0073] 1.4 Experimental methods

[0074] 1.4.1 Body weight and feeding efficiency experiments

[0075] Weigh the mice twice a week and take the average value as the weekly weight data. Record the food intake of each cage of mice twice a week and take the average value to calculate the average daily food intake. Calculate the feeding efficiency of mice according to the formula: feeding efficiency = total food intake / total body weight.

[0076] 1.4.2 Fasting blood glucose, glucose tolerance test and insulin sensitivity test

[0077] a. Fasting blood glucose test

[0078] After the last drug intervention, the mice in each group were fasted for 10 hours. For the mice in the compound essential oil inhalation administration group provided in Example 3 of the present invention, the tail vein blood glucose of the mice was tested using blood glucose test strips.

[0079] b. Glucose tolerance test

[0080] After the last drug intervention, the mice in each group were fasted for 10 h, and 50% high sugar solution was given at a dose of 2 mg / g to the mice in the transdermal administration group of the compound essential oil provided in Example 3 of the present invention for intraperitoneal injection. The tail vein blood glucose of the mice was detected using blood glucose test strips at 0 min, 30 min, 60 min, 90 min and 120 min after the injection.

[0081] c. Intraperitoneal insulin sensitivity test

[0082] After the last drug intervention in each group of mice, about 4-5 hours after eating, the mice in the transdermal administration group of the compound essential oil provided in Example 3 of the present invention were intraperitoneally injected with insulin solution according to the calculated given dose, and the tail vein blood glucose of the mice was detected using blood glucose test strips at 0 min, 30 min, 60 min, 90 min and 120 min after the injection.

[0083] 1.4.3 Liver and peritesticular white fat / brown fat weight experiment

[0084] After the last drug intervention, mice in each group were fasted for 10 hours, anesthetized with 0.3% sodium pentobarbital at a dose of 0.1 mL / 10 g, blood was collected from the retro-orbital vein, and mice were killed by cervical dislocation. Mice were dissected, and the hypothalamus, abdominal skin, liver, peritesticular white fat and brown fat were removed. The liver, peritesticular white fat and brown fat tissue were weighed and analyzed.

[0085] 1.4.4 HE staining experiment

[0086] 1) thawing of frozen sections; 2) hematoxylin staining; 3) eosin staining; 4) dehydration and sealing; 5) Image-Pro Plus 6.0 analysis software result interpretation: cell nucleus is blue and cytoplasm is red.

[0087] 1.4.5 Automatic biochemical analyzer or ELISA to detect serum biochemical indicators

[0088] After the last drug intervention, mice in each group were fasted for 10 h, anesthetized with 0.3% sodium pentobarbital at a dose of 0.1 mL / 10 g, and blood was collected by removing the eyeballs. The blood was left to stand at room temperature for 1 h, and then centrifuged at 4000 r·min-1 and 4°C for 20 min.

[0089] Take the upper serum and store it in -80℃ ultra-low temperature refrigerator for long-term storage. Perform the automatic biochemical analyzer or ELISA test according to the instructions of the kit to detect the following biochemical indicators:

[0090] Sugar metabolism pathway: GLU, INS

[0091] Lipid metabolism pathway: TC, TG, HDL-C, LDL-C

[0092] Liver function: AST, ALT

[0093] “Brain-gut axis” appetite pathway: LEP, GLP-1, GIP, GCG, GDF15

[0094] Bile acid metabolism pathway: FXR

[0095] Fat Thermogenesis Pathway: UCP1

[0096] 1.4.6 Immunofluorescence staining experiment

[0097] 1) Fresh tissue embedding; 2) Tissue sectioning; 3) Frozen section fixation; 4) Antigen retrieval; 5) Circle serum blocking; 6) Add primary antibody; 7) Add secondary antibody; 8) DAPI counterstaining of cell nuclei; 9) Sealing; 10) Microscopic examination and photography: The sections are observed under a fluorescence microscope and images are collected; 11) Image-Pro Plus 6.0 analysis software results interpretation: DAPI-stained cell nuclei are blue under ultraviolet excitation, and positive expression is red light labeled with the corresponding fluorescent marker.

