A method for evaluating the effect of a flavor on satiety based on animal ethology

Abstract

The application discloses a method for evaluating the satiety of spices based on animal behavior, and comprises the following steps: establishing an appetite intervention model (giving spices or drugs by gavage), using a modified elevated plus maze device (containing a detachable food trough and a sensor), synchronously recording the first contact food latency T, food area exploration frequency F and food intake I of the experimental object, and calculating the correlation coefficient r between the behavior parameters and the food intake by a Pearson correlation coefficient formula. When |r|>0.6, it is determined that the behavior change can reflect the satiety state. The method realizes multi-dimensional parameter cross-validation of appetite evaluation, and is suitable for the development of functional foods and drugs.

Description

Technical Field

[0001] This invention relates to animal behavioral analysis methods, and in particular to an experimental animal appetite evaluation system that integrates animal behavioral parameters and food intake indicators. It is applicable to the screening of drugs or functional foods for regulating appetite, the study of feeding behavior mechanisms, and the construction of metabolic disease models. Background Technology

[0002] Animal models are a simplified method for studying humans. They can help researchers indirectly analyze the complex activity of appetite and are of great value in the discovery, improvement, and biological mechanism research of weight loss drugs.

[0003] Current methods for evaluating appetite or satiety are limited to measuring only the amount of food consumed by experimental animals. They cannot reflect the impact of environmental stimuli (such as noise, stress, etc.) on appetite or satiety, and it is difficult to distinguish between physiological effects and stress interference, which may lead to evaluation results that are out of sync with the actual physiological situation.

[0004] However, in the treatment of diseases such as obesity and diabetes, or when appetite intervention programs are needed for specific environments (such as chronic stress and circadian rhythm disorders), it is necessary to evaluate the appetite-inhibiting or appetite-promoting effects of functional foods. Existing methods cannot meet the cross-validation requirements of behavioral data and food intake. It is necessary to combine behavioral data in different environments to analyze behavioral responses under environmental stress. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides a method for evaluating the regulation of satiety by spices based on animal behavior. This invention aims to provide a method for evaluating the regulation of satiety by spice intake based on animal behavior, and through structural modifications to the behavioral experimental apparatus, to achieve dynamic analysis of appetite under different environments, thereby more comprehensively assessing the appetite status of mice.

[0006] The technical solution of the present invention is as follows:

[0007] The purpose of this invention is to provide a method for evaluating the regulation of satiety by spices based on animal behavior, the method comprising the following steps:

[0008] (1) Establish an appetite intervention model;

[0009] (2) Construct an elevated cross maze, with a detachable feeding trough and sensor at one end of one open arm of the elevated cross maze;

[0010] (3) The subjects of the intervention were placed in an elevated cross maze, and the latency period T of the first food contact, the number of food area explorations F, and the amount of food consumed I during the experiment were recorded. A linear regression equation was fitted.

[0011] The linear regression equation was fitted based on the Pearson correlation coefficient, specifically:

[0012]

[0013] Among them, the incubation period T for first food exposure, in seconds;

[0014] F represents the number of times food areas are explored, in units of F.

[0015] Food intake (I), in grams.

[0016] In one embodiment of the present invention, in step (1), the method for establishing the appetite intervention model is as follows: the experimental subjects are given an appetite intervention substance by gavage, and the food intake of the experimental subjects is monitored every day. When the daily food intake is reduced by 20% compared with the blank group, the modeling is successful.

[0017] In one embodiment of the present invention, the appetite-intervening substance is a spice, a weight-loss drug, or a functional food.

[0018] The fragrance is either γ-terpinene (50ml, purchased from Ron Chemical Reagent Platform) or D-limonene (50ml, purchased from Aladdin Chemical Reagent Platform).

[0019] In one embodiment of the present invention, the experimental subjects are experimental inbred or outbred mice, rats or guinea pigs, and the weight difference between the experimental subjects is no more than 20%.

[0020] In one embodiment of the present invention, before appetite intervention, the experimental subjects are transported to a temporary cage in a behavioral laboratory in advance to adapt to the environment for at least 3 hours to reduce anxiety. They are then given gavage in groups 15 minutes before the start of the experiment.

[0021] In one embodiment of the present invention, in step (2), the elevated cross maze is composed of two open arms and two closed arms, which are intersected in a cross shape, with a connecting area in the center; neither of the two open arms has a side wall, and the closed arm has a high side wall; one end of one of the open arms is provided with a detachable feeding trough and a sensor.

