Compound essential oil for intervening obesity and application thereof

Abstract

The application relates to the technical field of traditional Chinese medicine, in particular to a compound essential oil for intervening in obesity and application. The compound essential oil for intervening in obesity comprises, in terms of weight fractions, 8-30 parts of agarwood essential oil; a first component, the first component comprising essential oil of Cyperus rotundus L. and essential oil of ginger; and a second component, the second component comprising essential oil of Citrus reticulata, essential oil of Citrus reticulata var. sarcodactylis, essential oil of Zanthoxylum bungeanum, essential oil of Atractylodes lancea and essential oil of Citrus aurantium; the mass ratio of the agarwood essential oil, the first component and the second component is 3:3:10. The application adjusts and improves the compatibility of traditional Chinese medicine essential oils in related research under the guidance of the theory of traditional Chinese medicine aromatherapy, and remarkable therapeutic effects are achieved on obesity through the synergistic effect between agarwood and common formula components.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine, and particularly relates to a compound essential oil for intervening obesity and application. BACKGROUND

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

[0003] Traditional Chinese medicine essential oil is a complementary and alternative therapy for treating obesity based on the rules of traditional Chinese medicine aromatherapy combined with western medicine mechanisms, and is also a potential effective way to treat obesity. In related research, compound essential oils generally have complex raw materials and are not specific enough in treating obesity, and the efficacy is not obvious.

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

[0005] To solve the above problems, the purpose of the present application is to provide a compound essential oil for intervening obesity and application. The present application adjusts and improves the compatibility of traditional Chinese medicine essential oil in related research, and achieves significant therapeutic effect on obesity through the synergistic effect between eaglewood and common formula components.

[0006] To this end, the present application adopts the following technical solutions:

[0007] A compound essential oil for intervening obesity, which is composed of the following components by weight fraction:

[0008] Eaglewood essential oil 10-21 parts, orange peel essential oil 9-13 parts, green peel essential oil 9-13 parts, Sichuan pepper essential oil 9-13 parts, atractylodes essential oil 9-13 parts, citron essential oil 9-13 parts, cyperus rotundus essential oil 6-10 parts, ginger essential oil 6-10 parts, magnolia officinalis essential oil 5-8 parts, and zedoary essential oil 5-8 parts.

[0009] In some embodiments, the preparation method of the eaglewood essential oil, the orange peel essential oil, the green peel essential oil, the Sichuan pepper essential oil, the atractylodes essential oil, the citron essential oil, the cyperus rotundus essential oil, the ginger essential oil, the magnolia officinalis essential oil, and the zedoary essential oil includes any one or more of steam distillation, pressing method, and supercritical extraction method.

[0010] Meanwhile, the application also provides application of the compound essential oil in preparation of a medicine for intervening obesity, characterized in that the medicine is an inhalation preparation or a body surface liniment, and the compound essential oil is any one of the compound essential oils for intervening obesity as described above.

[0011] Compared with related obesity prevention or treatment means, the application has obvious beneficial effects, and specifically, it is verified that after 6 or 8 weeks of intervention of obesity by sniffing and transdermal administration, the body weight, food intake efficiency and fasting blood glucose of db / db mice or HFD mice can be significantly reduced, and the glucose tolerance and insulin sensitivity of HFD mice can be significantly improved; by using the application, the liver and eWAT weight of db / db mice and HFD mice can be significantly reduced, and the BAT weight can be significantly increased; by using the application, the liver steatosis of db / db mice or HFD can be significantly improved, the white fat droplet accumulation can be reduced, the brown fat droplet accumulation can be increased, and the transdermal administration mode does not cause skin irritation; by using the application, the expression levels of the pharmacodynamic mechanism index 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 can be significantly improved. Under the guidance of the theory of traditional Chinese medicine aromatherapy, the essential oil compatibility of traditional Chinese medicine in related research is adjusted and improved, and through the synergistic effect between eaglewood and common formula components, a significant therapeutic effect on obesity is achieved. BRIEF DESCRIPTION OF DRAWINGS

[0012] In order to more clearly illustrate the beneficial effects of the embodiments of the application, the following will briefly introduce the drawings obtained after experimental verification of the embodiments. Obviously, the drawings described below are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0013] Figure 1 In order to respectively use two kinds of compound essential oils and western medicines on experimental mice, the results of the body weight and food intake efficiency experiments of the two kinds of mice are shown in the schematic diagram, wherein Figures A and C are the results of db / db mouse research, and Figures B and D are the results of HFD mouse research.

