TW202525318A - Use ofcitrus depressaextract for decreasing lipid accumulation and modulating gut microbiota - Google Patents

Abstract

The present disclosure provides a use of a Citrus depressapeel extract for decreasing lipid accumulation and modulating gut microbiota. The Citrus depressaextract of the present disclosure achieves the effect of decreasing lipid accumulation and modulating gut microbiota through various efficacy experiments.

Description

Use of Taiwanese lemon extract to reduce lipid accumulation and regulate intestinal flora

The present invention relates to the use of a Citrus depressa extract for reducing lipid accumulation and regulating intestinal flora.

Obesity is primarily caused by an imbalance in energy metabolism within the body, leading to an increase in fat cells and lipid accumulation. Since cells primarily consume glucose as energy, when energy intake exceeds energy consumption, in order to store the excess energy, cells not only convert the excess glucose into glycogen for storage but also convert some of the glucose into triglycerides, which are stored in adipose tissue. Consequently, the excess energy also promotes the differentiation of adipocyte precursor cells into adipocytes, further increasing the accumulation of fat cells and the formation of adipose tissue, leading to obesity.

In recent years, the human gut microbiome has garnered significant attention from scientists and businesses worldwide. Given that the human gut microbiome far outnumbers human cells, the human body effectively serves as a carrier for this microbiome, with our daily diet providing food for these microbes. The quantity and quality of the gut microbiome are crucial to overall gut health. It not only regulates gut health but also influences the brain's central nervous system by modulating signaling pathways.

Factors such as an unhealthy diet, high work pressure, stress, irregular lifestyles, and indiscriminate use of antibiotics can lead to an imbalance in intestinal flora, causing constipation, diarrhea, depression, and obesity. Once the intestinal flora is disrupted, it is difficult to restore balance.

Given that current drugs or food products that reduce lipid accumulation and regulate intestinal flora still have drawbacks such as side effects, cytotoxicity, chemical synthesis, and ineffectiveness with diet or exercise, researchers in this field urgently need to develop novel and effective pharmaceutical or food compositions that can reduce lipid accumulation and regulate intestinal flora to benefit the vast population in need.

In view of this, the present invention aims to provide a Citrus depressa extract for use in preparing a composition for reducing lipid accumulation and regulating gut microbiota, wherein the Citrus depressa extract is obtained by extracting Citrus depressa with a solvent, and the solvent is water, alcohol, an alcohol-water mixture, or a combination thereof.

In one embodiment of the present invention, the Taiwan bergamot extract is Taiwan bergamot peel extract.

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

In one embodiment of the present invention, the effective concentration of the Taiwan lemon extract is at least 2% (w/w).

In one embodiment of the present invention, the Taiwan lemon extract inhibits weight gain.

In one embodiment of the present invention, the Taiwan lemon extract reduces serum total cholesterol (TCHO), triacylglycerol (TG), abdominal fat weight, inguinal fat weight, and adipocyte size.

In one embodiment of the present invention, the Taiwan lemon extract upregulates the expression of phosphorylated AMP-activated protein kinase α (p-AMPKα) and decreases the expression of fatty acid synthase (FAS) protein.

In one embodiment of the present invention, the Taiwan lemon extract increases the abundance of Lactobacillus reuteri .

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

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

In one embodiment of the present invention, the composition has a powder, a granule, a solution, a gel or a paste form.

In summary, the efficacy of the Taiwan lemon extract of the present invention is to reduce lipid accumulation and regulate intestinal flora by inhibiting weight gain, lowering serum total cholesterol, triglycerides, abdominal fat weight, inguinal fat weight, and adipocyte size, upregulating AMPKα expression, reducing fatty acid synthase protein expression, and increasing the abundance of Lactobacillus reuteri.

The following further describes the implementation of the present invention. The following embodiments are intended to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone skilled in the art may make modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope of the attached patent application.

