Use of Taiwan bergamot extract for reducing lipid accumulation and regulating intestinal flora

Abstract

The invention provides an application of a Taiwan bergamot extract for reducing lipid accumulation and regulating intestinal flora. The Taiwan bergamot extract provided by the invention achieves the effects of reducing lipid accumulation and regulating intestinal flora through a plurality of efficacy experiments.

Description

Technical Field

[0001] The present invention relates to the use of an extract of Taiwanese calamondin (Citrus depressa) for reducing lipid accumulation and regulating gut microbiota. Background Art

[0002] The cause of obesity mainly stems from the imbalance of energy metabolism in the human body, resulting in an increase in adipocytes and lipid accumulation. Since cells mainly take glucose as the form of energy uptake, when the energy intake is greater than the energy consumption, in order to store the excess energy, in addition to converting the excess glucose into glycogen for storage, cells will also convert part of the glucose into triglycerides, which are stored in adipose tissue. Therefore, at the same time, the excess energy will also promote the differentiation of adipose precursor cells into adipocytes, thereby increasing the accumulation of adipocytes and the formation of adipose tissue, leading to the occurrence of obesity.

[0003] In recent years, the human gut microbiota has attracted the attention of global scientists and enterprises. In terms of the fact that the number of human gut microbiota far exceeds the number of human cells, the human body is actually a carrier of microbiota, and the daily diet of the human body provides food for the gut microbiota. The quantity and quality of gut microbiota are related to human gut health. It not only regulates gut health but also can affect the central nervous system of the brain by regulating signaling factors.

[0004] Factors such as unreasonable diet structure, high work pressure, mental stress, irregular living habits, and misuse of antibiotics cause gut microbiota dysbiosis, leading to problems such as constipation, diarrhea, depression, and obesity. Once the gut microbiota is damaged, it is very difficult to restore balance.

[0005] In view of the fact that current drugs or food products for reducing lipid accumulation and regulating gut microbiota still have disadvantages such as side effects, cytotoxicity, chemical synthesis, and ineffective diet or exercise effects. To solve the above problems, those skilled in the art urgently need to develop novel and effective pharmaceutical compositions or food compositions for reducing lipid accumulation and regulating gut microbiota to benefit the vast population with such needs. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide the use of an extract of Taiwanese calamondin (Citrus depressa) for preparing a composition for reducing lipid accumulation and regulating gut microbiota, wherein the Taiwanese calamondin extract is prepared by extracting a Taiwanese calamondin with a solvent, and the solvent is water, alcohol, an alcohol-water mixture, or a combination thereof.

[0007] In an embodiment of the present invention, the Taiwanese calamondin extract is a Taiwanese calamondin peel extract.

[0008] In one embodiment of the present invention, the alcohol is ethanol.

[0009] In one embodiment of the present invention, the effective concentration of the Taiwanese bergamot extract is at least 2% (w / w).

[0010] In one embodiment of the present invention, the Taiwanese bergamot extract inhibits weight gain.

[0011] In one embodiment of the present invention, the Taiwanese bergamot extract reduces serum total cholesterol (TCHO), triacylglycerol (TG), abdominal fat weight, inguinal fat weight, and adipocyte size.

[0012] In one embodiment of the present invention, the Taiwanese bergamot extract upregulates the expression of phosphorylated AMP-activated protein kinase α (p-AMPKα) and reduces the expression of fatty acid synthase (FAS) protein.

[0013] In one embodiment of the present invention, the Taiwanese bergamot extract increases the abundance of Lactobacillus reuteri.

[0014] In one embodiment of the present invention, the composition is a pharmaceutical composition or a food composition.

[0015] In one embodiment of the present invention, the composition further comprises a pharmaceutically acceptable carrier or an edible material.

[0016] In one embodiment of the present invention, the composition has a powder form, a granular form, a solution form, a gel form, or a paste form.

[0017] In summary, the efficacy of the Taiwanese bergamot extract of the present invention lies in: by inhibiting weight gain, reducing serum total cholesterol, triacylglycerol, abdominal fat weight, inguinal fat weight and adipocyte size, upregulating the expression of AMPKα and reducing the expression of fatty acid synthase protein, and increasing the abundance of Lactobacillus reuteri, achieving the efficacy of reducing lipid accumulation and regulating the intestinal flora.