[0014] Figure 2 In order to respectively use two kinds of compound essential oils and western medicines on experimental mice, the results of the fasting blood glucose, glucose tolerance and insulin sensitivity experiments of the two kinds of mice are shown in the schematic diagram, wherein Figures A, C and E are the results of db / db mouse research, and Figures B, D and F are the results of HFD mouse research.

[0015] Figure 3Figures 1A-1F are diagrams showing the results of weight analysis of liver and peritesticular white / brown fat of mice treated with two kinds of compound essential oils and western medicine, wherein Figures 1A, 1C, 1E are the results of db / db mice research, and Figures 1B, 1D, 1F are the results of HFD mice research.

[0016] Figure 4 Figures 2A-2B are diagrams showing the results of HE staining analysis of liver, peritesticular white / brown fat of mice treated with two kinds of compound essential oils and western medicine, and abdominal skin of db / db mice, wherein Figure 2A is the results of db / db mice research, and Figure 2B is the results of HFD mice research.

[0017] Figure 5 Figures 3A-3H are diagrams showing the results of serum pharmacodynamic index analysis of db / db mice treated with two kinds of compound essential oils and western medicine by automatic biochemical analyzer or ELISA, wherein Figure 3A shows GLU level, Figure 3B shows INS level, Figure 3C shows TC level, Figure 3D shows TG level, Figure 3E shows LDL-C level, Figure 3F shows HDL-C level, Figure 3G shows AST level, and Figure 3H shows ALT level.

[0018] Figure 6 Figures 4A-4H are diagrams showing the results of serum pharmacodynamic index analysis of HFD mice treated with two kinds of compound essential oils and western medicine by automatic biochemical analyzer or ELISA, wherein Figure 4A shows GLU level, Figure 4B shows INS level, Figure 4C shows TC level, Figure 4D shows TG level, Figure 4E shows LDL-C level, Figure 4F shows HDL-C level, Figure 4G shows AST level, and Figure 4H shows ALT level.

[0019] Figure 7 Figures 5A-5G are diagrams showing the results of serum mechanism index analysis of db / db mice treated with two kinds of compound essential oils and western medicine by automatic biochemical analyzer or ELISA, wherein Figure 5A shows LEP level, Figure 5B shows GLP-1 level, Figure 5C shows GIP level, Figure 5D shows GCG level, Figure 5E shows GDF15 level, Figure 5F shows FXR level, and Figure 5G shows UCP1 level.

[0020] Figure 8The diagram shows the results of immunofluorescence staining analysis of two types of mice after applying two compound essential oils and Western medicines respectively. Figure A is the immunofluorescence staining diagram, Figure B shows the expression level of LEPR positive cells, Figure C shows the expression level of GLP-1R positive cells, Figure D shows the expression level of GIPR positive cells, Figure E shows the expression level of GCGR positive cells, Figure F shows the expression level of GDF15 positive cells, Figure G shows the expression level of FXR positive cells, and Figure H shows the expression level of UCP1 positive cells.

[0021] Figure 9 The diagram shows the results of RT-qPCR analysis of two types of mice after applying two compound essential oils and Western medicines respectively. Figure A shows the expression level of LEPR mRNA, Figure B shows the expression level of GLP-1R mRNA, Figure C shows the expression level of GIPR mRNA, Figure D shows the expression level of GCGR mRNA, Figure E shows the expression level of DF15 mRNA, Figure F shows the expression level of FXR mRNA, and Figure G shows the expression level of UCP1 mRNA.

Detailed Implementation Methods

[0022] Embodiment 1 of the present invention

[0023] A compound essential oil for the intervention of obesity, comprising, by weight: 8-30 parts agarwood essential oil, 6-20 parts dried tangerine peel essential oil, 6-20 parts green tangerine peel essential oil, 6-20 parts Sichuan pepper essential oil, 6-20 parts Atractylodes lancea essential oil, 6-20 parts Citrus aurantium essential oil, 4-15 parts Cyperus rotundus essential oil, 4-15 parts fresh ginger essential oil, 2-10 parts Magnolia officinalis essential oil, and 2-10 parts Curcuma zedoaria essential oil.

[0024] It should be noted that, in this embodiment, the agarwood essential oil, the tangerine peel essential oil, the green tangerine peel essential oil, the Sichuan pepper essential oil, the Atractylodes lancea essential oil, the bitter orange peel essential oil, the Cyperus rotundus essential oil, the ginger essential oil, and the Curcuma zedoaria essential oil can be prepared by steam distillation; in other embodiments, they can also be obtained by pressing, supercritical extraction, and oil separation methods, or one or more of these methods.

