Natural product for reducing fat deposition and application

Abstract

The invention discloses application of aucubin in reducing fat deposition, and belongs to the field of biotechnology and animal husbandry application. The new application refers to application of aucubin in preparation of a composition for reducing fat deposition of non-human mammals. In particular, the composition can be used to induce and establish a standardized low fat deposition animal model or state, preferably a high fat diet induced obese mouse model (intragastric dose of 30 mg / kg body weight per day). In addition, the composition can also be used as a feed additive for non-human mammals. The aucubin is developed into a tool for constructing a standardized metabolism scientific research model for the first time, a brand new application scheme is provided for the aucubin as a safe and effective fat deposition regulating agent in livestock production, and the aucubin has important scientific research and industrial value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and animal husbandry applications, specifically relating to the novel functions and applications of the natural iridoid compound aucubin. More specifically, it relates to the use of aucubin in the preparation of compositions that reduce fat deposition in animals, including but not limited to its use in inducing a fat deposition inhibition phenotype to establish a standardized model and as a feed additive to improve animal production performance. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] The regulation of fat deposition is of core significance in both basic life science research and livestock production practices, but its specific goals and technical approaches are quite different.

[0004] In the livestock production sector, pork is one of the world's most consumed meats. Its carcass quality, especially the balance between lean meat percentage and fat deposition, directly determines the economic benefits of farming and the value of the meat. Moderate intramuscular fat (marbling) improves flavor and tenderness, but excessive subcutaneous and visceral fat reduces slaughter rate, increases feed costs, and may affect meat quality. Therefore, exploring safe and effective nutritional strategies to precisely control fat deposition in growing-finishing pigs and improve lean meat percentage is a key goal continuously pursued in the livestock industry. Currently, in addition to genetic selection, developing highly efficient and residue-free feed additives from natural sources is considered a feasible technical measure.

[0005] In the field of life science research, a deep understanding of lipid metabolism mechanisms is the theoretical foundation for developing the aforementioned applied technologies. Constructing stable, reliable, and phenotypically defined animal models of "lipid deposition inhibition" or "metabolic improvement" is an indispensable tool for related drug screening, efficacy evaluation, and mechanism studies. Some existing modeling methods (such as simple high-fat diet induction or the use of certain chemical inducers) may have limitations in terms of modeling cycle, model stability, or animal welfare. Therefore, developing a standardized modeling method based on clearly defined active substances that can induce a specific and reproducible phenotype of lipid deposition inhibition has significant scientific research value.

[0006] Aucupolinoside ( Aucubin) is an iridoid glycoside widely found in plants such as Eucommia ulmoides and Plantago asiatica. It is extracted from a variety of inexpensive plants, and the extraction process is relatively mature. It possesses numerous biological activities, including antiviral, anti-inflammatory, antibacterial, and antioxidant effects. Existing technologies (such as CN201911124660.X) disclose its application in treating a mouse model of type 2 diabetes. However, these existing technologies do not address, nor suggest, the following two non-therapeutic novel uses directly related to the objectives of this invention: Aucubanoside is used as a standardized core reagent to actively construct research animal models exhibiting a specific phenotype of "inhibition of lipid deposition," serving as a basis for fundamental mechanism research and screening of related bioactive substances. This application is fundamentally different from using compounds to "treat" an existing disease model.

[0007] Aucupin is being developed as a feed additive to improve lean meat percentage and carcass quality in healthy or productive animals during their production cycle, for direct application in livestock production. This application aims to improve production performance, not to prevent or treat diseases. Summary of the Invention

[0008] Based on the background technology, existing technologies lack a standardized research animal model that can stably construct a "lipid deposition inhibition" phenotype based on specific natural active substances. This lack of a solution limits efficient research on the mechanisms of lipid metabolism and the screening of related active substances. Simultaneously, in intensive livestock farming, how to precisely regulate fat deposition and optimize carcass lean meat percentage in late-growth animals through nutritional means while ensuring animal health is a key bottleneck in improving farming efficiency and meat quality. This invention aims to simultaneously solve the aforementioned non-therapeutic technical problems in research and production, providing a novel, non-therapeutic application of aucubin.

[0009] The technical solution adopted in this invention is as follows: To address the aforementioned technical problems, this invention provides the use of aucubin in the preparation of compositions for reducing fat deposition in non-human mammals.

[0010] As a specific and important technical direction, the composition is used to induce a lipid deposition inhibition phenotype in animal models to construct a standardized low-lipid deposition animal model or state. This use is characterized by its non-therapeutic nature.

[0011] The low-fat deposition animal model or state is a standardized physiological state established by administering an effective amount of aucubin to animals on a high-energy diet, and it is characterized by the following co-phenotypic features: (a) There was no statistically significant decrease in average daily food intake, but weight gain was significantly suppressed; (b) A significant reduction in the weight and / or volume of white adipose tissue; (c) The average size of adipocytes in white adipose tissue was significantly reduced.