[0098] 1.4.7 RT-qPCR experiments

[0099] 1) Extract total RNA from samples

[0100] TRizol total RNA extraction reagent was used to extract sample RNA, and the experimental operation was carried out according to the product instructions.

[0101] 2) Reverse transcription to synthesize cDNA

[0102] use All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit was used for cDNA reverse transcription, and the experimental operations were performed according to the product instructions.

[0103] 3) Real Time PCR Sample Testing

[0104] All cDNA samples were prepared into Realtime PCR reaction system using ChamQ Universal SYBR qPCR Master Mix kit. During the preparation of reaction solution and sample addition, all reagents and 96-well plates were placed on ice.

[0105] Add sample;

[0106] The 96-PCR plate was placed on a Realtime PCR instrument for PCR reaction. The PCR reaction program was: 95° C. for 10 minutes; 45 PCR cycles, wherein the parameters of the PCR cycle process were set to 95° C. for 15 seconds; and 60° C. for 60 seconds.

[0107] 4) Realtime PCR reaction was performed on the target gene and internal reference of each sample.

[0108] 2 Statistical analysis

[0109] The experimental results were expressed as Mean±SD and statistically analyzed using SPSS21.0 software. The inter-group comparisons were analyzed using the One-way ANOVA method, and P < 0.05 was considered statistically significant.

[0110] Note: Compared with the blank group, b P<0.05, ab P<0.01; compared with the model group, c P<0.05, ac P<0.01.

[0111] 3 Experimental results and discussion

[0112] 3.1 Body weight and feeding efficiency experimental results and discussion

[0113] like Figure 1 As shown, in the compound essential oil inhalation administration group provided in Example 3 of the present invention and the compound essential oil transdermal administration group provided in Example 3 of the present invention, intervention in obese db / db mice for 6 weeks can significantly reduce the body weight of the mice (P<0.01), and there is no significant difference in body weight compared with the western medicine group (P>0.05); it can reduce their feeding efficiency, and the feeding efficiency of the transdermal administration group is close to that of the western medicine group.

[0114] In the compound essential oil inhalation administration group provided in Example 3 of the present invention and the compound essential oil transdermal administration group provided in Example 3 of the present invention, the obese HFD mice were intervened for 8 weeks, and the body weight of the mice was also significantly reduced (P < 0.01). There was no significant difference in body weight between the transdermal administration group and the western medicine group (P > 0.05); the feeding efficiency was reduced, and the feeding efficiency of the transdermal administration group was close to that of the western medicine group.

[0115] After the above db / db mice and HFD mice were intervened with the compound essential oil provided in Example 3 of the present invention, their weight decreased by about 30%. Although the weight decreased after intervention with the comparison essential oil, the performance of weight change was not as good as that of the compound essential oil provided in Example 3 of the present invention. The performance difference was even greater when using the transdermal administration method. It is speculated that there is a synergistic effect between agarwood and common formula components in the present invention. The Western medicine group used semaglutide injection. The significant effects of semaglutide in lowering blood sugar and weight loss are widely recognized worldwide. The intervention effect of the compound essential oil provided in Example 3 of the present invention on obesity was not significantly different from that of the Western medicine group, and the present invention does not require invasive administration, is more cost-effective, and is naturally safe. The beneficial effects of the present invention are obvious.

[0116] 3.2 Results and discussion of fasting blood glucose, glucose tolerance test, and insulin sensitivity test

[0117] like Figure 2 As shown, the compound essential oil inhalation administration group provided in Example 3 of the present invention can significantly reduce the fasting blood glucose level of db / db mice and HFD mice (P<0.05), wherein the fasting blood glucose level of HFD mice was not significantly different from that of the western medicine group (P>0.05); the compound essential oil transdermal administration group provided in Example 3 of the present invention can significantly improve the glucose tolerance of db / db mice and HFD mice (P<0.05), and significantly improve their insulin sensitivity (P<0.05), and the performance is close to that of the western medicine group.

