Purpose and preparation method of perilla frutescens essential oil and active ingredients thereof

Abstract

The invention relates to application and a preparation method of the essential oil and the effective components thereof, and the essential oil and the effective components thereof can reduce fat, prevent and / or treat obesity, and have better effects on improving insulin resistance, improving glycometabolism, reducing blood sugar and treating diabetes mellitus. The invention further provides application of the east purple perilla essential oil in improving intestinal flora and serving as a scientific research reagent.

Description

Technical Field

[0001] The invention relates to the application and preparation method of oriental perilla essential oil and effective components thereof. Background Art

[0002] Elsholtzia bodinieri (Van.) is a perennial herbaceous plant of the genus Elsholtzia in the Lamiaceae family. It is also known as Phoenix Tail Tea, Small Elsholtzia, and Wild Camellia. The chemical components of Elsholtzia bodinieri are primarily volatile and non-volatile. Non-volatile components include flavonoids, steroids, triterpenes, phenols, tannins, amino acids, peptides, proteins, reducing sugars, polysaccharides, glycosides, and other compounds, as well as trace elements beneficial to the human body. The volatile components of Elsholtzia bodinieri are its essential oil. According to relevant research, the main chemical components of Elsholtzia bodinieri essential oil are α-myrcene, linalool, borneol, β-pinene, α-phellandrene, β-elemenone, thymol, and carvacrol. Terpenes are the most abundant, accounting for 87.64% of the total essential oil. Flavonoids are the most abundant active chemical components of Elsholtzia bodinieri. The total flavonoid content is highest in the root, up to 133.17 mg / g. So far, more than 30 flavonoids (glycosides) have been isolated and identified from Perilla frutescens, mainly flavonoids and dihydroflavonoids.

[0003] Current research on the biological activities of flavonoids from Perilla frutescens primarily focuses on antibacterial, antiviral, antioxidant, and anti-inflammatory properties. Among these, isolated luteolin and quercetin exhibit varying degrees of antibacterial and bactericidal effects against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. Foraminin exhibits significant anti-HCV activity (SI value >500). Total flavonoids from Perilla frutescens can effectively scavenge superoxide anion free radicals (O2). - , H2O2 and DPPH free radicals, and has good in vitro antioxidant effect.

[0004] Some scholars have studied the volatile oils of Perilla frutescens leaves from 15 populations in Yunnan Province. GC-MS detection found that the monoterpene content was 72.5% to 91.8%, the sesquiterpene content was 3.72% to 13.1%, and the main component of the volatile oil was 1,8-cineole. α-Pinpineol may be a precursor for the synthesis of 1,8-cineole and α-pinyl acetate. Moreover, the volatile oil extraction rate is highest in August. Currently, there are few studies on the activity of Perilla frutescens essential oil, mainly focusing on anti-influenza and antioxidant activities. It is reported that EBVEO shows a good dose-effect relationship for the scavenging rate of DPPH free radicals, with an IC50 value of 12.25 mg / mL. It has certain in vitro anti-influenza virus A / WSN / 33 (H1N1) activity. When the concentration is 6 mg·mL -1 The inhibition rate was (16.90±1.77)%.

[0005] Abnormal lipid metabolism is a significant risk factor for a variety of chronic diseases, increasing the morbidity and mortality of chronic conditions such as hyperlipidemia, obesity, fatty liver disease, type 2 diabetes, cardiovascular and cerebrovascular disease, and metabolic syndrome. The "2020 Report on the Nutrition and Chronic Disease Status of Chinese Residents" shows that the rates of overweight and obesity among adults in my country are 34.3% and 16.4%, respectively, and the prevalence of hyperlipidemia is as high as 35.6%. Diseases related to abnormal lipid metabolism have placed a heavy burden on both individual and public health. Maintaining a balanced lipid metabolism is key to preventing a variety of chronic and metabolic diseases.

[0006] Therefore, it is of great significance to focus on the flavonoids and essential oil components in Perilla frutescens and focus on studying their biological activity in regulating lipid metabolism and reducing fat content in the body, which can lay the foundation for the development and utilization of Perilla frutescens plant resources. Summary of the Invention

[0007] The present invention provides a use of Perilla frutescens essential oil in preparing products for preventing and / or treating metabolic diseases or metabolic abnormalities;

[0008] Preferably, the prevention and / or treatment of metabolic diseases or metabolic abnormalities include: fat reduction, lipid lowering, anti-inflammation, prevention and / or treatment of obesity, prevention and / or treatment of insulin resistance, prevention and / or treatment of abnormal glucose metabolism, and prevention and / or treatment of diabetes;

[0009] The use as described above is characterized in that: the fat reduction or lipid lowering refers to accelerating fat decomposition, reducing triglycerides, and / or lowering cholesterol; the prevention and / or treatment of obesity refers to promoting fat decomposition, reducing body weight, and / or improving blood lipids; and the anti-inflammatory refers to reducing the increased level of inflammatory factors.

[0010] The present invention provides a use of perilla essential oil in preparing products for improving intestinal flora, inhibiting fat synthesis-related SREBP1, ACC1 and / or FAS proteins, and promoting AMPK protein expression.

[0011] Preferably, the improvement of the intestinal flora refers to alleviating the increase in the Firmicutes / Bacteroidetes (F / B) ratio caused by a high-fat diet and reducing the relative abundance of Actinobacteria at the phylum level;

[0012] Preferably, at the genus level, the perilla essential oil can reduce the relative abundance of Lactobacillus, Bifidobacterium, Bacillus, and Candidatus_Saccharimonas.

[0013] Preferably, the relative abundance of Odoribacter, Mucispirillum, Blautia, and Ruminococcus_torques_group is increased.

[0014] The dominant bacteria in the perilla essential oil group are mainly Firmicutes (p__Firmicutes), Marinifilaceae (f__Marinifilaceae) and Odoribacter (g__Odoribacter).