[0018] The following will further illustrate the embodiments of the present invention. The following listed embodiments are used to clarify the present invention and are not intended to limit the scope of the present invention. Any person skilled in the art can make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to what is defined by the claims. Description of the Drawings

[0019] Figures 1A - 1E To show the effect of Citrus depressa ethanolic extract (CDEE) on adipose tissue weight. (1A) Retroperitoneal, (1B) mesenteric, epididymal, and (1D) inguinal adipose tissue weights, where Figure 1C To show the photographs of retroperitoneal, mesenteric, epididymal, and inguinal adipose tissues. (1E) Body fat percentage (perigonadal weight + retroperitoneal weight + mesenteric weight / final body weight). One-way analysis of variance (one-way ANOVA) with Duncan's test was used to detect statistical differences. Different letters (a - c) indicate significant differences between groups (p < 0.05). ND represents normal diet; NDCDEE represents ND supplemented with 2% CDEE; HFD represents high-fat diet; HFDCDEE represents HFD supplemented with 2% CDEE.

[0020] Figure 2A and 2B To show the effect of CDEE on adipose tissue size and the protein expression related to the AMP-activated protein kinase (AMPK) pathway. (2A) The size of epididymal adipocytes was evaluated by H&E staining, and representative images were taken at a magnification of 200×; (2B) Protein expression of AMP-activated protein kinase (AMPK), p-AMPK, and fatty acid synthase (FAS). One-way analysis of variance with Duncan's test was used to detect statistical differences. Different letters (a - c) indicate significant differences between groups (p < 0.05). ND represents normal diet; ND + CDEE represents ND supplemented with 2% CDEE; HFD represents high-fat diet; HFD + CDEE represents HFD supplemented with 2% CDEE; GADPH represents glyceraldehyde-3-phosphate dehydrogenase.

[0021] Figures 3A - 3C To show the effect of CDEE on gut microbiota. (3A) The top 10 bacteria at the phylum level; (3B) Firmicutes / Bacteroidetes ratio; (3C) The top 10 bacterial taxa at the genus level. ND represents normal diet; NDCDEE represents ND supplemented with 2% CDEE; HFD represents high-fat diet; HFDCDEE represents HFD supplemented with 2% CDEE.

[0022] Figures 4A - 4FShow the effects of CDEE on the α-diversity of gut microbiota. (4A) Species richness; (4B) Venn diagram; (4C) Chao1; (4D) ACE; (E) Shannon; and (4F) Simpson index. ND represents normal diet; NDCDEE represents ND supplemented with 2% CDEE; HFD represents high-fat diet; HFDCDEE represents HFD supplemented with 2% CDEE.

[0023] Figures 5A - 5D Show the effects of CDEE on specific gut bacteria. (5A) Analyze the bacterial abundances at the phylum level by Welch's t-test; (5B) Analyze the bacterial abundances at the phylum level by metagenomeSeq; (5C) Analyze the bacterial abundances at the genus level by Welch's t-test; (5D) Analyze the bacterial abundances at the genus level by metagenomeSeq.

[0024] Figure 6A and 6B Show the effects of CDEE on gut bacteria based on PICRUSt functional prediction. (6A) Clusters of orthologous groups (COG) annotated at Level 2; (6B) Kyoto Encyclopedia of Genes and Genomes (KEGG) annotated at Level 2. Detailed implementation

[0025] Definition

[0026] The numerical values used herein are approximate values, and all experimental data are represented within the range of ±20%, preferably within the range of ±10%, and most preferably within the range of ±5%.

[0027] Unless otherwise specified in the text, the terms "a", "the", and similar terms used in this specification (especially in the following claims) should be understood to include both singular and plural forms.

[0028] The "Taiwanese bergamot extract" described herein refers to the product obtained by extracting Taiwanese bergamot with a solvent for a specific time and temperature.

[0029] The pharmaceutical composition provided according to the present invention can be a medicine, a nutritional supplement, a health food, or any combination thereof, and can further include a pharmaceutically acceptable excipient, carrier, adjuvant, and / or food additive.

[0030] The pharmaceutical composition provided by the present invention can be in any suitable form without special limitation, and it will be in a corresponding suitable dosage form depending on the intended use. For example, but not limited thereto, the pharmaceutical composition can be administered to an individual in need by oral or parenteral administration.

[0031] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for parenteral or oral administration by techniques well-known to those skilled in the art, which includes, but is not limited to: injections [e.g., sterile aqueous solutions or dispersions], sterile powders, tablets, troches, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, and the like.

[0032] The pharmaceutical composition according to the present invention can be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, intralesional injection, sublingual administration, and transdermal administration.