[0012] In the experimental verification, this state was manifested as follows: when the experimental animals were fed a high-energy diet, compared with the control group fed the same diet but without aucubin, the experimental group animals simultaneously met the above phenotypic indicators (a), (b) and (c).

[0013] Optionally, the low-fat deposition animal model or state is also accompanied by an improved metabolic function phenotype, manifested as a significant improvement in glucose tolerance and / or insulin sensitivity in the experimental group animals.

[0014] The standardized system constructed through the above phenotypic combinations is specifically designed to serve research purposes such as studying lipid metabolism mechanisms and screening lipid-lowering active substances.

[0015] Furthermore, the animal model is preferably a high-fat diet (HFD) induced model.

[0016] Furthermore, the mammal is preferably a rodent, such as a mouse.

[0017] Furthermore, the dosage of aucubin in constructing the model is from 10 mg / kg body weight / day to 100 mg / kg body weight / day, preferably 30 mg / kg body weight / day.

[0018] As another specific technical direction, the composition can be used as a feed additive. Its purpose is to reduce fat deposition and increase lean meat percentage in the normal feeding process of non-human mammals (especially economic animals) without reducing their feed intake, thereby improving production performance and carcass quality.

[0019] Furthermore, the feed additive is used in non-human mammals, such as, but not limited to, laboratory animals (e.g., mice, rats) or commercial animals (e.g., pigs, cattle, sheep). This application provides an innovative solution based on clearly defined active substances to address the aforementioned challenge of regulating fat deposition in livestock production.

[0020] Based on the above uses, the present invention also provides a feed additive containing an effective amount of aucubin for reducing fat deposition in non-human mammals.

[0021] The aucubin described in this invention is a plant extract, its chemical name is β-D-glucopyranoside, it is an iridoid glycoside compound, and its molecular formula is C2. 15 H 22 O9, with a purity preferably greater than 98%.

[0022] Optionally, the plant extracts include Eucommia ulmoides extract, Plantago asiatica extract, Rehmannia glutinosa extract, etc.

[0023] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) For the first time, aucubin was clearly defined as a core reagent for inducing an anti-lipid deposition phenotype in animal models. The model constructed by this method has well-defined parameters (such as HFD induction and a dose of 30 mg / kg) and a reproducible composite phenotype (inhibition of body weight gain, reduction of adipose tissue weight, slowing down adipocyte expansion, and improvement of glucose metabolism), providing a stable and reliable new tool for life science research.

[0024] (2) The innovative application of aucubin as a feed additive for non-human mammals was proposed. This provides a new technical approach and potential solution for the safe and natural regulation of fat deposition and improvement of lean meat percentage in economic animals (such as pigs and cattle).

[0025] (3) Basic experiments have confirmed that aucubin can effectively inhibit fat deposition without causing a significant decrease in animal feed intake. This characteristic makes it a significant safety advantage whether it is used as a scientific research tool or a potential feed ingredient.

[0026] (4) Based on the clear biological effects verified in standard laboratory animals (mice), the application logic of this invention can be reasonably extended to other non-human mammals. Those skilled in the art can apply the dose-response relationship disclosed in this invention to specific production systems through routine experiments, such as developing feed additives to improve the lean meat percentage of pigs. Attached Figure Description

[0027] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0028] Figure 1 To investigate the regulatory effects of aucubin on daily food intake and weight gain in mice: (A) Statistical graph of daily food intake in mice, ns showed no statistical significance; (B) Statistical graph of weight gain in mice after 8 weeks of gavage, compared with the HFD group *** P <0.001, P <0.05 is statistically significant; (C) Body weight change curve of mice, compared with the HFD group* P <0.05,** P <0.01, *** P <0.001, P <0.05 is statistically significant.

[0029] Figure 2 The effects of aucubin on the volume and weight of adipose tissue and liver in mice: (A) Real-world images of adipose tissue and liver in mice after 8 weeks of gavage; (B) Statistical chart of the weight of adipose tissue and liver in mice after 8 weeks of gavage, compared with the HFD group*** P <0.001, P <0.05 is statistically significant.

[0030] Figure 3 The effects of aucubin on the size and density of mouse adipocytes: (A) HE (hematoxylin-eosin) staining of mouse adipose tissue after 8 weeks of gavage, scale bar 100 μm; (B) Statistical graph of adipocyte size and density in inguinal white adipose tissue (iWAT) of mice after 8 weeks of gavage, compared with the HFD group. P <0.05 is statistically significant; (C) Statistical graph of adipocyte size and density in the white adipose tissue (eWAT) of the epididymis of mice after 8 weeks of gavage, compared with the HFD group, P <0.05 is statistically significant.