Definition

The values used in this paper are approximate. All experimental data are expressed in 20% range, preferably within 10% range, the best is within the 5% range.

Unless otherwise specified herein, the terms "a", "an", "the" and similar terms used in this specification (especially in the following patent applications) should be understood to include both singular and plural forms.

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

The pharmaceutical composition provided by the present invention can be a pharmaceutical, a nutritional supplement, a health food, or any combination thereof, and can further comprise a pharmaceutically acceptable excipient, carrier, adjuvant, and/or food additive.

The pharmaceutical compositions provided by the present invention may be in any suitable form without particular limitation, and may be in a suitable dosage form depending on the intended use. For example, but not limited to, the pharmaceutical compositions may be administered orally or parenterally to a subject in need thereof.

According to the present invention, the pharmaceutical compositions can be formulated into dosage forms suitable for parenteral or oral administration using techniques well known to those skilled in the art, including, but 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.

The pharmaceutical composition according to the present invention can be administered via 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.

The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the group consisting of a solvent, an emulsifier, a suspending agent, a decomposer, a binding agent, an excipient, a stabilizing agent, a chelating agent, a diluent, a gelling agent, a preservative, a lubricant, an absorption delaying agent, a liposome, and the like. In addition, the topical composition of the present invention may further include an acceptable adjuvant widely used in topical product manufacturing technology. For example, the acceptable adjuvant may include one or more agents selected from the following: solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, chelating agents, surfactants, coloring agents, thickening agents, fillers, fragrances, and odor absorbers. The selection and amount of these agents are within the professional training and routine skills of those skilled in the art.

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

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 being administered. In the pharmaceutical composition provided by the present invention, the content ratio of the Taiwanese lemon extract in the composition can be adjusted according to actual application needs. In addition, the pharmaceutical composition can contain one or more other active ingredients (for example, drugs that reduce lipid accumulation and intestinal flora regulation products, etc.) as needed, or be used in combination with drugs containing one or more other active ingredients to further enhance the efficacy of the pharmaceutical composition or increase the flexibility and formulation of the preparation, as long as the other active ingredients do not adversely affect the benefits of the active ingredient of the present invention (i.e., Taiwanese lemon extract).

Optionally, the pharmaceutical compositions and food compositions provided by the present invention may contain appropriate amounts of additives, such as flavorings, coloring agents, and coloring agents that can enhance the palatability and visual experience of the pharmaceutical composition or food composition when taken, as well as buffers, preservatives, antiseptics, antibacterial agents, and antifungal agents that can improve the stability and storage properties of the pharmaceutical composition or food composition.

The food composition provided by the present invention can be a food product, and can be prepared with edible materials to include but not limited to beverages, fermented foods, bakery products, health foods, nutritional supplements, and dietary supplements.

According to the present invention, the edible material is selected from water, fluid milk products, milk, concentrated milk; fermented milk, 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; jelly; candies; infant formulas; health foods; foods); animal feeds; Chinese herbal medicines; and dietary supplements.

According to the present invention, the food product can be used as a food additive and can be added to the raw materials during preparation or during the food production process by known methods, and can be formulated with any edible material to form a food product for human and non-human animals to eat.

The beverages, fermented foods, baked products, health foods, nutritional supplements, and dietary supplements provided by the present invention can be consumed at varying frequencies, such as once daily, multiple times daily, or every few days, depending on the age, weight, and health status of the individual taking the beverage. The content of Taiwanese lemon extract in the beverages, fermented foods, baked products, health foods, nutritional supplements, and dietary supplements provided by the present invention can also be adjusted to meet the needs of specific populations, for example, to a recommended daily intake.

Beverages, fermented foods, baked products, health foods, nutritional supplements, and/or dietary supplements provided by this invention may be labeled on their packaging with recommended dosages, usage guidelines and conditions for specific groups (e.g., pregnant women), or recommendations for co-administration with other foods or medications. This allows users to safely consume these ingredients without the guidance of a physician, pharmacist, or other relevant personnel. The description and related applications of the Taiwanese lemon extract in the food ingredients provided by this invention are as described above.