[0118] After the above db / db mice and HFD mice were intervened by the compound essential oil provided in Example 3 of the present invention by sniffing administration, the fasting blood sugar level was reduced by an average of about 20%-30%, and the fasting blood sugar level was reduced after the intervention by the comparison essential oil by sniffing administration, but the performance of the reduction change was not as good as the compound essential oil provided in Example 3 of the present invention. It is speculated that there is a synergistic effect between the agarwood and the common formula components in the present invention. The Western medicine group used semaglutide injection, and the significant effects of semaglutide in lowering blood sugar and weight loss are widely recognized worldwide. The intervention effect of the compound essential oil provided in Example 3 of the present invention on obesity is not significantly different from that of the Western medicine group, and the present invention does not require invasive administration, the cost is more economical, and it is naturally safe. The beneficial effects of the present invention are obvious.

[0119] 3.3 Experimental results and discussion on liver and periteriate white fat / brown fat weight

[0120] like Figure 3 In the compound essential oil inhalation administration group provided in Example 3 of the present invention and the compound essential oil transdermal administration group provided in Example 3 of the present invention, the weight of the liver and peritesticular white fat (eWAT) of db / db mice was significantly reduced (P < 0.01), the weight of the liver and peritesticular white fat of HFD mice was significantly reduced (P < 0.01), and the weight of the brown fat (BAT) of db / db mice and HFD mice was significantly increased (P < 0.01); among them, the weight of the liver, peritesticular white fat and brown fat of the transdermal administration group of the compound essential oil provided in Example 3 of the present invention was close to that of the Western medicine group.

[0121] After the above db / db mice and HFD mice were intervened by the compound essential oil provided in Example 3 of the present invention, the weight of the liver and peritesticular white fat of the mice was reduced by an average of about 30-50%, and the weight of the brown fat was increased by an average of about 50%. After the intervention with the comparison essential oil, the weight of the liver and peritesticular white fat was reduced, and the weight of the brown fat was increased, but the performance of the changes was not as good as the compound essential oil provided in Example 3 of the present invention. It is speculated that there is a synergistic effect between the agarwood and the common formula components in the present invention. The Western medicine group used semaglutide injection, and the significant effects of semaglutide in reducing blood sugar and weight loss are widely recognized worldwide. The intervention effect of the compound essential oil provided in Example 3 of the present invention on obesity is not significantly different from that of the Western medicine group, and the present invention does not require invasive administration, the cost is more economical, and it is naturally safe. The beneficial effects of the present invention are obvious.

[0122] 3.4 HE staining experimental results and discussion

[0123] like Figure 4 As described, in the compound essential oil inhalation administration group provided in Example 3 of the present invention and the compound essential oil transdermal administration group provided in Example 3 of the present invention, the liver fatty degeneration problem of db / db mice and HFD mice was significantly improved, the accumulation of white fat droplets was reduced, and the accumulation of brown fat droplets was increased; the abdominal skin of db / db mice in Figure A, i.e., the administration site, did not show obvious damage and inflammatory cell infiltration, it can be inferred that the compound essential oil provided in Example 3 of the present invention did not cause skin irritation through transdermal administration, and the ingredients were safe and reliable.

[0124] 3.5 Results and discussion of serum biochemical indicators detected by fully automatic biochemical analyzer or ELISA

[0125] like Figures 5 to 7As shown, in the compound essential oil inhalation administration group provided in Example 3 of the present invention and the compound essential oil transdermal administration group provided in Example 3 of the present invention, the serum glucose metabolism pathway GLU and INS levels of db / db mice and HFD mice showed significant decreases (P<0.01), the serum lipid metabolism pathway TC, TG, HDL-C, and LDL-C levels showed extremely significant decreases (P<0.01), the liver function AST and ALT levels showed significant decreases (P<0.05), the serum "brain-gut axis" appetite pathway LEP, GLP-1, GIP, GCG, and GDF15 levels showed extremely significant decreases (P<0.01), the bile acid metabolism pathway FXR level showed an extremely significant decrease (P<0.01), and the serum fat heat production pathway UCP1 level showed an extremely significant decrease (P<0.01).