[0015] The present invention also provides the use of 1,8-cineole and α-terpineol in preparing products for reducing fat, lowering fat, preventing and / or treating obesity.

[0016] The use as described above is characterized in that:

[0017] The structure of 1,8-cineole is

[0018] The structure of α-terpineol is

[0019] The use as described above is characterized in that the product further comprises an acceptable carrier and / or excipient.

[0020] The use as described above is characterized in that the dosage forms of the product include: tablets, capsules, suspensions, solutions, injections, injectable powders, sustained-release agents, controlled-release agents, emulsions, and ointments.

[0021] The present invention also provides the use of perilla essential oil in products for upregulating the expression of nhr-49, acs-2, ech-1, and kat-1; regulating the nuclear hormone receptor pathway mediated by AAK-2 / NHR-49; increasing the expression of AMPK and PPARα; and / or reducing the expression of ACC1, FAS, and SREBP1 proteins.

[0022] The present invention also provides the use of perilla essential oil in a reagent for upregulating the expression of nhr-49, acs-2, ech-1, and kat-1; and / or regulating the nuclear hormone receptor pathway mediated by AAK-2 / NHR-49; and / or increasing the expression of AMPK and PPARα; and / or reducing the expression of ACC1, FAS, and SREBP1 proteins.

[0023] The reagent is a scientific research detection reagent or an experimental reagent.

[0024] The use as described above is characterized in that the product further comprises one or more other active ingredients.

[0025] The use as described above is characterized in that the content of the active ingredient in the product is 2-100%.

[0026] The use as described above, wherein the perilla essential oil contains terpineol acetate, geranyl propionate, palmitic acid, epollol, caryophyllene oxide, (Z)-5-dodecenyl acetate, α-terpineol, 1,8-cineole and thymol;

[0027] Preferably, the essential oil of Perilla frutescens contains 1-octen-3-ol, eucalyptol, linalool, fench alcohol, heptanol, (S)-cis-verbenol, β-terpineol, terpineol, p-methylphenylisopropyl alcohol, α-terpineol, L-carveol, nerol, p-isopropylbenzaldehyde, dextrorotatory carvone, geraniol, trans-2-dodecenal, isoborneol acetate, p-isopropylbenzyl alcohol, perillyl alcohol, thymol, (Z)-3,7-dimethyl-2,6-octadienoic acid methyl ester, terpineol acetate, neryl acetate, decanoic acid, acetic acid citric acid, ... One or more of leaf esters, cineole, decyl acetate, 1-caryophyllene, 4-(1-methylvinyl)-1-cyclohexene-1-methanol acetate, 4-isopropylbenzaldehyde, α-caryophyllene, α-ylang-ylangolene, torreyl alcohol, (S)-(+)-5-(1-hydroxy-1-methylethyl)-2-methyl-cyclohexene-1-one, epoxiol, caryophyllene oxide, (-)-eucalyptus globulus alcohol, (Z)-5-dodecenyl acetate, 2-phenyl-2-ethylbutyric acid, eicosanoic acid, phyton, geranyl propionate, and palmitic acid.

[0028] Preferred: Perilla essential oil contains 20-80% terpineol acetate, 2-10% geranyl propionate, 2-10% palmitic acid, 2-10% epollol, 2-10% caryophyllene oxide, 1-8% (Z)-5-dodecenyl acetate, 0-7% α-terpineol, 0-7% thymol, 0-1% 1,8-cineole

[0029] More preferably, the perilla essential oil contains 30-70% terpineol acetate, 3-7% geranyl propionate, 3-7% palmitic acid, 3-7% epollol, 3-7% caryophyllene oxide, 2-5% (Z)-5-dodecenyl acetate, 1-5% α-terpineol, 0.5-3% thymol, and 0-1% 1,8-cineole.

[0030] More preferably, the preparation method of the above-mentioned perilla essential oil is:

[0031] The dried eastern perilla is taken, crushed, extracted with a microwave solvent-free extractor, and the distillate is collected and allowed to stand for stratification to obtain the eastern perilla essential oil.

[0032] Furthermore, the preparation method is as follows: take 50g of sun-dried Perilla frutescens, crush it and extract it with a microwave solvent-free extractor, collect the distillate, let it stand to separate the layers, measure the volume, repeat each group of experiments 3 times, take the average value, and then store it in a sealed container at 4°C.

[0033] More preferably, the solid-liquid ratio is 1:6, the extraction time is 60 min, the soaking time is 2 h, the microwave power is 600 W, and the essential oil yield is 0.096 mL / 50 g.

[0034] More preferably, the above-mentioned Perilla frutescens essential oil contains the following ingredients:

[0035] The relative content of other detected components did not exceed 1%. In addition, there are various alcohols, lipids, aldehydes, terpenes, and ketones.

[0036] Chemical composition of Perilla frutescens essential oil extracted by microwave-assisted solvent-free extraction

[0037]

[0038]

[0039] The perilla essential oil provided by the present invention has a very good fat-reducing effect. It can upregulate the expression of NHR-49, ACS-2, ECH-1, and KAT-1, accelerating the decomposition of fat and triglycerides. The fat-reducing effect is achieved through the nuclear hormone receptor pathway mediated by AAK-2 / NHR-49.

[0040] The perilla essential oil provided by the present invention can reduce lipid accumulation and regulate through the AMPK / SIRT1 / PPARα signaling pathway.

[0041] The perilla essential oil provided by the present invention can reduce fat cells and lower the content of triglycerides and / or total cholesterol.

[0042] The perilla essential oil provided by the present invention can improve indicators related to abnormal lipid metabolism in mice, reduce body weight, and / or reduce ALT and AST levels.

[0043] The perilla essential oil provided by the present invention can be used in products for preventing and / or treating liver damage.