The present invention is further illustrated by the following examples. These examples are provided for illustrative purposes 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 attached patent application.

The chemicals used in the following examples according to the present invention are described below. Fatty acid synthase (FAS) antibody was purchased from Cell Signaling Technology (Beverly, MA, USA). AMP-activated protein kinase (AMPK) antibody was purchased from ABclonal (Woburn, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GADPH) control antibody was purchased from Proteintech (Rosemont, IL, USA). Rabbit and mouse secondary IgG antibodies were purchased from Croyez Bioscience Co. (Taipei, Taiwan). Ethanol and acetonitrile were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Hesperidin was purchased from Tokyo Chemical Industry (Tokyo, Japan). Taiwanese lemon ( Citrus depressa Hayata) fruit was purchased from Yung-Sin Cooperative (Pingtung, Taiwan). Nobiletin, tangeretin, 5-demethylnobiletin, 5-demethyltangeretin, and sinensetin were kindly provided by Dr. Guor-Jien Wei.

The sample preparation and flavonoid content analysis procedures are as follows. C. depressa peels were hand-collected, oven-dried at 50°C for 80 hours, ground into a powder, and extracted with 95% ethanol for 48 hours. The extract was evaporated in a rotary vacuum evaporator and freeze-dried to obtain C. depressa ethanolic extract (CDEE), which was stored at -20°C for further experiments. High-performance liquid chromatography (HPLC) was used with modifications (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). 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, and mobile phase B was acetonitrile.

The animal experimental procedures are as follows. Four-week-old male C57BL/6 mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and housed in a controlled environment (25 ± 1°C, 50% relative humidity) with a 12-hour light/12-hour dark cycle. Mice had free access to food and water throughout the experiment. After 1 week of acclimation, 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 was purchased from Young Li Trading Company (Taipei, Taiwan). Based on 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 is a modified normal diet (see Table 1). Food consumption was recorded daily, and body weight was recorded weekly. At 12 weeks, all animals were sacrificed by _CO₂ asphyxiation. Blood samples were collected by cardiac puncture. Epididymal, retroperitoneal, enterocolonic, and inguinal fat, 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). Table 1 Composition (calories; %) ND HFD protein 28.5 16.5 Fat 13.5 50 carbohydrate 58.0 33.5 Total 100 100

The protocol 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 aminotransferase (ALT), total cholesterol (TCHO), triglycerides (TG), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) were analyzed at the National Laboratory Animal Center (NLAC; Taipei, Taiwan) using a Hitachi 7080 biochemical analyzer (Tokyo, Japan) according to the manufacturer's instructions.

The procedures for histopathological examination and adipocyte size are 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 thicknesses, fixed in 10% buffered formalin, dehydrated with a series of ethanol solutions, and paraffin-embedded. The cells were then stained with hematoxylin and eosin (H&E) and observed under a microscope. Adipocyte size was measured at 100x magnification using Image J software (Bethesda, MD, USA).

The Western blotting protocol is as follows. Epididymal tissue was homogenized with RIPA buffer to extract total protein, as described by 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, with minor modifications. Cell lysates were centrifuged at 17,000 × g for 1 hour at 4°C. Total protein content was measured using the bicinchoninic acid assay. Cell lysates containing 35 μg of protein were heated at 95°C for 5 minutes and then subjected to SDS-PAGE. After 3–4 hours, the SDS-PAGE was transferred to a PVDF membrane using transfer buffer. FAS, AMPK, and primary antibodies against AMPK and GAPDH were applied. Blots were developed using a luminescence image analyzer (Fujifilm, Tokyo, Japan) and quantified using Image J software (Bethesda, MD, USA).