[0022] In one embodiment of the present invention, the sensor includes an automatic sensing column and a digital display disposed at the lower end of the removable feeding trough; the automatic sensing column transmits the weight of the removable feeding trough to the digital display.

[0023] In one embodiment of the present invention, the surface of the detachable feeding trough that comes into contact with food is coated with a hydrophobic material.

[0024] In one embodiment of the present invention, in step (3), a video recording system is used to record the activities of the experimental subject in the elevated cross maze.

[0025] In one embodiment of the present invention, the total recording time is 10 minutes.

[0026] In one embodiment of the present invention, the elevated cross maze of the experimental device is wiped with 75% ethanol before use to eliminate odor interference, the background noise in the laboratory is controlled below 40dB, and the light intensity is maintained at 10-20 lux; the interval between two adjacent experiments is ≥15min to ensure air circulation and renewal in the device.

[0027] In one embodiment of the present invention, when |r|>0.6, it is determined to be significantly correlated, p<0.05, indicating that the behavioral parameters T and F are significantly correlated with the food intake I, and the behavioral changes can reflect the state of satiety;

[0028] The larger the r value, that is, the closer it is to 1, the more obvious the synergistic change between behavioral parameters T and F and food intake I, and the stronger the effect of satiety regulation.

[0029] T: Latency period (seconds) of first food exposure, reflecting the degree of appetite suppression. The longer the latency period, the stronger the feeling of fullness.

[0030] F: Number of times food areas were explored, reflecting the intensity of appetite. The fewer the number of explorations, the stronger the feeling of fullness.

[0031] I: Food intake (g), directly indicates satiety; the lower the food intake, the stronger the feeling of satiety.

[0032] The beneficial technical effects of this invention are as follows:

[0033] This invention overcomes the limitations of traditional single-dimensional detection (only food intake or isolated behavioral parameters) by simultaneously collecting the latency period of first food contact and the number of times appetite areas are explored, and cross-validating this data with food intake data recorded by an automatic weighing sensor, thereby improving the accuracy of appetite assessment.

[0034] The method of this invention improves the resistance to environmental interference. The hydrophobic coating and detachable feed trough design (polytetrafluoroethylene coating) in the device effectively reduce food residue adhesion. Combined with environmental adaptation before the experiment (≥3 hours), odor elimination (wiping with 75% ethanol) and noise / light control (≤40dB, 10-20 lux), the impact of environmental stress on appetite is reduced.

[0035] The method of this invention utilizes a modularly designed detachable feeding trough, adaptable to elevated cross mazes, achieving "one machine for multiple uses." Compared to single-device solutions, experimental costs are reduced, while simultaneously supporting correlation analysis of multiple indicators such as appetite, anxiety, and exploratory behavior.

[0036] The method of this invention, through Pearson correlation analysis, confirmed a significant negative correlation between the latency period of first food exposure and food intake, verifying the direct link between behavioral parameters and appetite state. This indicates that prolonged latency and reduced exploration frequency can synergistically reflect increased satiety. Compared to the traditional single food intake indicator, the cross-validation of multiple parameters significantly improves data reliability and provides a quantitative basis for the dynamic analysis of appetite under environmental stress. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of an elevated cross-shaped maze.

[0038] In the picture, 1. Waterproof material, 2. Detachable feeding trough, 3. Automatic sensor column, 4. Digital display;

[0039] Figure 2 The results show the food intake of the mice in Example 1;

[0040] Figure 3 The results of the mouse behavioral tests in Example 1 are shown; a is the time when the mouse first arrived at the food area, and b is the number of times the mouse explored the food area.

[0041] Figure 4 The results show the food intake of the mice in Example 2;

[0042] Figure 5 The results of the mouse behavioral tests in Example 2 are shown; a is the time when the mouse first arrived at the food area, and b is the number of times the mouse explored the food area;

[0043] Figure 6 The results show the food intake of the mice in Example 3;

[0044] Figure 7 The results of the mouse behavior test in Example 3 are shown; a is the time when the mouse first arrived at the food area, and b is the number of times the mouse explored the food area. Detailed Implementation

[0045] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0046] Example 1

[0047] (1) Adaptive feeding: 25 six-week-old mice were selected and randomly grouped into groups of 5 mice each. They were placed in an SPF-grade experimental animal room for adaptive feeding under the same feeding conditions. The ambient temperature was 22-26℃, the humidity was 40-60%, and the light and darkness were alternated for 12 hours each. They were fed with SPF-grade feed and pure water for one week.