[0033] The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier may include one or more reagents selected from the group consisting of: solvent, emulsifier, suspending agent, decomposer, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, absorption delaying agent, liposome, and the like. In addition, the topical composition according to the present invention may further include an acceptable adjuvant widely used in topical manufacturing techniques. For example, the acceptable adjuvant may include one or more reagents selected from the following: solvent, gelling agent, active agent, preservative, antioxidant, screening agent, chelating agent, surfactant, coloring agent, thickening agent, filler, fragrance, and odor absorbent. The selection and quantity of these reagents fall within the professional competence and routine techniques of those skilled in the art.

[0034] According to the present invention, the pharmaceutically acceptable carrier includes a solvent selected from the group consisting of: water, normal saline, phosphate buffered saline (PBS), sugar solution, aqueous solution containing alcohol, and combinations thereof.

[0035] The pharmaceutical composition provided by the present invention can be administered at different dosing frequencies, such as once a day, multiple times a day, or once every few days, depending on the needs, age, weight, and health status of the individual to whom it is administered. In the pharmaceutical composition provided by the present invention, the content ratio of Taiwanese bergamot extract in the composition can be adjusted according to actual application requirements. In addition, the pharmaceutical composition may optionally contain one or more other active ingredients (for example: drugs for reducing lipid accumulation and skin care products for regulating the intestinal flora, etc.), or be used in combination with drugs containing the one or more other active ingredients, in order to further enhance the efficacy of the pharmaceutical composition or increase the flexibility and compatibility of the formulation, as long as the other active ingredients do not have an adverse effect on the benefits of the active ingredient of the present invention (i.e., Taiwanese bergamot extract).

[0036] Optionally, appropriate amounts of additives may be further contained in the pharmaceutical composition and food composition provided by the present invention, such as flavoring agents, color toners, coloring agents, etc. that can improve the taste and visual perception of the pharmaceutical composition or food composition during administration, and buffering agents, preservatives, antiseptics, antibacterial agents, antifungal agents, etc. that can improve the stability and storage properties of the pharmaceutical composition or food composition.

[0037] The food composition provided by the present invention can be a food product and is formulated with edible materials including but not limited to: beverages, fermented foods, bakery products, health foods, nutritional supplements, and dietary supplements.

[0038] According to the present invention, the edible material is selected from the group consisting of water, fluid milk products, milk, concentrated milk, fermented milk products such as yogurt, sour milk, frozen yogurt, lactic acid bacteria-fermented beverages, milk powder, ice cream, cream cheeses, dry cheeses, soybean milk, fermented soybean milk, vegetable-fruit juices, juices, sports drinks, confectionery, jellies, candies, infant formulas, health foods, animal feeds, Chinese herbals, and dietary supplements.

[0039] According to the present invention, the food product can be used as a food additive and added during the preparation of raw materials by conventional methods or during the production process of food, and formulated with any edible material into a food product for human and non-human animal consumption.

[0040] The beverages, fermented foods, baked products, health foods, nutritional supplements, and dietary supplements provided according to the present invention can be consumed at different frequencies such as once a day, multiple times a day, or once every few days, depending on the age, weight, and health status of the individual to whom they are administered. The content of Taiwanese bergamot extract in the beverages, fermented foods, baked products, health foods, nutritional supplements, and dietary supplements provided according to the present invention can also be adjusted according to the needs of specific populations, for example, adjusted to the daily dosage.

[0041] For the beverages, fermented foods, baked products, health foods, nutritional supplements, and / or dietary supplements provided according to the present invention, the recommended dosage, usage standards and conditions for specific population groups (such as pregnant women), or the recommended matters for taking with other foods or medicines can be marked on their outer packages, so that users can take them by themselves without the guidance of a doctor, pharmacist or relevant practitioners without safety concerns. In the food composition provided according to the present invention, the form of the Taiwanese bergamot extract and related applications are as described above.

[0042] The present invention will be further illustrated by the following examples. These examples are provided for illustration only and are not intended to limit the scope of protection of the present invention. The scope of protection of the present invention is as shown in the appended claims.

[0043] According to the present invention, the chemicals used in the following examples are described as follows. The fatty acid synthase (FAS) antibody was purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody against AMP-activated protein kinase (AMPK) was purchased from ABclonal (Woburn, MA, USA). The glyceraldehyde-3-phosphate dehydrogenase (GADPH) control antibody was purchased from Proteintech (Rosemont, IL, USA). The secondary antibodies IgG for rabbits and mice were purchased from Croyez Bioscience Co. (Taipei, Taiwan, China). Ethanol and acetonitrile were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Hesperidin was purchased from Tokyo Chemical Industry (Tokyo, Japan). The Taiwanese bergamot (Citrus depressa Hayata) fruits were purchased from Yongxin Cooperative (Pingtung, Taiwan, China). Nobiletin, tangeretin, 5-demethylnobiletin, 5-demethyltangeretin, and sinensetin were kindly provided by Dr. Guor-Jien Wei.

