A model for evaluating microtoxicity of evodia extract based on fruit fly behavior and a construction method and application thereof
By constructing a fruit fly behavior model and combining various experiments and linear regression analysis, the problems of accuracy and efficiency in the evaluation of trace toxicity of traditional Chinese medicine were solved, and efficient assessment of the toxicity of Evodia rutaecarpa extract was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ACADEMY OF MILITARY MEDICAL SCIENCES
- Filing Date
- 2024-01-10
- Publication Date
- 2026-07-07
AI Technical Summary
The evaluation of trace toxic substances in traditional Chinese medicine is difficult to be accurate, rapid, and efficient. Existing technologies cannot effectively utilize the olfactory and gustatory responses of fruit fly model organisms for the toxicity assessment of traditional Chinese medicine.
A trace toxicity model of Evodia rutaecarpa extract based on Drosophila behavior was constructed. The toxicity of Evodia rutaecarpa extract was evaluated by establishing a comprehensive evaluation index (POPI) through acute toxicity test, two-way selection feeding test, Y-type test and kissing test, combined with linear regression analysis.
This method enables accurate, rapid, and efficient assessment of the trace toxicity of Evodia rutaecarpa extract, reducing the variability of behavioral experiments and improving the accuracy and efficiency of the evaluation.
Smart Images
Figure CN117929639B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of toxicity evaluation technology, specifically relating to a model for evaluating the trace toxicity of Evodia rutaecarpa extract based on fruit fly behavior, its construction method, and its application. Background Technology
[0002] The evaluation of trace toxicity in traditional Chinese medicine (TCM) differs from that of chemical and biological drugs. TCM toxicity is characterized by minute levels of toxicity that are difficult to detect using modern biotechnology, making it one of the key challenges in TCM safety evaluation. How to accurately, rapidly, and efficiently evaluate the minute toxicity of TCM is a critical issue currently facing TCM toxicity research.
[0003] Fruit flies, as a model organism, possess sensitive olfactory and gustatory sensory systems, and can produce behavioral responses to faint toxic odors of traditional Chinese medicine. However, there are currently no reports on the in vivo and in vitro combined research using fruit flies as a model organism to study the odors of traditional Chinese medicine. Summary of the Invention
[0004] To accurately, rapidly, and efficiently evaluate the trace toxicity of Evodia rutaecarpa extract, avoid the potential fluctuations in the corresponding indices of each behavioral experiment in fruit flies, and better reflect the olfactory and gustatory behaviors of fruit flies, this invention constructs a model for evaluating the trace toxicity of Evodia rutaecarpa extract based on the behavior of fruit flies. This model is characterized by high accuracy, speed, and efficiency, and can be applied to the assessment of the toxicity of Evodia rutaecarpa extract.
[0005] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution:
[0006] The first objective of this invention is to provide a method for constructing a model for evaluating the trace toxicity of Evodia rutaecarpa based on Drosophila behavior, the method comprising the following steps:
[0007] S1. Evodia rutaecarpa extract solutions of different concentrations were prepared using 1% sucrose solvent. The mortality rate of fruit flies at different concentrations was detected using an acute toxicity test, and the LC-weighted average (LCL) values were obtained. 80 LC 65 LC 50 LC 35 LC 20 Corresponding concentration;
[0008] S2. Drosophila subjected to wet starvation treatment underwent a series of experiments consisting of a two-way selection feeding experiment, a Y-shaped experiment, and a proboscis test, in that order. The preference index PI, olfactory index OI, and proboscis test PERI were obtained, respectively. The concentration of the Evodia rutaecarpa extract solution used in the series experiments was the same as the LC50 concentration in S1. 80 LC 65 LC 50 LC 35 LC 20Corresponding concentration;
[0009] S3. Use SPSS Statistics 27 to perform linear regression and find the linear regression equation of the POPI (Polystic Opinion Index) for the comprehensive evaluation of olfactory and gustatory behaviors.
[0010] S4. Calculate LC based on the linear regression equation described in S3. 50 The Point of Interest Index (POPI) is used to evaluate whether the toxicity of the Evodia rutaecarpa extract solution at the test concentration has reached the intermediate lethal concentration by comparing the two POPIs.