[0044] The perilla essential oil provided by the present invention can lower blood sugar, improve sugar metabolism, prevent and / or treat insulin resistance, and can be used to treat diabetes.

[0045] The perilla essential oil provided by the present invention can improve oxidative stress, reduce the level of inflammatory factors, and / or has anti-inflammatory effects.

[0046] The perilla essential oil provided by the present invention can improve intestinal microbial flora, inhibit the reproduction of harmful microorganisms, reduce the abundance of harmful microorganisms, and promote the reproduction of beneficial microorganisms, thereby increasing the abundance of beneficial microorganisms.

[0047] The present invention also provides the use of 1,8-cineole and / or α-terpineol, the main components of Perilla frutescens essential oil, in preparing products for reducing fat, lowering lipids, preventing and / or treating obesity.

[0048] 1,8-cineole and / or α-terpineol can reduce the content of fat and triglyceride and can be used for preventing and / or treating obesity. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 ; RNA-seq sequencing and results (a) Biological process, cellular components, molecular function gene function annotation; (b) KEGG enrichment results of differentially expressed genes in Caenorhabditis elegans after treatment with Perilla frutescens essential oil.

[0050] Figure 2 (a) Oil Red O staining of C. elegans between normal and obese groups. (b) Comparison of Oil Red O staining intensity among different groups (Image J software). (c) Comparison of triglyceride (TG) content among different groups. (d) Comparison of nematode length among different groups. (e) Comparison of nematode width among different groups. Experiments were repeated three times. Results are expressed as mean ± SD. *, P < 0.05, **, P < 0.01.

[0051] Figure 3:(a) Effect of perilla essential oil on the expression of acs-2, ech-1, mdt-15, nhr-49 and kat-1 genes in normal nematodes (b) Effect of perilla essential oil on the expression of acs-2, ech-1, mdt-15, nhr-49 and kat-1 genes in obese nematodes (c) qRT-PCR detection of gene expression after RNAi interference of nhr-49, ech-1, acs-2 and kat-1 (d) Oil red O staining results of perilla essential oil in normal and obese nematodes (e) Comparison of Oil red O staining intensity in different groups (ImageJ software) (f) Effect of perilla essential oil on triglyceride content in normal and obese nematodes after RNAi interference. The experiment was repeated 3 times, and the results are expressed as mean ± standard deviation.

[0052] *,P<0.05,**,P<0.01.

[0053] Figure 4 : Effects of EBVEO essential oil (EBVEO) and orlistat on the viability of HepG2 cells.

[0054] Figure 5 : Effects of EBVEO on the fat content of HepG2 cells induced by PA.

[0055] Figure 6 : Effects of EBVEO on triglyceride and cholesterol levels in HepG2 cells induced by PA.

[0056] Figure 7 : Effects of EBVEO on the expression of lipid metabolism genes in HepG2 cells induced by PA.

[0057] Figure 8 : Effects of EBVEO on the expression of lipid metabolism proteins in HepG2 cells induced by PA.

[0058] Figure 9 Effects of Perilla frutescens essential oil on blood lipids and liver function (ALT and AST) in mice fed a high-fat diet. A. Serum TC content; B. Liver TG content; CD. Serum LDL-C and HDL-C levels; EF. Serum ALT and AST activity levels. Compared with the Con group, "*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001; compared with the HFD group, "#" indicates P < 0.05, "##" indicates P < 0.01, and "###" indicates P < 0.001; unmarked indicates P > 0.05.

[0059] Figure 10: A. Pathological section analysis of mouse liver HE staining under 100x microscope; B. HE staining of white fat under 100x microscope.

[0060] Figure 11 Effects of Perilla frutescens tea powder on glucose metabolism in mice fed a high-fat diet. A. Blood glucose levels in mice; B. Differences in the area under the glucose curve in the OGTT experiment; C. Glucose-6-phosphate dehydrogenase (G6PDH) levels in the liver. Compared with the Con group, "***" indicates P < 0.001; compared with the HFD group, "#" indicates P < 0.05, "###" indicates P < 0.001; unmarked indicates P > 0.05.

[0061] Figure 12 Effects of EBVEO on oxidative stress and inflammation in mice fed a high-fat diet. A. Liver malondialdehyde (MDA) content; B. Liver interleukin-6 (IL-6) content; C. Serum leptin (LEP) content. Compared with the Con group, "**" indicates P < 0.01, "***" indicates P < 0.001; compared with the HFD group, "#" indicates P < 0.05, "##" indicates P < 0.01, and "###" indicates P < 0.001; unmarked indicates P > 0.05.

[0062] Figure 13 : Effects of EBVEO on the intestinal microbiota of mice fed a high-fat diet. AC: Box plots of Ace, Chao, and Shannon indices for α-diversity evaluation at the ASV level; D. Principal coordinates analysis (PCoA) for β-diversity evaluation at the SAV level.

[0063] Figure 14 Effects of EBVEO on the intestinal microbial composition of mice. A. Phylum-level, species differences; B. Phylum-level, comparisons of Firmicutes, Bacteroidota, and Actinobacteriota species groups; E. Genus-level, Venn diagram; F. LEfSe multi-level species hierarchical dendrogram; G. LDA discriminant histogram.

[0064] Figure 15 LC-MS data preprocessing. A. QC sample evaluation plot; B. PLS-DA analysis plot; C. PLS-DA permutation test plot; D. Venn diagram; E. Volcano plot of HFD-HEO vs. HFD; F. Bubble plot of HFD-HEO vs. HFD; G. KEGG pathway differential abundance score plot of HFD-HEO vs. HFD.

[0065] Figure 16 : Heatmap diagram of correlation analysis between differential metabolites and intestinal microorganisms.