The following protocol was used to characterize the intestinal microbiota by next-generation sequencing (NGS). This protocol was adapted from 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 Fecal DNA Kit (Jena, Germany) according to the manufacturer's instructions. All PCR reactions were performed using the ^Phusion® High-Fidelity PCR Kit (MA, USA). The pooled PCR products were then purified using a Qiagen gel extraction kit (Hilden, Germany). Finally, the library was sequenced using the Illumina HiSeq 2500 platform, generating 250-bp paired-end reads. The operational taxonomic units were assigned based on the Greengenes database (https://greengenes.lbl.gov/).

The statistical analysis procedure is as follows. Data are expressed as mean ± SD, and significant differences between groups were determined using SAS (version 9.4, SAS Institute Inc., Cary, NC, USA) using one-way analysis of variance (ANOVA) and Duncan's multiple range test. A value of p < 0.05 was considered statistically significant. Example 1. Flavonoid Content of CDEE

HPLC analysis showed that the ethanol extract of Taiwan lemon peel contained a variety of flavonoid components, such as hesperidin (2844.6 mg/100 g dry extract), zeaxanthin (10283.4 mg/100 g dry extract), tangeretin (5622.5 mg/100 g dry extract), and tangeretin (627.9 mg/100 g dry extract), which are commonly found in citrus peel. In this example, 5-demethylzeaxanthin (667.3 mg/100 g dry extract) and, in particular, 5-demethyltangeretin (26 mg/100 g dry extract) were found, which is the first time it has been found in Taiwan lemon peel extract (see Table 2). Table 2 flavonoids (mg/100 g dry extract) Hesperidin 2844.6 Sinesentin 627.9 Nobiletin 10283.4 Tangeretin 5622.5 *5-OH nobiletin 667.3 ^# 5-OH Tangeretin 26.0 *5-Demethyltangeretin ^# 5-Demethyltangeretin Example 2. Effect of CDEE on body weight and food intake

At the beginning of the experiment, the initial weights of the four groups in the aforementioned animal experimental process did not show significant differences. After 12 weeks, the final weight of the HFD group was 30.2 g, which was significantly higher than that of the ND group (23.1 g). Mice fed a normal diet supplemented with CDEE (NDCDEE) did not show a final weight difference from 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, which was significantly lower than that of the HFD group. Throughout the experiment, the weight gain of the HFD group was significantly higher than that of the ND group (2.5%). The weight gain of the HFDCDEE group was significantly lower (0.8%) compared with the HFD group. The effect of food intake in the ND and NDCDEE groups was significantly greater than that in the HFD and HFDCDEE groups. However, no significant differences were observed between ND and NDCDEE groups or between HFD and HFDCDEE groups. Although food intake was significantly higher in the ND and NDCDEE groups than in the HFD and HFDCDEE groups, the food efficiency of HFD and HFDCDEE mice was significantly higher than that of ND and NDCDEE mice (see Table 3). In this example, CDEE can inhibit weight gain when mice are fed a high-energy diet. Table 3 Group ND NDCDEE HFD HFDCDEE Initial weight (g) 19.1 ± 1.1 ^a 19.0 ± 0.6 ^a 19.0 ± 1.0 ^a 18.3 ± 0.7 ^a Final weight (g) 23.1 ± 1.1 ^c 23.0 ± 0.7 ^c 30.2 ± 0.9 ^a 26.2 ± 0.9 ^b Weight gain (g) 4.2 ± 0.5 ^c 4.2 ± 0.3 ^c 10.7 ± 1.0 ^a 8.8 ± 0.7 ^b Food intake (g) 3.4 ± 0.02 ^a 3.3 ± 0.06 ^a 2.7 ± 0.15 ^b 2.8 ± 0.20 ^b Food efficiency (g/day/mouse) 0.1 ± 0.2 ^b 0.1 ± 0.2 ^b 2.7 ± 0.15 ^b 0.2 ± 0.2 ^ab Different letters (a, b, c, ab) for ND, NDCDEE, HFD, and HFDCDEE indicate significant differences. Repeated letters indicate no significant differences from that group. For example, if the initial weight for all four groups is a, it means there is no significant difference in the starting weights. If the final weight for ND and NDCDEE has the same c, it means there is no significant difference between the two groups. However, ND and NDCDEE are significantly different from HFD and HFDCDEE, and HFD and HFDCDEE are also significantly different from each other. Example 3. Effect of CDEE on blood biochemistry