[0025] It is understood that the agarwood essential oil, the dried tangerine peel essential oil, the green tangerine peel essential oil, the Sichuan pepper 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 purchased finished essential oils obtained through the above preparation methods or homemade essential oils obtained by oneself through the above preparation methods.

[0026] Embodiment 2 of the present invention

[0027] A compound essential oil for intervening obesity, comprising: 9-25 parts by weight of linaloe essential oil, 8-15 parts by weight of dried tangerine peel essential oil, 8-15 parts by weight of green tangerine peel essential oil, 8-15 parts by weight of Sichuan pepper essential oil, 8-15 parts by weight of Chinese atractylodes rhizome essential oil, 8-15 parts by weight of immature orange fruit essential oil, 5-12 parts by weight of Chinese cinnamon bark essential oil, 5-12 parts by weight of ginger essential oil, 3-9 parts by weight of magnolia officinalis essential oil and 3-9 parts by weight of zedoary essential oil.

[0028] It should be noted that in the present embodiment, the preparation method of the linaloe essential oil, the dried tangerine peel essential oil, the green tangerine peel essential oil, the Sichuan pepper essential oil, the Chinese atractylodes rhizome essential oil, the immature orange fruit essential oil, the Chinese cinnamon bark essential oil, the ginger essential oil and the zedoary essential oil can be steam distillation method; in other embodiments, any one or more of pressing method, supercritical extraction method and oil separation method can also be used.

[0029] It can be understood that the linaloe essential oil, the dried tangerine peel essential oil, the green tangerine peel essential oil, the Sichuan pepper essential oil, the Chinese atractylodes rhizome essential oil, the immature orange fruit essential oil, the Chinese cinnamon bark essential oil, the ginger essential oil and the zedoary essential oil can be purchased as finished essential oil obtained by the above preparation method, or can be self-made essential oil obtained by the above preparation method.

[0030] Embodiment 3 of the present application

[0031] A compound essential oil for intervening obesity, comprising: 10-21 parts by weight of linaloe essential oil, 9-13 parts by weight of dried tangerine peel essential oil, 9-13 parts by weight of green tangerine peel essential oil, 9-13 parts by weight of Sichuan pepper essential oil, 9-13 parts by weight of Chinese atractylodes rhizome essential oil, 9-13 parts by weight of immature orange fruit essential oil, 6-10 parts by weight of Chinese cinnamon bark essential oil, 6-10 parts by weight of ginger essential oil, 5-8 parts by weight of magnolia officinalis essential oil and 5-8 parts by weight of zedoary essential oil.

[0032] It should be noted that in the present embodiment, the preparation method of the linaloe essential oil, the dried tangerine peel essential oil, the green tangerine peel essential oil, the Sichuan pepper essential oil, the Chinese atractylodes rhizome essential oil, the immature orange fruit essential oil, the Chinese cinnamon bark essential oil, the ginger essential oil and the zedoary essential oil can be steam distillation method; in other embodiments, any one or more of pressing method, supercritical extraction method and oil separation method can also be used.

[0033] It can be understood that the linaloe essential oil, the dried tangerine peel essential oil, the green tangerine peel essential oil, the Sichuan pepper essential oil, the Chinese atractylodes rhizome essential oil, the immature orange fruit essential oil, the Chinese cinnamon bark essential oil, the ginger essential oil and the zedoary essential oil can be purchased as finished essential oil obtained by the above preparation method, or can be self-made essential oil obtained by the above preparation method.

[0034] The pathogenesis of obesity is multifactorial and multi-mechanism, involving genetics, environment, lifestyle, endocrine regulation, inflammatory response and intestinal flora, etc. Studies have shown that obesity increases the risk of type 2 diabetes by affecting beta cell function, adipose tissue biology and multi-organ insulin resistance, and these changes can be improved or even normalized by appropriate weight loss.

[0035] Obesity is a heterogeneous disease, and common related factors include genetics, epigenetics, neurobehavior, endocrinology, and metabolism. Current clinical drug candidates focus on four target areas: leptin (LEP), ghrelin, mitochondrial uncoupling agents, and growth differentiation factors. Studies have shown that GIPR / GLP-1R synergistic agonist tirzepatide can reduce the body weight of obese patients by more than 20%; GIPR / GLP-1R / GCGR agonists (triple agonists) that target appetite homeostasis regulators and intestinal insulin-promoting molecules in the gut-brain axis can further enhance the efficacy of dual GIPR and GLP-1R agonists, improve energy consumption, weight loss, and blood glucose control. GDF15 targets the regulation of calcium ion ineffective circulation in muscle tissue through the adrenergic receptor axis, resists the reduction of energy consumption caused by diet control, and thus solves the weight rebound bottleneck problem of obesity treatment. FXR forms a systematic regulation mechanism for systemic metabolism by activating the signaling pathway network of intestinal, liver, and neuronal hormones. UCP1 uncouples oxidative phosphorylation from ATP synthesis, thereby dissipating energy as heat.