[0066] Figure 17Effects of EBVEO on key proteins in hepatic fatty acid synthesis in mice fed a high-fat diet. A. AMPK protein expression level; B. SREBP1 protein expression level; C. ACC1 protein expression level; D. FAS protein expression level. Compared with the Con group, "*" indicates P < 0.05, "**" indicates P < 0.01; compared with the HFD group, "#" indicates P < 0.05, "##" indicates P < 0.01, and "###" indicates P < 0.001; unmarked indicates P > 0.05.

[0067] Figure 18 :Chemical structure diagram of 1,8-cineole and α-terpineol.

[0068] Figure 19 :Effects of 1,8-cineole on Oil Red O, TG, and TC in PA-induced AML-12 cells. D. Results of Oil Red O staining of AML-12 cells with 1,8-cineole (20×); E. Effects of 1,8-cineole on triglyceride content in AML-12 cells; F. Effects of 1,8-cineole on total cholesterol content in AML-12 cells

[0069] Figure 20 : Effects of α-terpineol on triglyceride and total cholesterol in PA-induced AML12 cells. DETAILED DESCRIPTION

[0070] The present invention will be further described below with reference to the examples.

[0071] Example 1: Extraction process of perilla essential oil

[0072] 50g of sun-dried Perilla frutescens was crushed and extracted using a microwave solvent-free extraction instrument. The distillate was collected, allowed to stand and stratified, and the volume was measured. Each experiment was repeated three times, and the average value was taken. The samples were then sealed and stored in a refrigerator at 4°C. Using microwave solvent-free extraction and an orthogonal experimental design, the optimal extraction process for essential oil was determined as follows: a solid-liquid ratio of 1:6, a soaking time of 2h, an extraction time of 60min, a microwave power of 600W, and an essential oil yield of 0.192mL / 100g.

[0073] Example 2: GC-MS analysis of chemical components of Perilla frutescens essential oil

[0074] Through GC-MS analysis, a total of 43 chemical components were detected. Terpineol acetate was the substance with the highest content in Perilla frutescens essential oil, with a relative content of 56.25%, followed by geranyl propionate (6.8%), palmitic acid (4.95%), epoxiol (4.83%), caryophyllene oxide (4.41%), (Z)-5-dodecenyl acetate (3.28%), α-terpineol (2.91%), and thymol (1.6%).

[0075]

[0076]

[0077] Example 3: Transcriptome Sequencing Using Caenorhabditis elegans as a Model Organism

[0078] Comparison of transcriptome data between the Perilla frutescens essential oil group and the control group. GO functional enrichment analysis revealed a high number of genes categorized as functional binding and catalytic activity in terms of molecular function; a high number of genes involved in redox processes and membrane transport in biological processes; and a high number of genes related to cells, cellular components, and cell membranes in terms of cellular components. KEGG enrichment analysis revealed upregulation of genes involved in two major detoxification and metabolic enzyme systems: xenobiotic cytochrome P450 metabolism and P450 drug metabolism, while downregulation of genes involved in ABC transporters. This suggests that cytochrome P450 and ABC transporters play a key role in essential oil metabolism. The top five molecular pathways for the differentially expressed genes are shown in Table 1.

[0079] Table 1 Top five molecular pathways of differentially expressed genes

[0080]

[0081]

[0082] Through transcriptome sequencing, we found that more differentially expressed genes were enriched in the fatty acid metabolism pathway, which guided us to conduct in-depth research and discussion on the fat-reducing effect of Perilla frutescens essential oil.

[0083] Example 4: Study on the fat-reducing effect and mechanism of Perilla frutescens essential oil using Caenorhabditis elegans as a model organism

[0084] After 48 hours of exposure to three concentrations of essential oil in normal and obese (5mM glucose induced) nematodes, Oil Red O staining and triglyceride determination experiments were performed. The results showed that compared with the blank and positive control groups (orlistat), the essential oil of Perilla frutescens had a fat-reducing effect in both states, and the effect of the experimental group with a concentration of 0.025mg / mL was the most significant. Figure 2 shown.

[0085] To determine how Perilla frutescens essential oil affects the lipid metabolism pathway in C. elegans, we studied several key genes associated with fat synthesis and β-oxidation in C. elegans. q-PCR results showed that exposure to Perilla frutescens essential oil at a concentration of 0.025 mg / mL for 48 hours significantly increased the expression of nhr-49, acs-2, ech-1, and kat-1 in both normal and obese nematodes. These genes are located in the nuclear hormone receptor NHR-49 pathway, which regulates lipolysis. This influences β-oxidation of fat in nematodes and regulates downstream genes involved in lipolysis. Figure 3 a, b)

[0086] To further verify our idea, we used RNAi to silence the four genes nhr-49, acs-2, ech-1, and kat-1. For normal and obese nematodes, Oil Red O staining results showed that after silencing the nhr-49, acs-2, ech-1, and kat-1 genes, compared with the control group fed with an empty vector, the ability of Perilla frutescens essential oil to reduce body fat in nematodes was reduced or even disappeared. We also found that the effect of silencing kat-1 was very significant, which may be related to kat-1 encoding the rate-limiting enzyme in the last step of β-oxidation in nematodes, which determines a very important step in fat catabolism. This is also consistent with the results of q-PCR, indicating that Perilla frutescens essential oil accelerates fat decomposition in nematodes ( Figure 3 c, d, e). At the same time, the changes in triglycerides also showed the same pattern. ( Figure 3 f)

[0087] Changes in protein expression can confirm changes in gene expression. Due to the difficulty in obtaining antibodies in nematodes, we used a PRM-targeted proteomics approach to examine changes in protein levels involved in lipolysis and oxidation in nematodes after treatment with Perilla frutescens essential oil. The results showed that compared with the blank control group, the levels of proteins encoded by NHR-49, ech-1, aak-2, B027.3, B0303.3, acox1.1, acox1.2, acox1.5, and acox-3 increased in both normal and obese nematodes after treatment with Perilla frutescens essential oil. B027.3 and B0303.3 are involved in fatty acid β-oxidation in nematodes, while acox1.1, acox1.2, acox1.5, and acox-3 are involved in the peroxidase enzyme pathway for fat breakdown in nematodes. These increases indicate accelerated lipolysis in nematodes. This suggests that Perilla frutescens essential oil promotes lipolysis through the same pathway in both normal and obese nematodes.