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). Group ND NDCDEE HFD HFDCDEE AST (U/L) 254.8 ± 56.1 ^a 324.7 ± 127.0 ^a 226.2 ± 57.8 ^a 313.1±37.4 ^a TCHO (mg/L) 104.8 ± 15.0 ^c 108.6 ± 8.0 ^c 192.2 ± 26.6 ^a 170.1 ± 6.8 ^b TG (mg/L) 46.4 ± 16.1 ^b 86.6 ± 17.3 ^a 43.8 ± 17.5 ^b 39.2 ± 4.8 ^b HDL-C (mg/L) 80.5 ± 13.9 ^b 89.7 ± 12.7 ^b 134.3 ± 21.5 ^a 127.9±5.4 ^a LDL-C (mg/L) 14.6 ± 2.1 ^b 9.9 ± 1.2 ^b 40.0 ± 9.2 ^a 40.2 ± 4.8 ^a Different English letters (a, b, c) for ND, NDCDEE, HFD, and HFDCDEE indicate significant differences. Repeated English letters indicate no significant differences from that group.

After ICR mice were fed an HFD containing 1.5% methanol extract of Taiwan lemon peel, serum TCHO and TG levels decreased. In this example, the serum TCHO level in the HFDCDEE group was significantly reduced compared with the HFD group, indicating that CDEE can alleviate the high serum cholesterol caused by HFD. Compared with the ND or HFD group, the serum AST levels in the NDCDEE and HFDCDEE groups did not show significant differences. AST is an important indicator of hepatotoxicity; therefore, the results obtained demonstrated that mice given CDEE did not suffer liver damage. Example 4. Effect of CDEE on adipose tissue weight and expression of lipogenesis-related proteins

Compared with the ND group, the HFD group had significantly increased epididymal, retroperitoneal, mesenteric, inguinal fat, and body fat percentage.

Figures 1A-1E show the effects of CDEE on adipose tissue weights. (1A) Retroperitoneal, (1B) perienteric, epididymal, and (1D) inguinal adipose tissue weights. Figure 1C shows photographs of retroperitoneal, perienteric, epididymal, and inguinal adipose tissue. (1E) Body fat percentage (periglandular weight + retroperitoneal weight + perienteric weight / final body weight). Statistical differences were tested using one-way ANOVA with Duncan's test. Groups with different letters (a-c) indicate significant differences (p < 0.05).

Compared with the HFD group, the epididymal, retroperitoneal, mesenteric, and inguinal fat weights, as well as body fat percentage, were significantly reduced in the HFDCDEE group.

The weight of peritoneal fat was significantly reduced in the NDCDEE group (Figure 1B). This mouse fat can represent one type of abdominal fat in humans.

Figures 2A and 2B show the effects of CDEE on adipose tissue size and the expression of proteins involved in the AMPK pathway. (2A) Epididymal adipocyte size was assessed by H&E staining, and representative images were captured at 200x magnification. (2B) Protein expression of AMPK, p-AMPK, and FAS. Statistical differences were tested by one-way analysis of variance with Duncan's test. Different letters (a–c) indicate significant differences between groups (p < 0.05).