[0036] In order to fully embody the beneficial effects of the technical scheme of the present application, the present application also uses the scheme provided in Embodiment 3 to prepare a compound essential oil from a certain formula selected therefrom, and compares and verifies the compound essential oil with a comparative essential oil. The components of the comparative essential oil are adopted in many related studies and are easily predictable by a person skilled in the art as a drug compatibility, including essential oils of pericarpium citri reticulatae, pericarpium citri vulgaris, zanthoxylum bungeanum, atractylodes lancea and fructus aurantii, each in an amount of 9-13 parts by weight.

[0037] After experimental verification, the obtained experiments are as follows:

[0038] 1 Experimental process

[0039] 1.1 Experimental animals

[0040] db / db mouse study: db / db mice, 5 weeks old, male, body weight 20-30 g.

[0041] HFD mouse study: C57BL / 6J mice fed with high-fat diet for 8 weeks, 13 weeks old, male, body weight 30-35 g.

[0042] Blank study: C57BL / 6J mice, 5 weeks old, male, body weight 15-20 g;

[0043] C57BL / 6J mice, 13 weeks old, male, body weight 25-30 g.

[0044] Experimental animal production license number: SCXK(Su) 2020-0009, Jiangsu Huacheng Xinnuo Pharmaceutical Technology Co., Ltd.

[0045] 1.2 Animal grouping and establishment of pathological model

[0046] The db / db mouse model is a genetic obesity model used to simulate human type 2 diabetes. In this experiment, 5-week-old male db / db mice were fed with ordinary feed to establish a db / db mouse model.

[0047] The HFD mouse model is an obesity model induced by 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 with high-fat feed until they were 13 weeks old to establish an HFD mouse model.

[0048] db / db mice and HFD mice were used as described above. Various mice were fed with ordinary feed or high-fat feed according to the grouping, and food and water were unlimited. The light and dark were 12 h, and the constant temperature was 22℃. Randomly divided into the following groups, n=7-9.

[0049] db / db mice were grouped as follows: blank group (CON), model group (MOD), western medicine group (SEMA), comparative essential oil sniffing administration group (CXEO_HD_I), compound essential oil sniffing administration group provided by Example 3 of the present application (CEO_HD_I), comparative essential oil transdermal administration group (CXEO_HD_T), and compound essential oil transdermal administration group provided by Example 3 of the present application (CEO_HD_T).

[0050] HFD mice were grouped as follows: blank group (CON), model group (MOD), western medicine group (SEMA), comparative essential oil sniffing administration group (CXEO_HD_I), compound essential oil sniffing administration group provided by Example 3 of the present application (CEO_HD_I), comparative essential oil transdermal administration group (CXEO_HD_T), and compound essential oil transdermal administration group provided by Example 3 of the present application (CEO_HD_T).

[0051] 1.3 Administration method and dosage

[0052] First, the efficacy experiment was conducted to screen the optimal dose of efficacy from low and high doses as the administration dose of the compound essential oil, i.e., the administration dose of the compound essential oil and the comparative essential oil provided by Example 3 of the present application.

[0053] 1.3.1 Sniffing administration

[0054] The main body of the aromatherapy sniffing device is a small transparent organic glass box with a size of 20x20x20 cm. The atomizer is placed in the middle of the small box, which is used to hold essential oil. The essential oil is the compound essential oil for intervening obesity provided in Example 3 of the application or the comparative essential oil. The essential oil is added to the spray pot of the atomizer, distilled water is added for dilution, and then the mice are placed in the small box. The atomizer continuously atomizes the essential oil. When the spray pot of the atomizer no longer produces mist, the small box is opened and the mice are taken out. At this time, the sniffing is completed, and the sniffing time of each group is 1 h.

[0055] Among them, for db / db mice and HFD mice, the administration concentration of the comparative essential oil sniffing administration group and the compound essential oil sniffing administration group provided in Example 3 of the application is 260 uL, once a day, and continuous administration for 8 weeks.

[0056] 1.3.2 Transdermal administration

[0057] 24 h before the experiment, the mice were depilated on the abdomen, and the pre-administration treatment was completed. The depilated area of the mouse was used as the administration site, and essential oil was applied to the depilated area. The essential oil was the compound essential oil provided in Example 3 of the application or the comparative essential oil.