[0088] The above experiments all prove that the molecular mechanism of Perilla frutescens essential oil in reducing fat in nematodes is through the nuclear hormone receptor pathway mediated by AAK-2 / NHR-49.

[0089] Example 5: Using HepG2 cells as a model, Perilla frutescens essential oil can reduce PA-induced lipid accumulation in HepG2 cells and regulate it through the AMPK / SIRT1 / PPARα signaling pathway

[0090] 5.1 Effects of different concentrations of essential oils and orlistat (weight loss positive control) on the survival rate of HepG2 cells

[0091] MTT assay was used to detect the effects of different concentrations of essential oils and orlistat on the survival rate of HepG2 cells. Figure 4 The results showed that compared with the blank control group, low concentrations of essential oil and orlistat had almost no significant effect on the viability of HepG2 cells. However, concentrations of 800 μg / mL to 1600 μg / mL of essential oil and 125 to 625 μg / mL of orlistat reduced the proliferation activity of HepG2 cells in a dose-dependent manner. Subsequent experiments on HepG2 cells were conducted using 50 μg / mL, 100 μg / mL, and 200 μg / mL of essential oil and 50 μg / mL of orlistat.

[0092] 5.2 Analysis of lipid accumulation by Oil Red O staining

[0093] The effects of essential oils and orlistat on lipids in HepG2 cells showed that adipogenic inducer (palmitic acid) caused different degrees of changes in the morphology and volume of HepG2 cells, and the number of lipid droplets and adipocytes increased. Figure 5 As shown in the figure: there were basically no lipid droplets or adipocytes in the blank group; in the PA-induced group, the adipocytes were stained red with Oil Red O and stained darker under the visual field; compared with the PA-induced group, the effect of low-concentration essential oil on HepG2 cells was not obvious, and adipocytes and lipid droplets stained darkly with Oil Red O were still visible after the action of medium-concentration essential oil; and the adipocytes in the high-concentration essential oil and orlistat treatment groups were reduced compared with the PA-induced group.

[0094] 5.3 Triglyceride (TG) and cholesterol (TC) content

[0095] The effects of essential oils and orlistat on triglyceride and total cholesterol levels in HepG2 cells showed that Figure 6 As shown in the results, the triglyceride and cholesterol contents in the PA-induced group were significantly increased compared with those in the blank group (P<0.001); the triglyceride and total cholesterol contents in the HepG2 cells in the essential oil and orlistat treatment groups were significantly reduced compared with those in the PA-induced group (P<0.05).

[0096] 5.4 qRT-PCR detection of lipid metabolism gene expression results

[0097] The effects of essential oils and orlistat on the mRNA expression of key factors in lipid metabolism in HepG2 cells showed that Figure 7 As shown in the results, compared with the blank group, the mRNA expressions of AMPK, PPARα, SIRT1, and CD36 in the PA-induced group of HepG2 cells were significantly decreased (P<0.01). Compared with the PA-induced group, the expressions of related factors in the essential oil-treated group were increased (P<0.01), but the expressions of AMPK and PPARα in the highest concentration essential oil-treated group were lower than those in the medium and low concentration essential oil-treated groups.

[0098] 5.5 Western-Blot detection of lipid metabolism-related protein expression results

[0099] The effects of essential oils and orlistat on the expression of key factors of lipid metabolism in HepG2 cells showed that compared with the blank group, the expression of key factors AMPK, PPARα, SIRT1, and CD36 proteins in the PA-induced group was reduced (P<0.05). Compared with the PA-induced group, the essential oil and orlistat treatment groups significantly increased the expression of AMPK, PPARα, SIRT1, and CD36 proteins (P<0.01, P<0.05). However, except for PPARα, there was no statistical difference in the other proteins in the 50 μg / ml essential oil treatment group ( Figure 8 ).

[0100] By using Caenorhabditis elegans and cells as models, it was preliminarily proved that Perilla frutescens essential oil has lipid-lowering effects.

[0101] Example 6: Using mice as an animal model, Perilla frutescens essential oil can prevent and improve indicators related to abnormal lipid metabolism in mice. 6.1 Effect of Perilla frutescens essential oil on body weight in mice fed a high-fat diet

[0102] After 15 weeks of intervention with perilla essential oil, the weight of the HFD group increased by nearly 45% compared with the Con group. According to the obesity standard, the body weight exceeded 20% of the standard value, which determined that the mice in the HFD group were obese (Table 2). During the intervention, the weight of mice in each group showed an upward trend. Compared with the Con group, the weight of the HFD group increased significantly, and the difference was statistically significant (P < 0.05). Compared with the HFD group, the weight of mice in the high-fat low-dose group (HFD-LEO), high-fat medium-dose group (HFD-MEO) and high-fat high-dose group (HFD-HEO) decreased significantly in a dose-dependent manner, and the difference was statistically significant (P < 0.05). Table 2 Effect of perilla essential oil on the body weight of mice fed a high-fat diet

[0103]

[0104] Note: Compared with the Con group, “***” means P < 0.001; compared with the HFD group, “##” means P < 0.01, “###” means P < 0.001; no mark means P > 0.05.