[0011] In one embodiment of the present invention, the method for detecting the mortality rate of fruit flies at different concentrations is to insert clean cotton balls into centrifuge tubes, and then add different concentrations of Evodia rutaecarpa extract solutions to different centrifuge tubes respectively, adding 800 μl of Evodia rutaecarpa extract solution to each tube. Then, 20 female fruit flies that have undergone 24 hours of wet starvation treatment are placed in each tube, and the mortality rate is counted after 24 hours.
[0012] In one embodiment of the present invention, the culture medium used in the bidirectional selective feeding experiment in S2 is prepared by taking a two-compartment cell culture dish and setting up sides A and B respectively. Side A consists of Evodia rutaecarpa extract solution, agarose, and Brilliant Blue FCF; side B consists of 1% sucrose solution in the same amount as the Evodia rutaecarpa extract solution in side A, agarose in the same amount as side A, and Sulforhodamine B.
[0013] In one embodiment of the present invention, the bidirectional selection feeding experiment in S2 involves placing 3-4 days of adult female fruit flies that have undergone 24 hours of wet starvation treatment into a bidirectional selection culture medium containing different concentrations of Evodia rutaecarpa extract solution, with 40 flies per group. Under dark room temperature conditions, bidirectional selection feeding is conducted for 90 minutes. After feeding, the flies are anesthetized with carbon dioxide, and the abdominal color of each group of fruit flies is observed under a stereomicroscope. The results are statistically analyzed, and the preference index PI is calculated as follows: PI = (number of red flies - number of blue flies) / (number of red flies + number of blue flies + number of purple flies).
[0014] In one embodiment of the present invention, the device used in the Y-type experiment in S2 is a Y-type experimental device formed by connecting three flat-bottomed test tubes with a glass U-shaped connecting tube and a rubber stopper. The Y-type experimental device has only one test tube at one end as the initial tube, and the other two test tubes at the other end as the stimulation tube and the control tube, respectively.
[0015] In one embodiment of the present invention, the Y-type experimental method in S2 involves placing a cotton ball in the stimulation tube and adding Evodia rutaecarpa extract solution, and placing a cotton ball in the control tube and adding an equal volume of 1% sucrose solution. Fruit flies that have undergone bidirectional selective feeding experiments at different concentrations are placed into the initial tubes of the corresponding concentrations. The Y-type experimental apparatus is then assembled, and the experiment is started with a timer. The number of fruit flies in the stimulation tube and control tube is counted every 30 minutes for a total of 4-5 times. The number of fruit flies at each concentration and time period is combined, and the average number of fruit flies in the stimulation tube and control tube at each concentration is calculated. Finally, the olfactory index OI is calculated, where OI = number of fruit flies in the stimulation tube / (number of fruit flies in the stimulation tube + number of fruit flies in the control tube).
[0016] In one embodiment of the invention, the kissing experiment in S2 involves anesthetizing fruit flies that have undergone the Y-type experiment with CO2, then confining the fruit flies within a pre-cut 200ml pipette tip to expose their heads and proboscis, while restricting their bodies and tarsi. After a 60-minute acclimatization period in a humidified chamber, the fruit flies are provided with water and allowed to drink freely until satisfied. Fruit flies that do not stop responding to water within 5 minutes are discarded. A pointed cotton swab is then soaked in a solution of Evodia rutaecarpa extract of the corresponding concentration, and then the swab containing the Evodia rutaecarpa extract is used to... The liquid was manually applied to the tip of the fruit fly's proboscis for 1-2 seconds, and the proboscis extension reflex was monitored. The proboscis test was scored using three values: 1.0 points indicated that the fruit fly extended its proboscis and ingested the food within 1.0 second of smelling the soaked swab; if the fly extended its proboscis and consumed the food in less than 1.0 second, the score was 0.5; if the fruit fly failed to extend its proboscis, the score was 0. Three consecutive proboscis tests were performed on each fruit fly, with a 1-minute interval. The PERI (Performance Index) for each test was calculated using the formula: PERI = Sum of scores / Number of fruit flies tested.