In addition, the epididymal fat of the HFD group had significantly larger adipocyte size compared to the ND group (Figure 2A). In contrast, the epididymal fat and adipocyte size caused by HFD in the HFDCDEE group were significantly reduced. In this example, p-AMPK protein expression was found to be significantly upregulated and FAS protein expression was significantly downregulated (Figure 2B). The results indicate that CDEE reduces lipid accumulation by regulating pAMPK and FAS protein expression. Example 5. Effect of CDEE on intestinal flora

Diet is one of the exogenous factors behind the differences in gut microbiota between individuals. After two weeks of HFD feeding, mice developed a gut microbiota composition that differed from that of the ND group. Firmicutes (F) predominated in the HFD group (33.0%), compared to 22.5% in the ND group. Firmicutes were present in 31.8% of the NDCDEE group and 41.2% of the HFDCDEE group. Bacteroidetes (B), another major phylum in the gut, were less abundant in the HFD group (55.77%) than in the ND group (74.8%).

Figures 3A-3C show the effects of CDEE on intestinal microbiota. (3A) Top 10 bacterial species at the phylum level; (3B) Firmicutes/Bacteroidetes ratio; (3C) Top 10 bacterial taxa at the genus level.

The NDCDEE group had 58.5% of the phylum Pseudomonas, and the HFDCDEE group had 52.8% (Figure 3A). The F/B ratios in the HFD and HFDCDEE groups were significantly higher than those in the ND group (Figure 3B). Previous studies have suggested that obese individuals have higher F/B ratios, suggesting that HFD diets may have higher ratios than ND diets, but this phenomenon remains unconfirmed. The HFD group had higher abundances of Akkermansia , Lachnospiraceae NK4A13 , and Desulfovibrio , while Muribaculum and Blautia were lower. The abundance of Oscillibacter and Ruminiclostridium 9 was higher in the HFDCDEE group (Figure 3C).

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.

Mice fed different diets had different bacterial compositions. A 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 HFD group had 8 OTUs, indicating that the OTUs were distinct among the groups (Figures 4A and 4B). Chao1 and ACE indices are used to estimate community richness; higher values indicate a richer intestinal 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 Chao1 and ACE indices were lower in the HFDCDEE group, no significant differences were observed among the other groups (Figures 4C and 4D). The Shannon Index and Simpson Index reflect the diversity of the gut microbiota; a higher Shannon Index indicates greater gut microbial diversity (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, a higher Simpson Index indicates lower gut microbial diversity. No significant differences were observed among the four groups (Figures 4E and 4F). These results suggest that different diets affect the richness and diversity of intestinal bacteria, but there are no statistically significant differences. The present invention further analyzed the overall bacterial composition in the intestine using Welch's t test and metagenomeSeq statistical methods.

Figures 5A-5D show the effects of CDEE on specific intestinal bacteria. (5A) Phylum-level bacterial abundance analyzed by Welch's t-test; (5B) Phylum-level bacterial abundance analyzed by metagenomeSeq; (5C) Genus-level bacterial abundance analyzed by Welch's t-test; (5D) Genus-level bacterial abundance analyzed by metagenomeSeq.