[0058] Among them, for HFD mice grouping, the transdermal administration concentration of the comparative essential oil transdermal administration group and the compound essential oil transdermal administration group provided in Example 3 of the application is 24% and 12%, respectively. The administration dose is 0.4 mL for the first 4 weeks and 0.8 mL for the last 4 weeks, once a day, and continuous administration for 8 weeks.

[0059] For db / db mice grouping, the transdermal administration concentration of the comparative essential oil transdermal administration group and the compound essential oil transdermal administration group provided in Example 3 of the application is 24% and 12%, respectively. The administration dose is 0.4 mL for the first 4 weeks and 0.8 mL for the last 4 weeks, once a day, and continuous administration for 8 weeks.

[0060] 1.3.3 Western medicine injection

[0061] For db / db mice, subcutaneous injection of semaglutide, according to the body surface area conversion of adult administration dose, the administration dose is 0.08 mg / kg, once every 3 days, and continuous administration for 6 weeks.

[0062] For HFD mice, subcutaneous injection of semaglutide, according to the body surface area conversion of adult administration dose, the administration dose is 0.08 mg / kg for the first 4 weeks and 0.16 mg / kg for the last 4 weeks, once every 3 days, and continuous administration for 8 weeks.

[0063] 1.4 Experimental methods

[0064] 1.4.1 Body weight, food intake efficiency experiment

[0065] The body weight of the mice was measured twice a week, and the average value was taken as the data of the body weight per week. The food intake of the mice in each cage was recorded twice a week, and the average value was taken to calculate the average daily food intake. The food intake efficiency of the mice was calculated according to the formula: food intake efficiency = total food intake / total body weight.

[0066] 1.4.2 Fasting blood glucose, glucose tolerance test and insulin sensitivity test

[0067] a. Fasting blood glucose test

[0068] After the last drug intervention, the mice in each group were fasted for 10 h, and the blood glucose of the mice in the compound essential oil sniffing and dosing group provided in Example 3 of the application was detected by using blood glucose test paper.

[0069] b. Glucose tolerance test

[0070] After the last drug intervention, the mice in each group were fasted for 10 h, and the mice in the compound essential oil transdermal dosing group provided in Example 3 of the application were given intraperitoneal injection of 50% high-sugar solution at a dose of 2 mg / g, and the tail vein blood glucose of the mice was detected by using blood glucose test paper at 0 min, 30 min, 60 min, 90 min and 120 min after injection, respectively.

[0071] c. Intraperitoneal insulin sensitivity test

[0072] After the last drug intervention, the mice in each group were fed for about 4-5 h, and the mice in the compound essential oil transdermal dosing group provided in Example 3 of the application were given intraperitoneal injection of insulin solution at the calculated given dose, and the tail vein blood glucose of the mice was detected by using blood glucose test paper at 0 min, 30 min, 60 min, 90 min and 120 min after injection, respectively.

[0073] 1.4.3 Liver, peritesticular white adipose / brown adipose weight experiment

[0074] After the last drug intervention, the mice in each group were fasted for 10 h, and the mice were anesthetized by using 0.3% sodium pentobarbital at a dose of 0.1 mL / 10 g, blood was taken through the retro-orbital vein, and the mice were sacrificed by cervical dislocation. The mice were dissected, and the hypothalamus, abdominal skin, liver, peritesticular white adipose tissue and brown adipose tissue were removed, and the liver, peritesticular white adipose tissue and brown adipose tissue were weighed and analyzed, respectively.

[0075] 1.4.4 HE staining experiment

[0076] 1) Rewarming of frozen sections; 2) hematoxylin staining; 3) eosin staining; 4) dehydration and mounting; 5) Image-Pro Plus 6.0 analysis software results interpretation: the cell nucleus is blue, and the cytoplasm is red.

[0077] 1.4.5 Automatic biochemical analyzer or ELISA for detecting serum biochemical indicators

[0078] After the last drug intervention, the mice were fasted for 10 h, anesthetized with 0.3% sodium pentobarbital at a dose of 0.1 mL / 10 g, and the blood was taken by enucleation. The obtained blood was placed at room temperature for 1 h, and then centrifuged at a speed of 4000 r·min-1 at 4 ℃ for 20 min

[0079] The upper serum was stored in a -80 ℃ ultra-low temperature refrigerator for long-term preservation. The automatic biochemical analyzer or ELISA was used to detect the following biochemical indicators according to the kit instructions.