[0017] In one embodiment of the present invention, the method for S4 to assess whether the toxicity of the Evodia rutaecarpa extract solution at the test concentration reaches the median lethal concentration is as follows: if the POPI of the Evodia rutaecarpa extract solution at the test concentration is greater than LC... 50 If the POPI of the test concentration of Evodia rutaecarpa extract solution is lower than the lethal median concentration, then the POPI of the test concentration of Evodia rutaecarpa extract solution is less than the LC50 concentration. 50 For the corresponding concentration of POPI, the concentration of Evodia rutaecarpa extract solution to be tested is higher than the lethal concentration.
[0018] A second objective of this invention is to provide a model for evaluating the trace toxicity of Evodia rutaecarpa extract based on the behavior of fruit flies, obtained by the above-described construction method.
[0019] A third objective of this invention is to provide the application of the above-mentioned model for evaluating the trace toxicity of Evodia rutaecarpa extract in evaluating the trace toxicity of Evodia rutaecarpa extract. Attached Figure Description
[0020] Figure 1 A graph showing the statistical results of mortality rates for different concentrations of Evodia rutaecarpa extract solutions in acute toxicity experiments;
[0021] Figure 2 A graph showing the statistical results of the concentration of Evodia rutaecarpa extract solutions corresponding to different lethal probabilities;
[0022] Figure 3 This is a graph showing the statistical results of the preference index of fruit flies for different lethal concentrations of Evodia rutaecarpa extract solutions in a two-way selection feeding experiment.
[0023] Figure 4 This is a schematic diagram of the Y-shaped experimental setup.
[0024] Figure 5 This is a graph showing the statistical results of the olfactory index of fruit flies to different lethal concentrations of Evodia rutaecarpa extract solutions in a Y-type experiment.
[0025] Figure 6 The figure shows the statistical results of the kissing index of fruit flies to different lethal concentrations of Evodia rutaecarpa extract solution in the kissing experiment;
[0026] Figure 7 The figure shows the results of the difference analysis between the POP index obtained from the cascade experiment and the POP index obtained from the linear regression equation. Detailed Implementation
[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, and instruments used are all conventional materials, reagents, and instruments in the art, which can be obtained commercially by those skilled in the art.
[0028] Laboratory animals and their breeding
[0029] The fruit flies used in this invention are wild-type Drosophila melanogaster (Canton-S, Fungene Biotech, China). The standard culture medium for rearing the fruit flies contained sucrose, glucose, anhydrous calcium chloride, agar, yeast, cornmeal, potassium sorbate, and nicotinic acid. The rearing conditions were a temperature of 24±1℃, a humidity of 60±5%, and a light / dark cycle of 12 hours. To ensure similar nutritional conditions, the same number of adult fruit flies were stored in each vial in all experiments.
[0030] Preparation of freeze-dried Evodia rutaecarpa powder
[0031] 185g of Evodia rutaecarpa (salted) was mixed with 1850ml of pure water (solid-liquid ratio 1g:10ml) and soaked for 8 hours. The mixture was then boiled and maintained at a gentle boil for 1 hour. The decoction was filtered through sixteen layers of gauze to separate the herb from the water extract. 1480ml of pure water (solid-liquid ratio 1g:8ml) was then added to the previously decocted herb and mixed thoroughly for a second decoction. After boiling, the mixture was maintained at a gentle boil for 1 hour, and then filtered again. Pure water was then added to the residue at a 1g:8ml solid-liquid ratio, and the above steps were repeated for a third decoction. The extracts obtained from the three decoctions were combined and rotary evaporated to a suitable volume. Finally, the extracts were freeze-dried to obtain freeze-dried powder. The weight of the freeze-dried powder was measured, and the extract yield was calculated to be 69.6%.
[0032] Data processing and analysis:
[0033] The acute toxicity LC value was calculated using SPSS Statistics 27 with Probit and the LC trend chart was plotted using GraghPad Prism 9. The experimental data of each group of PI, OI and PERI were statistically analyzed using Microsoft Excel and the trend charts of PI, OI and PERI were plotted using GraghPad Prism 9.