At the genus level, the abundance of Turicibacter , GCA 900066575, Faecalibaclum , and Bifidobacteria was significantly higher in the HFD group than in the ND group, while the abundance of Ruminococcaceae UCG013 was significantly lower. The abundance of ASF356 and Ruminococcaceae UCG013 was significantly lower in the NDCDEE group than in the ND group. The abundance of Acetatifactor in the HFDCDEE group was significantly higher than in the HFD group, while the abundance of Negativibacillus was significantly lower (Figure 5A). MetagenomeSeq results showed that the abundance of Ruminococcaceae UCG013 was significantly higher in the ND group than in the HFDCDEE group. The HFDCDEE group showed a higher abundance of Ruminococcus spp. UCG013 than the HFD group, but the difference was not significant. Ruminococcus spp. UCG013 is a butyrate-producing bacterium that can degrade cellulose and hemicellulose; therefore, this bacterium is found in higher numbers 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). The abundance of Ruminococcus spp. in the ND and NDCDEE groups was significantly lower than that in the HFD group. The abundance of Ruminococcus spp. in the HFDCDEE group was also lower than that in the HFD group, but the difference was not significant. The abundance of negative bacteria was lower in HFDCDEE and NDCDEE than in ND and HFD (Figure 5B). At the species level, the abundance of Lachnospiraceae 615 was significantly higher in the HFD and NDCDEE groups than in the ND group. The abundance of Bifidobacterium longum was significantly higher in the ND group than in the HFD group. The abundance of Lactobacillus reuteri was significantly higher in the HFDCDEE group than in the HFD group (Figure 5C). MetagenomeSeq results showed that compared with the ND and HFD groups, mice fed CDEE and ND or HFD diets had a higher abundance of Lachnospiraceae 615 and a lower abundance of Bifidobacterium longum. The abundance of Lactobacillus reuteri in the ND group was significantly higher than 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).

Lactobacillus reuteri is a well-studied probiotic that has demonstrated beneficial effects on host health (see Mu, Q.; Tavella, VJ; Luo, XM. Role of Lactobacillus reuteri in human health and diseases. Front Microbiol . 2018, 9 , 757). In this example, CDEE ameliorates the adverse effects of HFD-induced dysbiosis by interacting with Lactobacillus reuteri, demonstrating its potential as a prebiotic. In this example, the gut bacterial composition was input into PICRUSt functional prediction. Clusters of orthologous groups (COGs) revealed that the bacterial composition of the HFDCDEE group was higher in abundance than that of the HFD group, while lower in abundance in genes related to lipid transport and metabolism.

Figures 6A and 6B show the effects of CDEE on intestinal bacteria based on PICRUSt functional prediction. (6A) Level 2 annotation clusters of orthologous groups (COG); (6B) Level 2 annotations from the Kyoto Encyclopedia of Genes and Genomes (KEGG).

Furthermore, the results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that the bacterial composition of the HFDCDEE group was lower in abundance in genes related to amino acid metabolism and lipid metabolism (Figures 6A and 6B). The present invention discovered that the ethanol extract of Citrus bergamia peel (CDEE) contains a variety of flavonoids, including hesperidin, tangeretin, tangeretin, 5-demethyltangeretin, and 5-demethyltangeretin. Mice fed a high-fat diet (HFD) supplemented with CDEE had lower body weight and smaller epididymal adipose tissue weight and cell size due to higher energy intake. Blood cholesterol was also lower in the HFDCDEE group compared to the HFD group. Results on gut bacterial composition showed that CDEE supplementation increased the abundance of Lactobacillus reuteri in mice fed a HFD. Lactobacillus reuteri is a well-studied probiotic, suggesting that CDEE may have a prebiotic-like effect due to its flavonoid content, preventing lipid accumulation.

In summary, the Taiwanese lemon extract of the present invention reduces lipid accumulation and regulates intestinal flora by inhibiting weight gain, lowering serum total cholesterol, triglycerides, abdominal fat weight, inguinal fat weight, and adipocyte size, upregulating AMPKα expression, reducing fatty acid synthase protein expression, and increasing the abundance of Lactobacillus reuteri.

The above description is for illustrative purposes only and is not intended to be limiting. Any equivalent modifications or variations that do not depart from the spirit and scope of the present invention shall be included in the scope of the patent application attached hereto.