[0105] 6.2 Effects of Perilla frutescens Essential Oil on High-Fat Diet Blood Lipid Indexes and Liver Function ALT and AST

[0106] Compared with the Con group, the TC, TG, LDL-C, and HDL-C levels in the HFD group were significantly increased (P < 0.05), but compared with the HFD-Tween 80 group, these indicators were not significantly different, indicating that the solvent Tween 80 had no significant effect on the lipid metabolism indicators of mice fed a high-fat diet. However, after intervention with Perilla frutescens essential oil, the TC and TG levels in the HFD-MEO and HFD-HEO groups were significantly reduced (P < 0.05), and although the LDL-C level showed a downward trend, the difference did not reach statistical significance (P > 0.05) ( Figure 9 AD). Compared with the Con group, high-fat diet feeding increased the serum ALT and AST levels of mice (P<0.05), while the serum ALT and AST levels of mice in the Perilla frutescens essential oil intervention group were significantly reduced (P<0.05) ( Figure 9 EF). In conclusion, Perilla frutescens essential oil may improve the levels of TC, TG, ALT, and AST in mice induced by a high-fat diet, thereby improving the blood lipid levels and liver function damage in mice.

[0107] 6.3 Effects of Perilla frutescens Essential Oil on Histopathology of Liver and White Fat in Mice Fed a High-Fat Diet

[0108] The liver and white fat of the mice were stained with HE. The liver results showed that compared with the Con group, the liver cells in the HFD group and HFD-Tuween80 group were disordered, with obvious fat vacuoles and inflammatory cell infiltration in the cells. After the intervention of Perilla frutescens essential oil, the fat infiltration and inflammatory cell infiltration of the liver were improved ( Figure 10 A). The results of white fat showed that the adipocytes in the HFD and HFD-Tuween80 groups were significantly enlarged, while the white adipocytes in the low, medium, and high dose essential oil groups were significantly reduced in a dose-dependent manner ( Figure 10 B). In conclusion, Perilla frutescens essential oil may improve hepatic fatty infiltration and white fat enlargement in mice induced by a high-fat diet.

[0109] 6.4 Effects of EBVEO essential oil on blood glucose and glucose metabolism-related indicators in mice fed a high-fat diet

[0110] OGTT results showed that compared with the Con group, the tail tip blood glucose level and blood glucose area under the curve (AUC) of the HFD group mice at each time point were increased (P < 0.05). However, the tail tip blood glucose and AUC of the EBVEO intervention groups (HFD-MEO, HFD-HEO) were lower than those of the HFD group (P < 0.05) ( Figure 11 AB). Compared with the HFD group, the G6PDH level in the EBVEO HFD-HEO group was decreased (P<0.05) ( Figure 11 C). In summary, EBVEO may improve glucose metabolism by ameliorating some insulin resistance indicators in mice induced by a high-fat diet.

[0111] 6.5 Effects of EBVEO essential oil on oxidative stress and inflammatory factors in mice fed a high-fat diet

[0112] Compared with the Con group, the levels of liver MDA, liver IL-6 and serum LEP in the HFD group were significantly increased (P<0.05), and EBVEO intervention could reduce the levels of MDA, IL-6 and LEP (P<0.05) ( Figure 12 AC), the results showed that the EBVEO intervention group could improve the increased MDA level in mice fed a high-fat diet, thereby improving the oxidative stress level to a certain extent, and may reduce the increased level of inflammatory factors induced by a high-fat diet, and improve the inflammatory level in mice fed a high-fat diet.

[0113] Effects of 6.6EBVEO on the intestinal microbiota of mice fed a high-fat diet

[0114] 6.6.1 Analysis of EBVEO colony diversity and composition in mice fed a high-fat diet

[0115] A total of 18 fecal samples from 6 mice in each group were randomly collected for intestinal microbial analysis. The α diversity results showed that the median values of the Ace index and Chao index in the Con group (Ace=682.3, Chao=673) were higher, indicating that the species richness of this group was higher. The medians of the two indices in the HFD group (Ace=454.8, Chao=449.4) were lower, indicating that the species richness was reduced. The medians of the two indices in the EBVEO intervention group (Ace=520.5, Chao=515.4) were between the Con group and the HFD group, indicating that EBVEO may have a certain alleviating effect on the reduction in species richness caused by a high-fat diet, but the difference was not statistically significant (P>0.05) ( Figure 13AB). Shannon is one of the indices used to estimate the diversity of microorganisms in the sample. The larger the Shannon value, the higher the community diversity. The Shannon index of the HFD group (Shannon = 3.92) and the HFD_HEO group (Shannon = 3.71) was generally lower than that of the Con group (Shannon = 4.23), which may mean that the high-fat diet and EBVEO reduced the diversity of the intestinal flora ( Figure 13 C).

[0116] The principal coordinate analysis (PCoA) analysis of β diversity at the SAV level evaluated the community structure of the intestinal microbiota. The Con group, HFD group, and HFD-HEO group were clustered, indicating that there were certain differences in the community composition of the three groups ( Figure 13 D) The HFD group significantly changed the structure of the intestinal flora, and EBVEO intervention may help alleviate this change.

[0117] 6.6.2 Effects of EBVEO on the Gut Microbial Composition of Mice Fed a High-Fat Diet

[0118] At the phylum level, the top 20 species with the highest abundance were analyzed. Firmicutes, Bacteroidota, Actinobacteriota, and Desulfobacterota were the dominant phyla in the intestinal flora of mice, among which Firmicutes and Bacteroidota were the dominant phyla. Figure 14 A). Compared with the Con group, the relative abundance of Firmicutes increased and the relative abundance of Bacteroidetes decreased in the HFD group, but the differences were not statistically significant. The Actinobacteria increased significantly (P < 0.05). After EBVEO intervention, the relative abundance of Firmicutes and Bacteroidetes did not differ, while the relative abundance of Actinobacteria decreased (P < 0.05). ( Figure 14 BD). In summary, EBVEO intervention may help regulate the reduction of Actinobacteria in mice induced by high-fat diet.