[0031] Figure 4 The effects of aucubin on glucose metabolism in mice: (A) glucose tolerance; (B) area under the glucose tolerance curve; (C) insulin tolerance; (D) area under the insulin tolerance curve. Compared with the HFD group* P <0.05,** P <0.01, P <0.05 is statistically significant. Detailed Implementation

[0032] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0033] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0034] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0036] Example: Verification of aucubin's role in inducing lipid deposition inhibition phenotype and its application as a core ingredient in functional feeds Experimental Design Forty 8-week-old male C57BL / 6J mice were randomly divided into 4 groups (n=10) after one week of free access to water and normal diet under constant temperature and humidity conditions: NCD group (basal feed group): fed with ordinary feed (NCD) and administered the corresponding volume of sterile saline by gavage daily.

[0037] NCD+AU group (basic functional feed validation group): fed with ordinary feed (NCD), and administered aucubin solution by gavage at a dose of 30 mg / kg body weight daily.

[0038] HFD group (high-fat model control group): fed high-fat diet (HFD) and administered physiological saline by gavage.

[0039] HFD+AU group (high-fat model construction group / functional feed validation group): fed with high-fat diet (HFD), and administered aucubin solution by gavage at a dose of 30 mg / kg body weight daily.

[0040] The feeding period was 8 weeks. During this period, the weight of the remaining feed and the body weight of the mice were weighed every other day to calculate the average daily feed intake and monitor changes in body weight.

[0041] Model building and efficacy verification (1) Body weight, food intake and macroscopic fat deposition phenotype At the end of the 8th week of feeding, mice in each group were weighed and dissected after fasting for 16 hours. White adipose tissue (iWAT) from the groin, white adipose tissue (eWAT) from the epididymis, brown adipose tissue (BAT) from the liver were collected, photographed, weighed, and then washed with pre-cooled physiological saline to remove blood stains. The mice were then fixed in 10% paraformaldehyde or stored at -80°C for later use.

[0042] The results are as follows Figure 1 , 2 As shown: Figure 1 As shown in (A), intervention with aucubin did not affect the average daily food intake of mice. Figure 1 As shown in (B) and (C), the body weight of mice in the HFD+AU group was significantly lower than that in the HFD group. This indicates that aucubin statistically significantly controlled the body weight of obese mice with minimal side effects.

[0043] Compared to obese mice in the HFD group, mice in the HFD+AU group showed reduced adipose tissue volume and weight, with significantly decreased iWAT and eWAT content (see [reference needed]). Figure 2 (A)(B). Furthermore, the livers of obese mice in the HFD group were significantly enlarged, with tense capsules and a milky white color, while the livers of mice in the HFD+AU group were similar in condition to those in the NCD and NCD+AU groups fed with normal diets, exhibiting normal size, dark red color, and soft texture. (See...) Figure 2 (A) indicates that aucubin has a significant lipid-lowering effect.

[0044] (2) Phenotypic analysis of adipose tissue cells To quantitatively characterize the fat deposition inhibition effect at the microscopic level, the collected iWAT and eWAT samples were processed and analyzed as follows: The iWAT and eWAT samples from each group were cut into thin sections, stained with hematoxylin and eosin (HE), and the morphology of adipocytes was observed under a microscope, with photographs taken to record the sections. In addition, the density of adipocytes of different sizes was statistically analyzed, and adipocyte distribution curves were plotted.

[0045] The results are as follows Figure 3 As shown: Figure 3 As shown in (A), the volume of adipocytes in obese mice in the HFD group increased, while the number of cells per unit area decreased. This phenomenon was caused by the increased accumulation of lipid droplets in the cells. In contrast, the volume of adipocytes in mice in the HFD+AU group decreased significantly, and the cell density increased significantly. It can be seen that the intervention of aucubin can restore the size and density of adipocytes to a certain extent. Figure 3 (B)(C) Quantitative data verified that aucubin has a significant inhibitory effect on high-fat diet-induced adipocyte expansion.

[0046] (3) Phenotypes associated with glucose metabolism function To assess the metabolic integration of the model, intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (IPITT) were performed at weeks 6 and 7, respectively.

[0047] IPGTT: The glucose tolerance test (IPGTT) was conducted on mice during the 6th week of rearing to assess their glucose tolerance. The bedding in the mouse cages was changed the day before the experiment, and the mice were fasted for 16 hours until the next day for measurement. During the fasting period, the mice had free access to water. First, approximately 1 mm of the mouse's tail tip was cut off with sterile scissors until blood flowed out. The tail was gently squeezed to concentrate the blood into a single drop, and fasting blood glucose was measured using a glucometer, recorded as the 0-minute blood glucose value. Subsequently, a glucose solution was injected intraperitoneally into the mice, typically at a dose of 2 g / kg (based on mouse body weight). For example, the injection volume of a 0.25 g / mL glucose solution dissolved in physiological saline was 8 mL / kg. Blood was collected from the tail tip at 30, 60, 90, and 120 minutes, and the blood glucose values ​​of each mouse at these time points were measured to plot the IPGTT curve.