[0034] Example 1:
[0035] (1) Acute toxicity test
[0036] Newly emerged female adults (3-4 days old) were collected from standard culture medium and placed in 25×90 mm fruit fly tubes containing only cotton. The cotton was then evenly sprinkled with purified water, and the collected fruit flies were placed inside for a 24-hour wet starvation treatment. Different concentrations of Evodia rutaecarpa extract solutions were prepared: These solutions were prepared using Evodia rutaecarpa extract powder as the solute and a 1% sucrose solution as the solvent. The concentrations of the Evodia rutaecarpa extract, converted according to the extract yield, were 1.4 g / ml, 1.1 g / ml, 0.72 g / ml, 0.51 g / ml, 0.36 g / ml, 0.14 g / ml, 0.072 g / ml, 0.014 g / ml, 0.0086 g / ml, and 0.0028 g / ml. A 1% sucrose solution was set as the negative control group, and a 2.5% lambda-cyhalothrin solution diluted 5,000 times was set as the positive control group. Acute toxicity experiments were conducted on fruit flies subjected to over-wet starvation treatment to detect the mortality rate of fruit flies under different concentrations of Evodia rutaecarpa extract solution.
[0037] The preparation method of the fruit fly culture medium tubes used in the acute toxicity test is as follows: a clean cotton ball is inserted into a 15ml centrifuge tube, and then the prepared solutions of Evodia rutaecarpa extract of different concentrations, negative control solution and positive control solution are added to different centrifuge tubes respectively, with 800μl added to each tube. Three replicates are set up, and 20 female fruit flies are placed in each tube.
[0038] Fruit flies subjected to wet starvation were placed in fruit fly culture tubes of corresponding concentrations of Evodia rutaecarpa. The number of fruit flies that died after 24 hours was recorded, and the mortality rate was calculated (see [link to relevant documentation]). Figure 1 ),from Figure 1 It can be seen that the mortality rate of fruit flies gradually increases with the increase of Evodia rutaecarpa extract concentration. The mortality rate of the normal control group with 1% sucrose and the Evodia rutaecarpa extract solution with 0.0028g / ml of raw drug was 0, indicating that the toxicity of Evodia rutaecarpa at this concentration is insufficient to kill fruit flies, and the toxicity is within the safe range. However, the mortality rate of the positive control group with 2.5% high-efficiency cyhalothrin solution diluted 5,000 times reached 100%, indicating that the 2.5% high-efficiency cyhalothrin solution diluted 5,000 times is extremely toxic to fruit flies. When the concentration of Evodia rutaecarpa extract exceeds 0.72g / ml of raw drug, the mortality rate is greater than 50%.
[0039] Linear regression models of different concentrations of Evodia rutaecarpa extract solutions and their corresponding lethalities were obtained using SPSS Statistics 27's Probit algorithm. The model fit was assessed using the model summary R (R>0.7) or R-squared (R-squared>0.5), indicating a high degree of fit. The Probit Pearson goodness-of-fit test showed a significance greater than 0.05, indicating acceptance of the null hypothesis and a good fit between the data and the model. ANOVA analysis showed a significance less than 0.05, proving the model's regression was significant. Based on the obtained linear regression model, the LC... 80 LC 65 LC 50 LC 35 LC 20 The corresponding concentration of Evodia rutaecarpa extract (see...) Figure 2 ), LC 80 LC 65 LC 50 LC 35 LC 20 The corresponding concentrations of Evodia rutaecarpa extract were 1.442 g raw drug / ml, 1.127 g raw drug / ml, 0.861 g raw drug / ml, 0.594 g raw drug / ml, and 0.279 g raw drug / ml, respectively. The figure shows that as the concentration of Evodia rutaecarpa decreases, the LC also gradually decreases, indicating less toxicity to Drosophila.