[0044] The procedures for sample preparation and flavonoid content analysis are as follows. The pericarp of Citrus depressa was hand-collected, dried in an oven at 50 °C for 80 h, ground into powder, and extracted with 95% ethanol for 48 h. The extract was evaporated in a rotary vacuum evaporator and freeze-dried to obtain the Citrus depressa ethanolic extract (CDEE), which was stored at -20 °C for further experiments. The high-performance liquid chromatography (HPLC) method (see Lin, Z.-H.; Chan, Y.-F.; Pan, M.-H.; Tung, Y.-C.; Su, Z.-Y. Aged citrus peel (chenpi) prevents acetaminophen-induced hepatotoxicity by epigenetically regulating Nrf2 pathway. Am J Chin Med. 2019, 47, 1833-1851 and Lou, S.-N.; Lai, Y.-C.; Hsu, Y.-S.; Ho, C.-T. Phenolic content, antioxidant activity and effective compounds of kumquat extracted by different solvents. Food Chem. 2016, 197, 1-6) was used with modifications. Briefly, the CDEE extract was applied to a Shimadzu LC-10AT HPLC system equipped with a C18 column (250 mm × 4.6 mm, 5 μm) and a photodiode array (PDA) at a wavelength of 280 nm (Shimadzu, Kyoto, Japan). The injection volume was 20 μL, and the flow rate was maintained at 1.0 mL / min. Mobile phase A consisted of deionized water, while mobile phase B was acetonitrile.

[0045] The procedures for animal experiments are as follows. Male C57BL / 6 mice at four weeks of age were purchased from the Experimental Animal Center (Taipei, Taiwan, China) and housed in a controlled environment (25 ± 1 °C, 50% relative humidity) with a 12-hour light / 12-hour dark cycle. Throughout the experiment, the mice had free access to food and water. After one week of adaptation, the animals were randomly divided into four groups (n = 7): normal diet (ND, 15% energy from fat), high-fat diet (HFD, 50% energy from fat), ND supplemented with 2% CDEE (NDCDEE), and HFD supplemented with 2% CDEE (HFDCDEE); the experiment lasted for 12 weeks. LabDiet (Laboratory Rodent Diet) 5001 was used as the ND and purchased from Young Li Trading Company (Taipei, Taiwan, China). According to previous studies (see Tung, Y.-C.; Chou, R.-F.; Nagabhushanam, K.; Ho, C.-T.; Pan, M.-H. 3′-hydroxydaidzein improves obesity through the induced browning of beige adipose and modulation of gut microbiota in mice with obesity induced by a high-fat diet. J Agric Food Chem. 2020, 68, 14513-14522), the HFD was a modified normal diet (see Table 1). Food consumption was recorded daily and body weight was recorded weekly. At the 12th week, all animals were sacrificed by CO2 asphyxiation. Blood samples were collected by cardiac puncture. Epididymal, retroperitoneal, mesenteric, and inguinal fats as well as feces were immediately removed and stored at -80 °C until further analysis. All animal experimental protocols used in this invention were approved by the Institutional Animal Care and Use Committee of Chung Yuan Christian University (IACUC, approval number: 11005).

[0046] Table 1

[0047]

[0048] The procedure for serum analysis is as follows. This analysis was performed according to Tung, Y.-C.; Chou, R.-F.; Nagabhushanam, K.; Ho, C.-T.; Pan, M.-H. 3′-hydroxydaidzein improves obesity through the induced browning of beige adipose and modulation of gut microbiota in mice with obesity induced by a high-fat diet. J Agric Food Chem. 2020, 68, 14513-14522. Blood samples were centrifuged at 4,000×g for 10 minutes at 4 °C and stored at -20 °C until analysis. Serum levels of alanine transaminase (ALT), total cholesterol (TCHO), triglyceride (TG), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) were analyzed at the Experimental Animal Center (Taipei, Taiwan, China) using a Hitachi 7080 biochemical analyzer (Tokyo, Japan) according to the manufacturer's instructions.

[0049] The procedure for histopathological examination and adipocyte size is as follows. Histopathological examination was performed according to Tung, Y.-C.; Chou, R.-F.; Nagabhushanam, K.; Ho, C.-T.; Pan, M.-H. 3′-hydroxydaidzein improves obesity through the induced browning of beige adipose and modulation of gut microbiota in mice with obesity induced by a high-fat diet. J Agric Food Chem. 2020, 68, 14513-14522. Epididymal and inguinal adipose tissues were cut into 4-μm thick sections, fixed in 10% buffered formalin, dehydrated with a series of ethanol solutions, and processed for paraffin embedding. Then, they were stained with hematoxylin and eosin (H&E) and examined under a microscope. Adipocyte size was measured at a magnification of 100× and determined using Image J software (Bethesda, MD, USA).