[0119] At the genus level, there were 108 common genera between the groups, 13 unique genera in both the blank control (Con group) and the high-fat diet (HFD) group, and 14 unique genera in the EBVEO group (HFD-HEO). Figure 14E) Unique genera in the EBVEO group primarily include candidate Arthromitus (g__Candidatus_Arthromitus), Frisingicoccus (g__Frisingicoccus), unclassified Butyricicoccaceae (g__unclassified_f__Butyricicoccaceae), and Chlamydia (g__Chlamydia). LEfSe (Learning-Analysis-of-Search) shows the dominant bacteria in the three groups (from phylum to genus level). LDA linear discriminant analysis estimated that these differential species significantly contributed to the distinction between groups. Only groups with LDA values greater than 2 are displayed. Circles indicate phylogenetic levels; the diameter and color of each circle represent its abundance and grouping, respectively. f__Muribaculaceae (LDA=4.98) and g__norank_f__Muribaculaceae (LDA=4.97) had the greatest impact on the species difference effect of the blank control group (Con), o__Lactobacillales (LDA=4.79), g__Lactobacillus (LDA=4.78) and f__Lactobacillaceae (LDA=4.78) had the greatest impact on the species difference effect of the high-fat model group, p__Firmicutes (LDA=4.83), f__Marinifilaceae (LDA=4.21) and g__Odoribacter (LDA=4.21) had the greatest impact on the species difference effect of the EBVEO group ( Figure 14 FH).

[0120] 6.7 Effects of Perilla frutescens Essential Oil (EBVEO) on Liver Metabolites in Mice Fed a High-Fat Diet

[0121] Metabolite differences

[0122] For the overall data, if RSD < 0.3 and the cumulative proportion of ion peaks is > 70%, the overall data is qualified. When RSD ≤ 30%, the cumulative proportion of peaks ranges from 75% before data preprocessing to 79% after preprocessing, and the data after processing is good ( Figure 15 A), the partial least squares regression (PLS-DA) results showed that the three groups of samples were separated to a greater extent, indicating that the classification effect was better ( Figure 15 B), and with the decrease of permutation retention, R2 and Q2 decrease, and the regression line shows an upward trend, which also shows that the permutation test passes and the model does not have overfitting phenomenon ( Figure 15 C).

[0123] Venn diagram analysis of the number of common and unique metabolites in different groups showed that there were 1848 common metabolites, 27, 2, and 6 unique metabolites in Con, HFD, and HFD-HEO, respectively. The HFD-HEO group had 44 unique metabolites compared with the HFD group. The 6 unique metabolites in the EBVEO intervention group were N(6)-(Octanoyl)lysine, monocrotaline, D-digitotoxin (15-hexadecanolide), 7-ketolithocholic acid, Porson, and 1H-perfluorooctane (1H-perfluorooctane). Figure 15 D).

[0124] The difference volcano plot compared the metabolites between the HFD-HEO group and the HFD group. Compared with the high-fat model group, 109 metabolites in the EBVEO group were up-regulated and 89 metabolites were down-regulated. Among the top 8 differential metabolites, 1H-Perfluorooctane, 5-AminovalericAcid, Pg (363), and Guanidinoethylsulfonate were up-regulated, and Mevalonicacid, Glu ala asn, 4-GuanidinobutanoicAcid, and Zanamivir were down-regulated ( Figure 15 E).

[0125] The top five pathways enriched in the differential metabolic set were biosynthesis of cofactors, vitamin digestion and absorption, histidine metabolism, choline metabolism in cancer, and primary bile acid biosynthesis. Except for the choline metabolism in cancer pathway, which showed a down-regulated expression trend of the differential metabolites annotated in the pathway, the rest were up-regulated. Figure 15 F—G).

[0126] Association analysis between differential metabolites and differential intestinal microorganisms

[0127] The top 15 differential metabolites between the EBVEO and high-fat diet groups were screened and analyzed for correlation with the top 10 species at the genus level of intestinal microbial species abundance. The results showed that Allobaculum was positively correlated with 1H-Perfluorooctane and Guanidinoethyl sulfonate; Lactobacillus was positively correlated with Mevalonic acid and negatively correlated with Pyridoxal 5'-Phosphate; norank_f__Muribaculaceae was negatively correlated with 5-Aminovaleric Acid; Ileibacterium was negatively correlated with 1H-Perfluorooctane and Pg(36:3); unclassified_f__Lachnospiraceae was positively correlated with Zanamivir and Glu ala asn was negatively correlated with methacholine, and positively correlated with 5-Aminovaleric acid, pyridoxal5'-phosphate, N5-formyl-N5-hydroxy-L-ornithine, and 2-[(4-Oxo-3-phenyl-4H-chromen-7-YL)oxy]propanoic acid, and positively correlated with trans-4-aminocyclohexanecarboxylic acid; norank_f__norank_o__Clostridia_UCG-014 was negatively correlated with N5-formyl-N5-hydroxy-L-ornithine and positively correlated with 7-Methylguanosine 5'-phosphate; Bacteroides was positively correlated with Pg(36:3) and N5-formyl-N5-hydroxy-L-ornithine ( Figure 16 ).

[0128] 6.8 Effects of EBVEO essential oil on fat synthesis-related proteins in mice fed a high-fat diet

[0129] Western Blot was used to detect the expression levels of fatty acid synthesis-related proteins AMPK, SREBP1, FAS, and ACC1. Compared with the Con group, the HFD group showed a decrease in AMPK grayscale expression, while the HFD-LEO, HFD-MEO, and HFD-HEO groups showed a recovery in AMPK expression with the increase of the dose of Perilla frutescens ( Figure 17A). The expression of SREBP1 protein in the HFD group was higher than that in the Con group (P<0.05). However, with the increase of EBVEO dose, the expression of SREBP1 protein in the HFD-LEO, HFD-MEO and HFD-HEO groups showed a trend of gradual decrease ( Figure 17 B). In terms of ACC1 protein expression, the HFD group showed an increasing trend compared with the Con group. Although the ACC1 protein expression in the HFD-LEO and HFD-MEO groups was lower than that in the HFD group, the difference was not statistically significant (P>0.05). It is worth noting that the ACC1 protein expression in the HFD-HEO group was lower than that in the HFD group (P<0.05), indicating that high-dose EBVEO treatment may have an inhibitory effect on ACC1 protein expression ( Figure 17 C). Finally, HFD increased the expression of FAS (P < 0.05), while HFD-LEO, HFD-MEO, and HFD-HEO treatments all decreased the expression of FAS (P < 0.05, Figure 17 D).