[0126] The levels of various pharmacodynamic mechanism indicators of the transdermal administration group of the compound essential oil provided in Example 3 of the present invention are close to those of the western medicine group, and the western medicine group uses semaglutide injection. The significant effects of semaglutide in lowering blood sugar and reducing weight are widely recognized worldwide. The intervention effect of the compound essential oil provided in Example 3 of the present invention on obesity is not significantly different from that of the western medicine group. In addition, the present invention does not require invasive administration, is more cost-effective, and is naturally safe. The beneficial effects of the present invention are obvious.

[0127] 3.6 Immunofluorescence staining results and discussion

[0128] like Figure 8 As shown, the transdermal administration group of the compound essential oil provided in Example 3 of the present invention can significantly increase the expression level of LEPR-positive cells in the hypothalamus of the appetite pathway of the "brain-gut axis" of HFD mice (P < 0.01); increase the expression level of GLP-1R and GIPR-positive cells in the hypothalamus of db / db mice (P > 0.05), significantly increase the expression level of GCGR and GDF15-positive cells in the liver (P < 0.05), significantly increase the expression level of FXR-positive cells in the liver of the bile acid metabolism pathway (P < 0.05), and increase the expression level of UCP1-positive cells in the white fat of the fat thermogenesis pathway (P > 0.05).

[0129] 3.7 RT-qPCR experimental results and discussion

[0130] like Fig. 9As shown, the transdermal administration group of the compound essential oil provided in Example 3 of the present invention can significantly increase the expression level of LEPR mRNA in the hypothalamus of the appetite pathway of the "brain-gut axis" of HFD mice (P < 0.01); significantly increase the expression level of GCGR and GDF15 mRNA in the liver of db / db mice (P < 0.05), increase the mRNA expression level of GLP-1R and GIPR (P > 0.05), significantly increase the expression of FXR mRNA positive cells in the liver of the bile acid metabolism pathway (P < 0.05), and increase the expression of UCP1 mRNA in the brown fat of the fat thermogenesis pathway (P > 0.05).

[0131] In summary, according to the experimental results and discussions of 3.1 to 3.3, by adopting the present invention, after 6 or 8 weeks of intervention in obesity by inhalation and transdermal administration, the body weight, feeding efficiency and fasting blood sugar of db / db mice or HFD mice can be significantly reduced, and the glucose tolerance and insulin sensitivity of HFD mice can be significantly increased; by adopting the present invention, the weight of liver and peritesticular white fat of db / db mice and HFD mice is significantly reduced, and the weight of brown fat is significantly increased; these experimental results are better than the experimental results of the comparative essential oils, and it is speculated that there is a synergistic effect between agarwood and common formula components in the present invention. In the conventional technical means of professionals in this field, agarwood essential oil is generally used for relaxation, anti-depression and mood regulation, and has obvious effects on poor sleep quality or anxiety resistance. The present invention utilizes the synergistic effect between agarwood and other components to achieve unexpected results in the intervention of obesity.

[0132] The experimental results of the compound essential oil groups provided in Example 3 of the present invention were compared with those of the western medicine group, showing no significant difference or close to the experimental performance of the western medicine group. The western medicine group used semaglutide injection. The significant effects of semaglutide in lowering blood sugar and reducing weight are widely recognized worldwide. In addition, the present invention does not require invasive administration, is more cost-effective, and is naturally safe. It can still show a similar efficacy to semaglutide. The beneficial effects of the present invention are obvious.

[0133] According to the experimental results and discussions in 3.4 to 3.7, by adopting the present invention, the fatty degeneration of the liver of db / db mice or HFD is significantly improved, the accumulation of white fat droplets is reduced, the accumulation of brown fat droplets is increased, and no skin irritation is observed by transdermal administration; by adopting the present invention, the expression levels of the pharmacodynamic mechanism indicators related to the glucose and lipid metabolism pathway, liver function, "brain-gut axis" appetite pathway, bile acid metabolism pathway, and fat heat production pathway of db / db mice and HFD mice are significantly improved.