[0050] The procedure for Western blotting is as follows. The epididymal tissue was homogenized in RIPA buffer for total protein extraction, with slight modification as described in Lin, Z.-H.; Chan, Y.-F.; Pan, M.-H.; Tung, Y.-C.; Su, Z.-Y. Aged citrus peel (chenpi) prevents acetaminophen-induced hepatotoxicity by epigenetically regulating Nrf2 pathway. Am J Chin Med. 2019, 47, 1833-1851. The cell lysate was centrifuged at 17,000×g for 1 hour at 4 °C. The total protein content was measured using the bicinchoninic acid assay. The cell lysate containing 35 μg of protein was heated at 95 °C for 5 minutes and then subjected to SDS-PAGE. After 3-4 hours, SDS-PAGE was transferred to a PVDF membrane using transfer buffer. Primary antibodies against the target proteins FAS, AMPK, and AMPK and GAPDH were applied. The blots were developed using a chemiluminescent imaging analyzer (Fujifilm, Tokyo, Japan) and quantified using Image J software (Bethesda, MD, USA).

[0051] The procedure for classifying gut microbiota by next-generation sequencing (NGS) is as follows. This procedure was performed according to Tung, Y.-C.; Chou, R.-F.; Nagabhushanam, K.; Ho, C.-T.; Pan, M.-H. 3′-hydroxydaidzein improves obesity through the induced browning of beige adipose and modulation of gut microbiota in mice with obesity induced by a high-fat diet. J Agric Food Chem. 2020, 68, 14513-14522. Fecal DNA was extracted using the innuPREP Stool DNA Kit (Jena, Germany) according to the manufacturer's instructions. All PCR reactions were performed using Performed using a High-Fidelity PCR kit (MA, USA). Then, the mixed PCR products were purified using a Qiagen gel extraction kit (Hilden, Germany). Finally, the library was sequenced using an Illumina HiSeq 2500 platform; 250 bp paired-end reads were generated, and the operational taxonomic units were classified based on the Greengenes database (https: / / greengenes.lbl.gov / ).

[0052] The procedure for statistical analysis was as follows. Data were expressed as mean ± SD, and significant differences between groups were determined using SAS (version 9.4, SAS Institute Inc., Cary, NC, USA) via one-way analysis of variance (ANOVA) and Duncan's multiple range test. A p < 0.05 value was considered statistically significant.

[0053] Example 1. Flavonoid content of CDEE

[0054] HPLC analysis showed that the ethanol extract of Taiwanese citron peel contained various flavonoid components, such as hesperidin (2844.6 mg / 100 g dry extract), nobiletin (10283.4 mg / 100 g dry extract), tangeretin (5622.5 mg / 100 g dry extract), and sinensetin (627.9 mg / 100 g dry extract), which are commonly found in citrus peels. In this example, 5-demethylnobiletin (667.3 mg / 100 g dry extract) was found, especially 5-demethyltangeretin (26 mg / 100 g dry extract), which was found in the Taiwanese citron peel extract for the first time (see Table 2).

[0055] Table 2

[0056] Flavonoid (mg / 100 g dry extract) Hesperidin 2844.6 Sinesentin 627.9 Nobiletin 10283.4 Tangeretin 5622.5 *5 - OH Nobiletin 667.3 <![CDATA # 5-OH Hesperetin]]> 26.0

[0057] *5-Demethylnobiletin

[0058] # 5-Demethyltangeretin

[0059] Example 2. Effects of CDEE on body weight and food intake

[0060] At the beginning of the experiment, the initial weights of the four groups in the aforementioned animal experiment procedure did not show significant differences. After 12 weeks, the final weight of the HFD group was 30.2 g, significantly higher than that of the ND group (23.1 g). Mice fed a normal diet supplemented with CDEE (NDCDEE) did not show a difference in final weight compared to the ND group. On the other hand, the final weight of mice fed a high-fat diet supplemented with CDEE (HFDCDEE) was 26.2 g, significantly decreased compared to the HFD group. During the entire experiment, the weight gain of the HFD group was significantly higher than that of the ND group (2.5%). Compared to the HFD group, the weight gain of the HFDCDEE group was significantly lower (0.8%). The effects of food intake in the ND and NDCDEE groups were significantly greater than those in the HFD and HFDCDEE groups. However, there was no significant difference between ND and NDCDEE or between HFD and HFDCDEE. Although the food intake of the ND and NDCDEE groups was significantly higher than that of the HFD and HFDCDEE groups, the food efficiency of the HFD and HFDCDEE mice was significantly higher than that of the ND and NDCDEE mice (see Table 3). In this example, when mice were fed a high-energy diet, CDEE could inhibit weight gain.