[0080] Sugar metabolism pathway: GLU, INS

[0081] Lipid metabolism pathway: TC, TG, HDL-C, LDL-C

[0082] Liver function: AST, ALT

[0083] “Brain-gut axis” appetite pathway: LEP, GLP-1, GIP, GCG, GDF15

[0084] Bile acid metabolism pathway: FXR

[0085] Fat heat production pathway: UCP1

[0086] 1.4.6 Immunofluorescence staining experiment

[0087] 1) Fresh tissue embedding; 2) tissue sectioning; 3) frozen section fixation; 4) antigen repair; 5) drawing a circle to block serum; 6) adding a primary antibody; 7) adding a secondary antibody; 8) DAPI re-staining the cell nucleus; 9) mounting; 10) microscopic examination and photography: observing the section under a fluorescence microscope and collecting images; 11) Image-Pro Plus 6.0 analysis software results interpretation: the cell nucleus stained by DAPI is blue under ultraviolet excitation, and the positive expression is red light corresponding to the fluorescence label.

[0088] 1.4.7 RT-qPCR experiment

[0089] 1) Extraction of total RNA from samples

[0090] The total RNA extraction reagent TRizol was used for sample RNA extraction, and the experimental operation was performed according to the product instructions.

[0091] 2) Reverse transcription to synthesize cDNA

[0092] cDNA reverse transcription was performed using the TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit, and the experimental procedures were performed according to the product instructions.

[0093] 3) Real-Time PCR Sample Detection

[0094] Using the ChamQ Universal SYBR qPCR Master Mix kit, all cDNA samples were prepared into Realtime PCR reaction systems. During the preparation of the reaction solution and sample loading, all reagents and 96-well plates were placed on ice.

[0095] Adding samples;

[0096] The 96-PCR plate was placed on a Realtime PCR instrument for PCR reaction. The PCR reaction program was: 95 ℃ for 10 minutes; 45 PCR cycles, wherein the parameters of the PCR cycle were set to 95 ℃ for 15 s; 60 ℃ for 60 s.

[0097] 4) The target gene and internal control of each sample were subjected to Realtime PCR reaction.

[0098] 2. Statistical Analysis

[0099] All experimental results are expressed as Mean ± SD. Statistical analysis was performed using SPSS 21.0 software. One-way ANOVA was used for intergroup comparisons, and P < 0.05 was considered statistically significant.

[0100] Note: Compared with the control group, bP < 0.05, abP < 0.01; compared with the model group, cP < 0.05, acP < 0.01.

[0101] 3. Experimental Results and Discussion

[0102] 3.1 Results and Discussion of Experiments on Body Weight and Feeding Efficiency

[0103] like Figure 1 As shown, in the compound essential oil inhalation administration group and the compound essential oil transdermal administration group provided in Example 3 of the present invention, intervention with obese db / db mice for 6 weeks can significantly reduce the weight of the mice (P<0.01), and there is no significant difference in 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.

[0104] The compound essential oil inhalation administration group provided by the embodiment 3 of the present application and the compound essential oil transdermal administration group provided by the embodiment 3 of the present application can significantly reduce the weight of the HFD mice (P<0.01) after 8 weeks of intervention, and the weight of the transdermal administration group has no significant difference (P>0.05) compared with the western medicine group; and can reduce the food intake efficiency, and the food intake efficiency of the transdermal administration group is close to that of the western medicine group.

[0105] After the above db / db mice and HFD mice are intervened by the compound essential oil provided by the embodiment 3 of the present application, the weight is reduced by about 30%, while after the intervention by the comparative essential oil, the weight is also reduced, but the weight change is not as good as that of the compound essential oil provided by the embodiment 3 of the present application, and the difference is greater when the transdermal administration is used, it is speculated that there is a synergistic effect between the eaglewood and the common formula components in the present application. The western medicine group uses semeglu peptide injection, and the significant effect of semeglu peptide in reducing blood sugar and weight is widely recognized worldwide, the intervention effect of the compound essential oil provided by the embodiment 3 of the present application on obesity has no significant difference compared with the western medicine group, and the present application does not need invasive administration, is more economical in cost, is safe and natural, and the beneficial effects of the present application are obvious.

[0106] 3.2 Fasting blood glucose, glucose tolerance test and insulin sensitivity test results and discussion

[0107] As shown in Figure 2 , the compound essential oil inhalation administration group provided by the embodiment 3 of the present application can significantly reduce the fasting blood glucose level of the db / db mice and the HFD mice (P<0.05), and the fasting blood glucose level of the HFD mice has no significant difference (P>0.05) compared with the western medicine group; the compound essential oil transdermal administration group provided by the embodiment 3 of the present application can significantly improve the glucose tolerance of the db / db mice and the HFD mice (P<0.05), and significantly improve the insulin sensitivity (P<0.05), and the performance is close to that of the western medicine group.