[0048] like Figure 4 As shown in (A)(B), the area under the blood glucose curve in the HFD+AU group was significantly smaller than that in the HFD group, indicating that aucubin can significantly improve glucose tolerance in obese mice and ensure their glucose metabolism balance.

[0049] IPITT: An insulin tolerance test (IPITT) was performed on mice at week 7 of feeding to assess their insulin sensitivity. The procedure for IPITT was the same as that for IPGTT, with the insulin dose typically being 0.5 U / kg (based on mouse body weight). Blood glucose levels were recorded for each mouse at various time points, and an IPITT curve was plotted.

[0050] like Figure 4 As shown in (C)(D), the area under the blood glucose curve in the HFD+AU group was significantly smaller than that in the HFD group, indicating that aucubin can significantly improve insulin sensitivity in obese mice.

[0051] 3. Application Conclusions This embodiment demonstrates, through a complete experimental system, that: In terms of scientific model construction: using aucubin at 30 mg / kg / day as the core intervention method, it is not used to treat metabolic abnormalities that may be caused by diet, but to successfully transform it into a standardized experimental system with high consistency and reproducibility of "low fat deposition and active metabolism".

[0052] The inventiveness of this invention lies in the first-time disclosure of novel uses of aucubin in the following non-therapeutic areas: (1) Reagent as a standardized research tool: This protocol creates a model of “inhibition of lipid deposition state” defined by well-defined parameters (HFD background, 30 mg / kg dose) and a composite phenotype (see definition above). The core value of this model lies in its active constructability and phenotypic stability, providing a dedicated tool for mechanism research and substance screening that is superior to the traditional “treatment-improvement” model.

[0053] (2) As a potential feed additive for regulating production performance: The above results also demonstrate that aucubin can regulate animal feeding behavior without affecting normal feeding behavior. Figure 1 Under the premise of A), safe and effective regulation of fat deposition can be achieved. This characteristic fully meets the core requirements of livestock production for feed additives, providing direct and reliable experimental evidence for its development into a functional feed ingredient that improves lean meat percentage.

[0054] Compared with the existing use of aucubin in the treatment of metabolic diseases such as type 2 diabetes, the present invention has fundamental differences in application purpose, key points of technical solution design (such as emphasizing constant feed intake) and the ideal endpoint state pursued. It is not a simple conversion that is obvious to those skilled in the art.

[0055] 4. Application Extension Based on the clearly defined biological activity of aucubin in regulating lipid deposition revealed in this embodiment, those skilled in the art can reasonably foresee that this core discovery can provide a theoretical basis and research starting point for developing similar functional products for other economic animals (such as pigs and poultry). Its application parameters in specific production systems can be further optimized and determined through routine feeding trials.

[0056] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of aucubin in the preparation of compositions for reducing fat deposition in non-human mammals.

2. The use according to claim 1, characterized in that, The composition is used to induce a lipid deposition inhibition phenotype to establish a standardized low-lipid deposition animal model or state.

3. The use according to claim 2, characterized in that, The low-fat deposition animal model or state is a standardized physiological state established by administering an effective amount of aucubin to animals on a high-energy diet, and it is characterized by the following co-phenotypic features: (a) There was no statistically significant decrease in average daily food intake, but weight gain was significantly suppressed; (b) A significant reduction in the weight and / or volume of white adipose tissue; (c) The average size of adipocytes in white adipose tissue was significantly reduced.

4. The use according to claim 3, characterized in that, The low-fat deposition animal model or state is also accompanied by an improved metabolic function phenotype, manifested as a significant improvement in glucose tolerance and / or insulin sensitivity in the experimental group animals.

5. The use according to claim 2, characterized in that, The animal model was a high-fat diet (HFD) induced model, and the gavage dose of aucubin was 10 mg / kg body weight / day to 100 mg / kg body weight / day.

6. The use according to claim 5, characterized in that, The oral dose of aucubin is 30 mg / kg body weight / day.

7. The use according to claim 1, characterized in that, The non-human mammal in question is the mouse.

8. The use according to claim 1, characterized in that, The composition is a feed additive.

9. The use according to claim 8, characterized in that, The feed additive is intended for use in non-human mammals.

10. A feed additive comprising an effective amount of aucubin for reducing fat deposition in non-human mammals.

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