[0134] Therefore, it can be inferred that the compound essential oil for intervening in obesity provided in the embodiment of the present invention can achieve an effect better than the compound essential oil in related studies and close to that of semaglutide in the intervention of obesity, and its pharmacodynamic mechanism is based on the "brain-gut axis" appetite pathway LEP / GLP-1 / GIP / GCG / GDF15, bile acid metabolism pathway FXR and fat heat production pathway UCP1 to regulate the homeostasis of glucose and lipid metabolism. Combined with the theoretical basis described above, the beneficial effects of the present invention are scientific and reliable, and are not obtained by professionals in this field through simple association by arbitrarily adding or subtracting drugs based on conventional formulas, but are obtained through the inventor's repeated research and practice.

[0135] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be adjusted, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A compound essential oil for intervening obesity, characterized in that: Includes by weight: 8-30 parts of agarwood essential oil; A first component, the first component comprising cyperus rotundus essential oil and ginger essential oil; A second component, wherein the second component includes tangerine peel essential oil, green peel essential oil, Zanthoxylum essential oil, atractylodes essential oil and fructus aurantii essential oil; The mass ratio of the agarwood essential oil, the first component and the second component is 3:3:

10.

2. The compound essential oil for intervening obesity according to claim 1, characterized in that: It also includes a third component by weight, wherein the third component includes Magnolia bark essential oil and Curcuma zedoaria essential oil, and the mass ratio of the third component to the agarwood essential oil, the first component and the second component is 2:3:3:

10.

3. The compound essential oil for intervening obesity according to claim 2, characterized in that: Includes by weight: 6-20 parts of the tangerine peel essential oil, 6-20 parts of the green peel essential oil, 6-20 parts of the Zanthoxylum essential oil, 6-20 parts of the atractylodes essential oil, 6-20 parts of the fructus aurantii essential oil, 4-15 parts of the cyperus rotundus essential oil, 4-15 parts of the ginger essential oil, 2-10 parts of the magnolia bark essential oil and 2-10 parts of the zedoary turmeric essential oil.

4. The compound essential oil for intervening obesity according to claim 2, characterized in that: Includes by weight: 9-25 parts of the agarwood essential oil, 8-15 parts of the tangerine peel essential oil, 8-15 parts of the green peel essential oil, 8-15 parts of the Zanthoxylum essential oil, 8-15 parts of the atractylodes essential oil, 8-15 parts of the fructus aurantii essential oil, 5-12 parts of the cyperus rotundus essential oil, 5-12 parts of the ginger essential oil, 3-9 parts of the magnolia bark essential oil and 3-9 parts of the zedoaria essential oil.

5. The compound essential oil for intervening obesity according to claim 2, characterized in that, Includes by weight: 10-21 parts of the agarwood essential oil, 9-13 parts of the tangerine peel essential oil, 9-13 parts of the green peel essential oil, 9-13 parts of the Zanthoxylum essential oil, 9-13 parts of the atractylodes essential oil, 9-13 parts of the fructus aurantii essential oil, 6-10 parts of the cyperus rotundus essential oil, 6-10 parts of the ginger essential oil, 5-8 parts of the magnolia bark essential oil and 5-8 parts of the zedoaria essential oil.

6. The compound essential oil for intervening obesity according to claim 2, characterized in that: The preparation methods of the agarwood essential oil, the tangerine peel essential oil, the green peel essential oil, the Zanthoxylum bungeanum essential oil, the atractylodes lancea essential oil, the fructus aurantii essential oil, the cyperus rotundus essential oil, the ginger essential oil, the magnolia bark essential oil and the zedoaria essential oil include any one or more of steam distillation, pressing, supercritical extraction and oil separation.

7. Use of a compound essential oil in preparing a drug for intervening obesity, characterized in that: The type of the medicine is an inhalation preparation or a body surface ointment, and the compound essential oil is the compound essential oil for intervening obesity as described in any one of claims 1-5.

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