[0061] Table 3

[0062]

[0063] Example 3. Effects of CDEE on blood biochemistry

[0064] The levels of TCHO, HDL-C, and LDL-C in the HFD group were significantly higher than those in the ND and NDCDEE groups (see Table 4).

[0065] Table 4

[0066]

[0067] After ICR mice were fed an HFD containing 1.5% methanol extract of Taiwanese bergamot peel, the serum levels of TCHO and TG decreased. In this example, compared to the HFD group, the serum TCHO level in the HFDCDEE group was significantly decreased, indicating that CDEE could alleviate high serum cholesterol caused by HFD. Compared to the ND or HFD groups, there was no significant difference in the serum AST level between the NDCDEE and HFDCDEE groups. AST is an important indicator of liver toxicity; therefore, the obtained results proved that the mice given CDEE had no liver damage.

[0068] Example 4. Effects of CDEE on adipose tissue weight and expression of proteins related to adipogenesis

[0069] Compared to the ND group, the epididymal, retroperitoneal, mesenteric, inguinal fat, and body fat ratios in the HFD group were significantly increased.

[0070] Figures 1A - 1E Show the effect of CDEE on adipose tissue weight. (1A) Retroperitoneal, (1B) mesenteric, epididymal and (1D) inguinal adipose tissue weights, where Figure 1C Show photographs of retroperitoneal, mesenteric, epididymal and inguinal adipose tissues. (1E) Body fat percentage (perigonadal weight + retroperitoneal weight + mesenteric weight / final body weight). Statistical differences were detected by one-way ANOVA with Duncan's test. Different letters (a-c) indicate significant differences between groups (p < 0.05).

[0071] Compared with the HFD group, the epididymal, retroperitoneal, mesenteric and inguinal fat weights and body fat percentage in the HFDCDEE group were significantly decreased.

[0072] The mesenteric fat weight in the NDCDEE group was significantly decreased ( Figure 1B ), and this mouse fat can represent one of human abdominal fats.

[0073] Figure 2A and 2B Show the effect of CDEE on adipose tissue size and the expression of proteins related to the AMPK pathway. (2A) Assess the size of epididymal adipocytes by H&E staining and take representative images at a magnification of 200 times; (2B) Protein expression of AMPK, p-AMPK and FAS. Statistical differences were detected by one-way ANOVA with Duncan's test. Different letters (a-c) indicate significant differences between groups (p < 0.05).

[0074] In addition, compared with the ND group, the epididymal fat in the HFD group had significantly larger adipocyte sizes ( Figure 2A ). In contrast, the epididymal fat and adipocyte sizes caused by HFD in the HFDCDEE group were significantly reduced. In this example, it was found that the protein expression of p-AMPK was significantly up-regulated and the protein expression of FAS was significantly down-regulated ( Figure 2B ). The results indicate that CDEE reduces lipid accumulation by regulating the protein expression of pAMPK and FAS.

[0075] Example 5. Effect of CDEE on gut microbiota

[0076] Diet is one of the exogenous factors behind the differences in gut microbiota among different individuals. After mice were fed an HFD for 2 weeks, the gut microbiota composition was different from that of the ND group. Firmicutes (F) dominated in the HFD group (33.0%), while the proportion observed in the ND group was 22.5%. The NDCDEE group had 31.8% Firmicutes, and the HFDCDEE group had 41.2%. Bacteroidetes (B) is another major bacterial phylum in the gut, and the abundance in the HFD group (55.77%) was lower than that in the ND group (74.8%).

[0077] Figures 3A - 3C Show the effects of CDEE on gut microbiota. (3A) The top 10 bacteria at the phylum level; (3B) The Firmicutes / Bacteroidetes ratio; (3C) The top 10 bacterial taxa at the genus level.

[0078] The NDCDEE group had 58.5% Bacteroidetes, and the HFDCDEE group had 52.8% ( Figure 3A ). The F / B ratios in the HFD and HFDCDEE groups were significantly higher than that in the ND group ( Figure 3B ). Previous studies have pointed out that obese individuals have a higher F / B ratio. Therefore, the HFD was expected to be higher than the ND, but this phenomenon has not been conclusive yet. The abundances of Akkermansia, Lachnospiraceae NK4A13, and Desulfovibrio were higher in the HFD group, while the abundances of Muribaculum and Blautia were lower. The abundances of Oscillibacter and Ruminiclostridium 9 were higher in the HFDCDEE group ( Figure 3C ).