[0040] (2) Series Experiment
[0041] Based on the analysis of preliminary experimental results, the corresponding indices of each behavioral experiment in Drosophila may exhibit fluctuations. To reduce the impact of these fluctuations and better reflect the olfactory and gustatory behaviors of Drosophila, this invention sequentially strings together the bidirectional selection feeding experiment, the Y-shaped experiment, and the proboscis experiment. Each experiment in this sequential experiment is based on the sensitive olfactory and gustatory behavioral responses of Drosophila to explore the toxicity of different concentrations of Evodia rutaecarpa extract solution. LC50 values were prepared according to the lethal concentration (LC50) for acute toxicity. 80 LC 65 LC 50 LC 35 LC 20 Evodia rutaecarpa extract solutions of corresponding concentrations were prepared using a 1% sucrose solution. Using LC values to correspond to concentrations, a two-way selection feeding experiment, a Y-shaped experiment, and a kissing experiment were conducted sequentially. Behavioral indices were calculated for each experiment, and a comprehensive olfactory and gustatory behavior evaluation index (POPI, Olfactory index, Proboscis extension response index) was constructed.
[0042] ① Two-way selection feeding experiment:
[0043] Preparation of the culture medium used in the two-way selection feeding experiment:
[0044] Use 90mm bicompartment cell culture dishes, setting up sides A and B respectively. Experimental group setup: Side A: 2ml Evodia rutaecarpa extract solution, 2ml 1% agarose, 0.5ml Brilliant Blue FCF; Side B (control): 2ml 1% sucrose solution, 2ml 1% agarose, 0.5ml Sulforhodamine B. Positive control setup: Side A: 2ml 2.5% lambda-cyhalothrin diluted 5000 times, 2ml 1% agarose, 0.5ml Brilliant Blue FCF; Side B: 2ml 1% sucrose solution, 2ml 1% agarose, 0.5ml Sulforhodamine B. Negative control setup: Sides A and B each contain 2ml 1% sucrose and 2ml 1% agarose; Side A contains 0.5ml Brilliant Blue FCF, and Side B contains 0.5ml Sulforhodamine B. When preparing the solid culture medium for the bidirectional selective feeding experiment, the order of adding the solution is as follows: first add the Evodia rutaecarpa extract solution, then add the dye, and finally add the heated 1% agarose. Let it stand until the mixed solution solidifies, and the preparation is complete.
[0045] Procedure for a two-way selection feeding experiment:
[0046] Adult female fruit flies, pre-treated with 24-hour wet starvation for 3-4 days, were placed in bidirectional selection culture media containing different concentrations of Evodia rutaecarpa extract solution, with 40 flies per group. Under dark, room temperature conditions, they underwent bidirectional selective feeding for 90 minutes. After feeding, they were anesthetized with carbon dioxide, and the abdominal color of each group of fruit flies was observed under a stereomicroscope. The results were statistically analyzed, and the preference index (PI) was calculated: PI = (number of red flies - number of blue flies) / (number of red flies + number of blue flies + number of purple flies). In this invention, the bidirectional selective feeding experiment involves mixing the test drug with red dye; therefore, the above formula is used to calculate the preference index. If the test drug is mixed with blue dye, the following formula is used to calculate PI: (number of blue flies - number of red flies) / (number of red flies + number of blue flies + number of purple flies). Red indicates that the fruit fly ate the red-dyed side of the culture medium, resulting in a red abdomen; blue indicates that the fruit fly ate the blue-dyed side of the culture medium, resulting in a blue abdomen; purple indicates that the fruit fly ate both the red and blue sides of the culture medium. A PI of 0 indicates that fruit flies have no preference for the tested drug, while a PI between 0 and 1 indicates that fruit flies have a preference for the tested drug, with the preference increasing as the PI approaches 1. Conversely, a PI between -1 and 0 indicates that fruit flies have no preference for the tested drug, with the aversion increasing as the PI approaches -1. Behavioral data were collected under double-blind conditions, and the raters were unaware of the identity of each sample being rated.