[0130] Example 7: The main components of Perilla frutescens essential oil, 1,8-cineole and α-terpineol, have lipid-lowering effects on normal mouse liver cells AML-12

[0131]

[0132] The orange-red lipid deposition area in the 1,8-cineole intervention group was less than that in the model group (P<0.01). The analysis of lipid metabolism indicators showed that different concentrations of 1,8-cineole reduced TC levels (P<0.01), showing a clear dose-effect relationship ( Figure 19 E). In terms of triglyceride (TG) regulation, the TG level in the 1,8-cineole intervention group decreased gradually (P<0.001), and its lipid-lowering effect was not statistically different from that of orlistat (P>0.05) ( Figure 19 F).

[0133] The TG and TC contents of AML12 cells increased under the induction of palmitic acid, while they decreased after being treated with 50μg / mL, 100μg / mL and 200μg / mL of α-terpineol.

[0134] The effect of α-terpineol on lipids in AML12 cells demonstrates that PA inducers can successfully establish a lipid accumulation model, with a significant increase in the number of adipocytes and lipid droplets compared to the blank control group. Oil Red O staining results after treatment with 50 μg / mL, 100 μg / mL, and 200 μg / mL of α-terpineol are shown in the figure: Almost no adipocytes were observed in the blank control group; adipocytes in the PA-induced group were stained with Oil Red O. Compared with the PA-induced group, 50 μg / mL α-terpineol had no significant effect on AML12 cells. However, after treatment with 100 μg / mL α-terpineol, Oil Red O-stained adipocytes and lipid droplets were still observed. However, after treatment with 200 μg / mL α-terpineol, the number of adipocytes decreased.

[0135] The above description is a general description of the present invention. Variations in form and substitutions of equivalents may be made as appropriate. Although specific terminology is employed herein, such terminology is intended to be descriptive, not restrictive. Those skilled in the art may make various changes or modifications to the present invention, and such equivalents are intended to fall within the scope of the appended claims.

Claims

1. Use of Perilla frutescens essential oil in the preparation of products for preventing and / or treating metabolic diseases or metabolic abnormalities; Preferably, the prevention and / or treatment of metabolic diseases or metabolic abnormalities includes: Fat reduction, lipid lowering, anti-inflammation, prevention and / or treatment of obesity, prevention and / or treatment of insulin resistance, prevention and / or treatment of abnormal glucose metabolism, prevention and / or treatment of diabetes.

2. The use according to claim 1, wherein the perilla essential oil comprises terpineol acetate, geranyl propionate, palmitic acid, epollol, caryophyllene oxide, (Z)-5-dodecenyl acetate, α-terpineol, thymol and 1,8-cineole; Preferably, the essential oil of Perilla frutescens contains 20-80% terpineol acetate, 2-10% geranyl propionate, 2-10% palmitic acid, 2-10% epollol, 2-10% caryophyllene oxide, 1-8% (Z)-5-dodecenyl acetate, 0-7% α-terpineol, 0-7% thymol and 0-1% 1,8-cineole; More preferably, the essential oil of Perilla frutescens contains 30-70% terpineol acetate, 3-7% geranyl propionate, 3-7% palmitic acid, 3-7% epollol, 3-7% caryophyllene oxide, 2-5% (Z)-5-dodecenyl acetate, 1-5% α-terpineol, 0.5-3% thymol and 0-1% 1,8-cineole.

3. The use according to any one of claims 1 to 2, characterized in that: The fat reduction or lipid lowering refers to accelerating fat decomposition, reducing triglycerides, and / or lowering cholesterol; the prevention and / or treatment of obesity refers to promoting fat decomposition, reducing body weight, and / or improving blood lipids; and the anti-inflammatory refers to reducing the increased levels of inflammatory factors.

4. Use of perilla essential oil in the preparation of products for improving intestinal flora, inhibiting SREBP1, ACC1 and / or FAS proteins, and promoting AMPK protein expression. Preferably, the improvement of intestinal flora refers to alleviating the increase in the Firmicutes / Bacteroidetes (F / B) ratio caused by a high-fat diet and reducing the relative abundance of Actinobacteria; Preferably, the perilla essential oil can reduce the relative abundance of Lactobacillus, Bifidobacterium, Bacillus, and Candidatus_Saccharimonas; Preferably, the relative abundance of Odoribacter, Mucispirillum, Blautia, and Ruminococcus_torques_group is increased.

5. Use of 1,8-cineole and α-terpineol in the preparation of products for reducing fat, lowering lipids, preventing and / or treating obesity.

6. The use according to claim 5, characterized in that: The structure of 1,8-cineole is The structure of α-terpineol is 7. The method according to any one of claims 1 to 6, wherein the method is characterized in that The product further comprises an acceptable carrier and / or excipient.

8. The method according to any one of claims 1 to 6, wherein the method is characterized in that The dosage forms of the product include: tablets, capsules, suspensions, solutions, injections, injectable powders, sustained-release preparations, controlled-release preparations, emulsions, and ointments.

9. The method according to any one of claims 1 to 6, wherein the method is characterized in that The product may also contain one or more other active ingredients.

10. The use according to any one of claims 1 to 6, characterized in that The content of active ingredients in the product is 2-100%.

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