[0079] Figures 4A - 4F Show the effects of CDEE on gut microbial α-diversity. (4A) Species richness; (4B) Venn diagram; (4C) Chao1; (4D) ACE; (E) Shannon; and (4F) Simpson index.

[0080] Mice fed different diets had different bacterial compositions. The Venn diagram showed that the ND group had 4 Operational Taxonomic Units (OTUs), the NDCDEE group had 17 OTUs, the HFD group had 10 OTUs, and the HFDCDEE group had 8 OTUs. The OTUs of each group were different ( Figure 4A and4B )。Chao1 and ACE indices were used to estimate community richness; the higher the value, the richer the gut microbiota (see Yan, J.; Nie, Y.; Liu, Y.; Li, J.; Wu, L.; Chen, Z.; He, B. Yiqi-Bushen-Tiaozhi recipe attenuated high-fat and high-fructose diet induced nonalcoholic steatohepatitis in mice via gut microbiota. Front Cell Infect Microbiol. 2022, 12, 432). Although the Chao1 and ACE indices in the HFDCDEE group were lower, there were significant differences in other groups ( Figure 4C and 4D ). Shannon and Simpson indices were used to reflect the diversity of the gut microbiota; the higher the Shannon index, the higher the diversity of the gut microbiota (see Yan, J.; Nie, Y.; Liu, Y.; Li, J.; Wu, L.; Chen, Z.; He, B. Yiqi-Bushen-Tiaozhi recipe attenuated high-fat and high-fructose diet induced nonalcoholic steatohepatitis in mice via gut microbiota. Front Cell Infect Microbiol. 2022, 12, 432). In contrast, the higher the Simpson index, the lower the diversity of the gut microbiota. Here, there were no significant differences among the four groups ( Figure 4E and 4F ). These results indicate that different diets affect the richness and diversity of gut bacteria, but there are no significant differences statistically. The present invention further analyzed the overall bacterial composition in the gut using Welch's t-test and metagenomeSeq statistical methods.

[0081] Figures 5A - 5D Show the effects of CDEE on specific gut bacteria. (5A) Analyze the bacterial abundances at the phylum level using Welch's t-test; (5B) Analyze the bacterial abundances at the phylum level through metagenomeSeq; (5C) Analyze the bacterial abundances at the genus level using Welch's t-test; (5D) Analyze the bacterial abundances at the genus level through metagenomeSeq.

[0082] At the genus level, the abundances of Turicibacter, GCA 900066575, Faecalibaclum, and Bifidobacteria in the HFD group were significantly higher than those in the ND group, while the abundance of Ruminococcaceae UCG013 was significantly lower than that in the ND group. The abundances of ASF356 and Ruminococcaceae UCG013 in the NDCDEE group were significantly lower than those in the ND group. The abundance of Acetatifactor in the HFDCDEE group was significantly higher than that in the HFD group, while the abundance of Negativibacillus was significantly lower than that in the HFD group ( Figure 5A ). The results of MetagenomeSeq showed that the abundance of Ruminococcaceae UCG013 in the ND group was significantly higher than that in the HFDCDEE group. HFDCDEE showed a higher abundance of Ruminococcaceae UCG013 than the HFD group, but the difference was not significant. Ruminococcaceae UCG013 is a butyrate-producing bacterium that can degrade cellulose and hemicellulose; therefore, this bacterium is more abundant in people who consume a variety of fruits and vegetables (see Feng, J.; Ma, H.; Huang, Y.; Li, J.; Li, W. Ruminococcaceae_UCG-013 Promotes Obesity Resistance in Mice. Biomedicines. 2022, 10, 3272). Compared with the HFD group, the abundance of Turicibacter in the ND and NDCDEE groups was significantly reduced. The abundance of Turicibacter in HFDCDEE was also lower than that in the HFD group, but the difference was not significant. The abundances of Negativibacillus in HFDCDEE and NDCDEE were lower than those in ND and HFD ( Figure 5B ). At the species level, the abundance of Lachnospiraceae bacterium 615 in the HFD and NDCDEE groups was significantly higher than that in the ND group. The abundance of Bifidobacterium longum in the ND group was significantly higher than that in the HFD group. The abundance of Lactobacillus reuteri in the HFDCDEE group was significantly higher than that in the HFD group ( Figure 5C ). The results of MetagenomeSeq showed that compared with the ND and HFD groups, mice fed a CDEE and ND or HFD diet had a higher abundance of Lachnospiraceae bacterium 615 and a lower abundance of Bifidobacterium longum. The abundance of Lactobacillus reuteri in the ND group was significantly higher than that in the HFD and HFDCDEE groups. The abundance of Lactobacillus reuteri in the HFDCDEE group was also significantly higher than that in the HFD group ( Figure 5D ).