[0108] The fasting blood glucose levels of the above db / db mice and HFD mice are reduced by about 20-30% on average after intervention by the compound essential oil provided in Example 3 of the present application through sniffing administration, while the fasting blood glucose levels are reduced after intervention by the comparative essential oil through sniffing administration, but the change is not as good as that of the compound essential oil provided in Example 3 of the present application, which is presumed that there is a synergistic effect between the eaglewood and the common formula components in the present application. The western medicine group uses semeglu-tide injection, and the significant effect of semeglu-tide in reducing blood sugar and weight is widely recognized worldwide. The intervention effect of the compound essential oil provided in Example 3 of the present application on obesity has no significant difference compared with the western medicine group, and the present application does not need invasive administration, is more economical, and is naturally safe, and the beneficial effects of the present application are obvious.

[0109] 3.3 Experimental results and discussion of liver, peritesticular white fat / brown fat weight

[0110] As Figure 3 mentioned above, in the sniffing administration group of the compound essential oil provided in Example 3 of the present application and the transdermal administration group of the compound essential oil provided in Example 3 of the present application, the liver and peritesticular white fat (eWAT) weights of the db / db mice are significantly reduced (P<0.01), the liver and peritesticular white fat weights of the HFD mice are significantly reduced (P<0.01), and the brown fat (BAT) weights of the db / db mice and HFD mice are significantly increased (P<0.01); wherein the liver, peritesticular white fat and brown fat weights of the transdermal administration group of the compound essential oil provided in Example 3 of the present application are close to those of the western medicine group.

[0111] As mentioned above, after intervention by the compound essential oil provided in Example 3 of the present application, the liver and peritesticular white fat weights of the db / db mice and HFD mice are reduced by about 30-50% on average, and the brown fat weight is increased by about 50%, while after intervention by the comparative essential oil, the liver and peritesticular white fat weights are reduced, and the brown fat weight is increased, but the change is not as good as that of the compound essential oil provided in Example 3 of the present application, which is presumed that there is a synergistic effect between the eaglewood and the common formula components in the present application. The western medicine group uses semeglu-tide injection, and the significant effect of semeglu-tide in reducing blood sugar and weight is widely recognized worldwide. The intervention effect of the compound essential oil provided in Example 3 of the present application on obesity has no significant difference compared with the western medicine group, and the present application does not need invasive administration, is more economical, and is naturally safe, and the beneficial effects of the present application are obvious.

[0112] 3.4 Experimental results and discussion of HE staining

[0113] As Figure 4In the compound essential oil inhalation administration group and the compound essential oil transdermal administration group provided in Example 3 of the present invention, the liver steatosis problem of db / db mice and HFD mice was significantly improved, the accumulation of white fat droplets decreased, and the accumulation of brown fat droplets increased. In Figure A, the abdominal skin of db / db mice, i.e. the administration site, did not show obvious damage or inflammatory cell infiltration, which suggests that the compound essential oil provided in Example 3 of the present invention did not cause skin irritation when administered transdermally, and the ingredients are safe and reliable.

[0114] 3.5 Results and Discussion of Serum Biochemical Indicators Detected by Fully Automated Biochemical Analyzer or ELISA

[0115] like Figures 5 to 7 As shown, in the compound essential oil inhalation administration group 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 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 extremely significant decreases (P < 0.01), and the serum fat thermogenesis pathway UCP1 level showed extremely significant decreases (P < 0.01).

[0116] The efficacy indicators of the compound essential oil transdermal administration group provided in Example 3 of this invention are close to those of the Western medicine group. The Western medicine group used smegglutide injection. Smegglutide's significant effects in lowering blood sugar and weight loss are widely recognized globally. The intervention effect of the compound essential oil provided in Example 3 of this invention on obesity is not significantly different from that of the Western medicine group. Moreover, this invention does not require invasive drug delivery, is more economical, natural and safe, and the beneficial effects of this invention are obvious.

[0117] 3.6 Results and Discussion of Immunofluorescence Staining Experiment

[0118] like Figure 8As shown, the compound essential oil transdermal administration group provided in Example 3 of this invention can significantly increase the expression level of LEPR-positive cells in the hypothalamus of the appetite pathway in 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 white adipose tissue in the fat thermogenesis pathway (P > 0.05).