without

Figures 1A–1E show the effects of C. depressa ethanolic extract (CDEE) on adipose tissue weights. (1A) Retroperitoneal, (1B) perineural, epididymal, and (1D) inguinal adipose tissue weights. Figure 1C shows photographs of retroperitoneal, perineural, epididymal, and inguinal adipose tissue. (1E) Body fat percentage (periglandular weight + retroperitoneal weight + perineural weight / final body weight). Statistical differences were tested using one-way ANOVA with Duncan's test. Groups with different letters (a–c) indicate significant differences (p < 0.05). ND indicates normal diet; NDCDEE indicates ND supplemented with 2% CDEE; HFD indicates high-fat diet; and HFDCDEE indicates HFD supplemented with 2% CDEE. Figures 2A and 2B show the effects of CDEE on adipose tissue size and the expression of proteins involved in the AMP-activated protein kinase (AMPK) pathway. (2A) Epididymal adipocyte size was assessed by H&E staining, and representative images were taken at 200x magnification; (2B) Protein expression of AMP-activated protein kinase (AMPK), p-AMPK, and fatty acid synthase (FAS). Statistical differences were detected by one-way analysis of variance with Duncan's test. Different letters (a–c) indicate significant differences between groups (p < 0.05). ND denotes normal diet; ND+CDEE denotes ND supplemented with 2% CDEE; HFD denotes high-fat diet; HFD+CDEE denotes HFD supplemented with 2% CDEE; GADPH denotes glyceraldehyde-3-phosphate dehydrogenase. Figures 3A–3C show the effects of CDEE on the intestinal microbiota. (3A) Top 10 bacteria at the phylum level; (3B) Firmicutes/Bacteroidetes ratio; (3C) Top 10 bacterial groups at the genus level. ND denotes normal diet; NDCDEE denotes ND supplemented with 2% CDEE; HFD denotes high-fat diet; HFDCDEE denotes HFD supplemented with 2% CDEE. Figures 4A–4F show the effects of CDEE on intestinal microbial α-diversity. (4A) Species richness; (4B) Venn diagram; (4C) Chao1; (4D) ACE; (E) Shannon; and (4F) Simpson index. ND indicates normal diet; NDCDEE indicates ND supplemented with 2% CDEE; HFD indicates high-fat diet; and HFDCDEE indicates HFD supplemented with 2% CDEE. Figures 5A–5D show the effects of CDEE on specific intestinal bacteria. (5A) Phylum-level bacterial abundance analyzed by Welch's t-test; (5B) Phylum-level bacterial abundance analyzed by metagenomeSeq; (5C) Genus-level bacterial abundance analyzed by Welch's t-test; (5D) Genus-level bacterial abundance analyzed by metagenomeSeq. Figures 6A and 6B show the effects of CDEE on intestinal bacteria based on PICRUSt functional prediction. (6A) Level 2 annotation clusters of orthologous groups (COG); (6B) Level 2 annotations from the Kyoto Encyclopedia of Genes and Genomes (KEGG).

Claims (11)

A Citrus depressa extract is used to prepare a composition for reducing lipid accumulation and regulating gut microbiota, wherein the Citrus depressa extract is obtained by extracting Citrus depressa with a solvent, and the solvent is water, alcohol, an alcohol-water mixture, or a combination thereof. The use according to claim 1, wherein the Taiwan bergamot extract is Taiwan bergamot peel extract. The use as described in claim 1, wherein the alcohol is ethanol. The use of claim 1, wherein the effective concentration of the Taiwan lemon extract is at least 2% (w/w). The use according to claim 1, wherein the Taiwan lemon extract inhibits weight gain. The use of claim 1, wherein the Taiwan lemon extract reduces serum total cholesterol (TCHO), triacylglycerol (TG), abdominal fat weight, inguinal fat weight, and adipocyte size. The use as described in claim 1, wherein the Taiwan lemon extract upregulates the expression of phosphorylated AMP-activated protein kinase α (p-AMPKα) and reduces the expression of fatty acid synthase (FAS) protein. The use of claim 1, wherein the Taiwan lemon extract increases the abundance of Lactobacillus reuteri . The use as described in claim 1, wherein the composition is a pharmaceutical composition or a food composition. The use according to claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier or an edible material. The use as described in claim 1, wherein the composition has a powder, a granular, a solution, a gel or a paste form.