[0083] Lactobacillus reuteri is a well-studied probiotic that has shown beneficial effects on host health (see Mu, Q.; Tavella, V. J.; Luo, X. M. Role of Lactobacillus reuteri in human health and diseases. Front Microbiol. 2018, 9, 757). In this example, CDEE improves the adverse effects caused by HFD by reacting with Lactobacillus reuteri, demonstrating its prebiotic potential. In this example, the gut bacterial composition was input into PICRUSt functional prediction, and the results of clusters of orthologous groups (COG) showed that compared with the HFD group, the bacterial composition in the HFDCDEE group had a higher abundance related to carbohydrate transport and metabolism and a lower abundance related to lipid transport and metabolism.

[0084] Figure 6A and 6B Show the effects of CDEE on gut bacteria based on PICRUSt functional prediction. (6A) Clusters of orthologous groups (COG) annotated at Level 2; (6B) Kyoto Encyclopedia of Genes and Genomes (KEGG) annotated at Level 2.

[0085] In addition, the results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation showed that the bacterial composition in the HFDCDEE group had a lower abundance related to amino acid metabolism and lipid metabolism ( Figure 6A and 6B ). The present invention found that Citrus depressa peel ethanol extract (CDEE) contains various flavonoids, including hesperidin, nobiletin, tangeretin, sinensetin, 5-demethylnobiletin, and 5-demethyltangeretin. Mice fed a high-fat diet (HFD) supplemented with CDEE had lower body weights, and due to higher energy intake, the weights and cell sizes of epididymal adipose tissue were also smaller. Compared with the HFD group, the blood cholesterol in the HFDCDEE group was also lower. The results of gut bacterial composition showed that supplementing CDEE increased the abundance of Lactobacillus reuteri in mice fed HFD. Lactobacillus reuteri is a well-studied probiotic, indicating that CDEE may have a prebiotic-like effect due to its flavonoid content and can prevent lipid accumulation.

[0086] In summary, the Taiwanese bergamot extract of the present invention inhibits weight gain, reduces serum total cholesterol, triglycerides, abdominal fat weight, inguinal fat weight and adipocyte size, up-regulates the expression of AMPKα and reduces the expression of fatty acid synthase protein, and increases the abundance of Lactobacillus reuteri, achieving the effects of reducing lipid accumulation and regulating the intestinal flora.

[0087] The above description is only illustrative and not restrictive. Any equivalent modifications or changes made without departing from the spirit and scope of the present invention shall be included in the scope defined by the claims.

Claims

1. Use of an extract of Citrus depressa for preparing a composition for reducing lipid accumulation and regulating the composition of gut microbiota, characterized in that, The Taiwanese bergamot extract is prepared by extracting a Taiwanese bergamot with a solvent, and the solvent is water, alcohol, an alcohol-water mixture, or a combination thereof.

2. The use according to claim 1, characterized in that, The Taiwanese bergamot extract is a Taiwanese bergamot peel extract.

3. The use according to claim 1, wherein The alcohol is ethanol.

4. The use according to claim 1, wherein The effective concentration of the Taiwanese bergamot extract is at least 2% (w / w).

5. The use according to claim 1, characterized in that, The Taiwanese bergamot extract inhibits weight gain.

6. The use according to claim 1, characterized in that, The Taiwanese bergamot extract reduces serum total cholesterol (TCHO), triacylglycerol (TG), abdominal fat weight, inguinal fat weight, and adipocyte size.

7. The use according to claim 1, wherein, The Taiwanese bergamot extract upregulates the expression of phosphorylated AMP-activated protein kinase α (p-AMPKα) and reduces the expression of fatty acid synthase (FAS) protein.

8. The use according to claim 1, characterized in that, The Taiwanese bergamot extract increases the abundance of Lactobacillus reuteri.

9. The use according to claim 1, wherein The composition is a pharmaceutical composition or a food composition.

10. The use according to claim 1, characterized in that, The composition further comprises a pharmaceutically acceptable carrier or an edible material.

11. The use according to claim 1, wherein The composition has a dosage form of powder, granule, solution, gel, or paste.

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