[0048] (2) After the adaptive feeding in step (1), the mice were randomly divided into 5 groups using a random number method: a normal blank control group, a high-fat blank control group, and γ-terpinene groups (10 mg / kg, 50 mg / kg, and 100 mg / kg). Mice body weight was measured before administration. Each group was administered 0.3 mL of the corresponding dose via gavage, while the control group received NaCl (0.9%) once daily for four weeks. Food intake was measured. The results of food intake recording are shown in Table 1 and plotted. Figure 2 The results of the food intake records showed that the food intake of mice fed a high-fat diet was significantly reduced after the spice intervention, with the most significant reduction observed in the group fed a dose of 50 mg / kg of γ-terpinene.

[0049] (3) Behavioral testing of mice: The control group and experimental group were subjected to the elevated cross maze test after being fed in step 2.

[0050] Behavioral testing in the elevated cross maze: Mice were removed from their cages, placed with their backs to the experimenter, and gently positioned in the closed arm of the elevated cross maze. The experimenter then quickly and quietly left. A video recording system was activated to record the mice's activities within the maze for a total of 10 minutes. During this time, food was placed in the open arm area of ​​the maze to observe the mice's exploration and ingestion behavior. The latency period T(s) for first food exposure, the number of times the food area was explored F(times), and the amount of food ingested I(g) were recorded. The results are shown in Table 2 and plotted. Figure 3 The test results showed that, compared with the control group, the number of times mice explored the food area decreased after spice intervention, with the group receiving 100 mg / kg of γ-terpinene showing the greatest reduction in exploration frequency.

[0051] Pearson correlation analysis yielded the following values: r = 0.42 for the control group, r = 0.55 for the high-fat control group, r = 0.76 for the 100 mg / kg γ-terpinene group, r = 0.77 for the 50 mg / kg γ-terpinene group, and r = 0.605 for the 10 mg / kg γ-terpinene group. The r values ​​for each spice intervention group were generally higher than those for the control group, indicating a stronger correlation between behavioral parameters and food intake, and the r values ​​for all intervention groups were > 0.6.

[0052] This demonstrates that the spice-treated mice of the present invention showed a significant reduction in appetite after feeding.

[0053] Table 1

[0054] Ordinary blank group 2.915g 3.377g 3.058g 3.179g 3.234g High-fat blank group 13.846g 14.628g 15.594g 15.18g 18.17g 100 mg / kg γ-terpinene group 5.04g 5.4g 5.49g 5.472g 5.85g 50 mg / kg γ-terpinene group 3.757g 3.783g 3.952g 3.926g 4.446g 10 mg / kg γ-terpinene group 4.185g 4.38g 4.335g 4.575g 4.8g

[0055] Table 2

[0056]

[0057]

[0058] Example 2

[0059] (1) Same as Example 1, and will not be repeated.

[0060] (2) After the adaptive feeding in step (1), the mice were randomly divided into 5 groups using a random number method: a normal blank control group, a high-fat blank control group, and D-limonene groups (10 mg / kg, 50 mg / kg, and 100 mg / kg). Mice were weighed before administration and administered 0.3 mL of the corresponding dose via gavage. The control group received NaCl (0.9%) once daily for four weeks. Food intake was measured. The results of food intake recording are shown in Table 3 and plotted. Figure 4 The results of the food intake records showed that the food intake of mice fed a high-fat diet was significantly reduced after the spice intervention, with the most significant reduction observed in the group fed a dose of 50 mg / kg of γ-terpinene.

[0061] (3) Same as in Example 1, the detection data results are shown in Table 4 and plotted. Figure 5 The test results showed that, compared with the control group, mice explored the food area less frequently after D-limonene intervention, with the group receiving 100 mg / kg of D-limonene showing the greatest reduction in exploration frequency.

[0062] Pearson correlation analysis yielded the following values: r = 0.42 for the control group, r = 0.55 for the high-fat control group, r = 0.75 for the 100 mg / kg D-limonene group, r = 0.665 for the 50 mg / kg γ-terpinene group, and r = 0.8 for the 10 mg / kg D-limonene group. The r values ​​for each spice intervention group were generally higher than those for the control group, indicating a stronger correlation between behavioral parameters and food intake. Furthermore, the r values ​​for all intervention groups were > 0.6.

[0063] This demonstrates that the spice-treated mice of the present invention showed a significant reduction in appetite after feeding.