[0119] 3.7 Results and Discussion of RT-qPCR Experiments

[0120] like Figure 9 As shown, the compound essential oil transdermal administration group provided in Example 3 of this invention can significantly increase the expression level of LEPR mRNA in the hypothalamus of the appetite pathway in 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 expression level of GLP-1R and GIPR mRNA (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 brown adipose tissue in the fat thermogenesis pathway (P > 0.05).

[0121] In summary, based on the experimental results and discussion in sections 3.1 to 3.3, by employing this invention, after 6 or 8 weeks of inhalation and transdermal administration intervention for obesity, the body weight, feeding efficiency, and fasting blood glucose levels in db / db mice or HFD mice were significantly reduced, while glucose tolerance and insulin sensitivity in HFD mice were significantly increased. Furthermore, by employing this invention, the weight of white adipose tissue in the liver and peritoneal space was significantly reduced, while the weight of brown adipose tissue was significantly increased in both db / db mice and HFD mice. These experimental results are superior to those of the comparative essential oils, suggesting a synergistic effect between agarwood and common formulation components in this invention. In the conventional techniques used by those skilled in the art, agarwood essential oil is commonly used for relaxation, antidepressant effects, and mood regulation, showing significant effects on poor sleep quality or anxiety. This invention utilizes the synergistic effect between agarwood and other components to achieve unexpected results in the intervention of obesity.

[0122] The experimental results of the compound essential oil groups provided by the embodiment 3 of the present application and the western medicine group are compared, and it is shown that there is no significant difference or the experimental performance is close to that of the western medicine group, and the western medicine group uses semeglutide injection. The significant effect of semeglutide in reducing blood sugar and weight is widely recognized worldwide, and under the advantages of non-invasive administration, lower cost and natural safety, the present application can still show similar efficacy to semeglutide. The beneficial effects of the present application are obvious.

[0123] According to the experimental results and discussion of 3.4 to 3.7, by using the present application, the liver steatosis of db / db mice or HFD is significantly improved, the accumulation of white fat lipid droplets is reduced, the accumulation of brown fat lipid droplets is increased, and no skin irritation is found by transdermal administration. By using the present application, the expression levels of the related pharmacodynamic mechanism indicators of the glucose and lipid metabolism pathway, liver function, "brain-gut axis" appetite pathway, bile acid metabolism pathway and fat thermogenesis pathway of db / db mice and HFD mice are significantly improved.

[0124] Therefore, it can be inferred that the compound essential oil for intervening obesity provided by the embodiment of the present application can achieve better effect than the compound essential oil in related research and close to semeglutide in the intervention of obesity. The pharmacodynamic mechanism is based on the regulation of glucose and lipid metabolism homeostasis by "brain-gut axis" appetite pathway LEP / GLP-1 / GIP / GCG / GDF15, bile acid metabolism pathway FXR and fat thermogenesis pathway UCP1. Combined with the theoretical basis described above, the beneficial effects of the present application are scientific and reliable, and are not obtained by simply adding or subtracting drugs based on conventional formulations by professional personnel in the field through simple association, but are obtained through continuous and repeated research and practice by the inventors.

[0125] The above-described embodiments are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still adjust the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A compound essential oil for intervening obesity, characterized in that, consists of the following components by weight parts: Lignum aquilariae resinatum essential oil 10-21 parts, Pericarpium citri reticulatae viride essential oil 9-13 parts, Pericarpium citri reticulatae essential oil 9-13 parts, Zanthoxylum bungeanum essential oil 9-13 parts, Atractylodes lancea essential oil 9-13 parts, Citrus aurantium essential oil 9-13 parts, Cyperus rotundus essential oil 6-10 parts, Zingiber officinale essential oil 6-10 parts, Magnolia officinalis essential oil 5-8 parts, and Curcuma zedoary essential oil 5-8 parts.

2. The compound essential oil for intervening obesity according to claim 1, wherein, The preparation method of the lignum aquilariae resinatum essential oil, the Pericarpium citri reticulatae viride essential oil, the Pericarpium citri reticulatae essential oil, the Zanthoxylum bungeanum essential oil, the Atractylodes lancea essential oil, the Citrus aurantium essential oil, the Cyperus rotundus essential oil, the Zingiber officinale essential oil, the Magnolia officinalis essential oil, and the Curcuma zedoary essential oil includes any one or more of a water vapor distillation method, a pressing method, and a supercritical extraction method.

3. Use of a complex essential oil in the preparation of a medicament for intervening in obesity, characterized in that, The type of the medicine is an inhalation preparation or a body surface liniment, and the compound essential oil is the compound essential oil for intervening obesity according to any one of claims 1-2.

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