[0064] Table 3

[0065] Ordinary blank group 2.915g 3.377g 3.058g 3.179g 3.234g High-fat blank group 13.846g 14.628g 15.594g 15.18g 18.17g 100 mg / kg D-limonene group 4.432g 4.816g 4.672g 4.88g 5.072g 50 mg / kg D-limonene group 3.85g 4.228g 4.256g 4.396g 4.452g 10 mg / kg D-limonene group 6.094g 6.776g 6.71g 6.996g 7.04g

[0066] Table 4

[0067]

[0068]

[0069] Example 3

[0070] (1) Same as Example 1, and will not be repeated.

[0071] (2) After the adaptive feeding in step (1), the mice were randomly divided into four groups using a random number method: a normal blank control group, a high-fat blank control group, a 50 mg / kg dose D-limonene group, and a 50 mg / kg dose γ-terpinene group. The mice's weight was measured before administration. Each group was administered 0.3 mL of the corresponding dose via gavage, while the control group received NaCl (0.9%) once daily for four weeks. The food intake of the mice was measured. The results of the food intake records are shown in Table 5 and plotted. Figure 6 The results of food intake records showed that the food intake of mice fed a high-fat diet was significantly reduced after the spice intervention, with the γ-terpinene group showing a more significant reduction compared to the limonene group.

[0072] (3) Same as in Example 1, the detection data results are shown in Table 6 and plotted. Figure 7 The test results show that γ-terpinene has a more significant effect on the appetite of mice among different spices.

[0073] Table 5

[0074] 50 mg / kg γ-terpinene group 3.757g 3.783g 3.952g 3.926g 4.446g 50 mg / kg D-limonene group 3.85g 4.228g 4.256g 4.396g 4.452g

[0075] Table 6

[0076]

[0077]

[0078] Pearson correlation analysis yielded r = 0.77 for the 50 mg / kg γ-terpinene group and r = 0.665 for the 50 mg / kg D-limonene group. The difference in r values ​​among different spices at the same concentration indicates variations in their effects on food intake and behavior in mice. This result suggests a direct correlation between behavioral parameters and appetite status, and can serve as an objective indicator for quantitatively evaluating the effect of spices on satiety.

[0079] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for evaluating the effect of spices on satiety based on animal behavior, characterized in that, The method includes the following steps: (1) Establish an appetite intervention model; The method for establishing the appetite intervention model is as follows: the subjects are given appetite intervention substances by gavage, and their food intake is monitored daily. The model is considered successful when the daily food intake is reduced by 20% compared with the control group. (2) Construct an elevated cross maze, with a detachable feeding trough and sensor at one end of one open arm of the elevated cross maze; (3) The subjects of the intervention were placed in an elevated cross maze, and the subjects' latency period T for first contact with food, the number of times they explored the food area F, and the amount of food they consumed I during the experiment were recorded. A linear regression equation was fitted. The linear regression equation was fitted based on the Pearson correlation coefficient, specifically: Among them, the incubation period T for first food exposure, in seconds; F represents the number of times food areas are explored, in units of F. Food intake (I), in grams.

2. The method according to claim 1, characterized in that, Appetite-intervention substances include spices, weight-loss drugs, or functional foods.

3. The method according to claim 1, characterized in that, The experimental subjects were inbred or outbred mice, rats or guinea pigs, with a body weight difference of no more than 20% between the subjects.

4. The method according to claim 1, characterized in that, In step (2), the elevated cross maze consists of two open arms and two closed arms, which intersect in a cross shape, with the central area being the connecting area; neither of the two open arms has side walls, while the closed arms have high side walls; one end of one of the open arms is equipped with a detachable feeding trough and a sensor.

5. The method according to claim 4, characterized in that, The sensor includes an automatic sensing column and a digital display located at the bottom of the removable feeder; the automatic sensing column transmits the weight of the removable feeder to the digital display.

6. The method according to claim 4, characterized in that, The removable food trough has a hydrophobic coating on the surface that comes into contact with food.

7. The method according to claim 1, characterized in that, In step (3), a video recording system is used to record the activities of the experimental subjects in the elevated cross maze.

8. The method according to claim 7, characterized in that, The total recording time was 10 minutes.

9. The method according to claim 1, characterized in that, When |r|>0.6, it was considered to be significantly correlated, p<0.05, indicating that the behavioral parameters T and F are significantly correlated with food intake I, and behavioral changes can reflect the state of satiety; The larger the r value, that is, the closer it is to 1, the more obvious the synergistic change between behavioral parameters T and F and food intake I, and the stronger the effect of satiety regulation.

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