[0047] Calculate LC using the preference index formula. 80 LC 65 LC 50 LC 35 LC 20 The corresponding preference indices are -0.48, -0.54, -0.21, 0, and 0.53 (retaining two significant figures for ease of representation in the chart) (see... Figure 3 ), where LC 20 The corresponding fruit fly preference index was the highest, indicating that fruit flies showed a preference for this concentration of Evodia rutaecarpa extract solution, and the degree of preference was greater than that of other groups; LC 35 The preference index was 0, indicating that fruit flies exhibited neither aversion nor preference towards this concentration of Evodia rutaecarpa extract solution; while the LC... 80 LC 65 LC 50 The negative values of the preference index indicate that fruit flies exhibit aversion to the Evodia rutaecarpa extract solution at the above concentration, which is the same as the aversion exhibited by fruit flies to the positive control group of 2.5% high-efficiency cyhalothrin solution.
[0048] ②Y-shaped experiment:
[0049] This experiment uses a glass U-shaped connecting tube and rubber stoppers to connect three flat-bottomed test tubes, each approximately 10 cm long and 2.5 cm wide, forming a Y-shaped experimental setup (see...). Figure 4 Initial tubes (I), stimulation tubes (S), and control tubes (C) were set up. Fruit flies were placed in tube I, cotton balls with Evodia rutaecarpa extract solution were placed in tube S, and cotton balls with 1% sucrose solution were placed in tube C. Fruit flies that had undergone a two-way selection feeding experiment were placed in tube I for each concentration. The Y-shaped experimental setup was then assembled, and the experiment was started. The number of fruit flies in tubes S and C was counted every 30 minutes, for a total of 4-5 times. The number of fruit flies at each concentration and time period was combined, and the average number of fruit flies in tubes S and C for each concentration was calculated. Finally, the olfactory index (OI) was calculated: OI = number of fruit flies in S / (number of fruit flies in S + number of fruit flies in C). This index is used to assess the degree of fruit fly olfactory response to a sample. An OI between 0 and 0.5 indicates that the fruit fly has an aversion to the sample, with the closer to 0 being the degree of aversion. An OI between 0.5 and 1 indicates that the fruit fly has a preference for the test sample, with the closer to 1 being the degree of preference. An OI value of 0.5 can correspond to two different situations: no odorant was detected or a response between attraction and aversion.
[0050] Calculate LC using the olfactory index formula. 80 LC 65 LC 50 LC 35 LC 20 The corresponding olfactory indices are 0.35, 0.25, 0.20, 0.55, and 0.30 (two significant figures are retained for ease of representation in the graph) (see... Figure 5 OI > 0.5 is the Drosophila preference criterion, determined by... Figure 5 It is known that LC 35 A corresponding olfactory index greater than 0.5 indicates that at this concentration of Evodia rutaecarpa extract, more fruit flies entered the stimulation tube in the Y-type experiment than the control tube, showing a preference for the Evodia rutaecarpa extract solution in the stimulation tube. LC... 20 LC 50 LC 65 LC 80 An olfactory index less than 0.5 indicates that, at the above concentration of Evodia rutaecarpa extract solution, the number of fruit flies entering the control tube in the Y-type experiment was greater than that in the stimulation tube, indicating aversion to the Evodia rutaecarpa extract solution in the stimulation tube. Theoretically, the olfactory index should decrease with increasing concentration, compared to LC... 20 And LC 35 LC 80 The olfactory index fluctuates; analysis of LC35 LC 80 The corresponding concentration may have fluctuating attraction to fruit flies or abnormal olfactory behavior in fruit flies.
[0051] ③ Kissing experiment:
[0052] Drosophila undergoing the Y-type experiment were anesthetized with CO2 and then confined to a pre-cut 200ml pipette tip to expose their head and proboscis, while restricting their body and tarsi. After a 60-minute acclimatization period in a humidified chamber, the flies were provided with water and allowed free access to drink until satisfied. Flies that did not stop responding to water within 5 minutes were discarded. A pointed cotton swab was soaked in a solution of Evodia rutaecarpa extract of the corresponding concentration, and then manually applied to the tip of the fly's proboscis for 1-2 seconds, monitoring the proboscis extension reflex. The proboscis extension test was scored using three values. A score of 1.0 indicates that the fly extended its proboscis and ingested the food for 1.0 second upon smelling the soaked swab. A score of 0.5 was given if the fly extended its proboscis and consumed the food for less than 1.0 second. A score of 0 was given if the fly failed to extend its proboscis. Three consecutive proboscis extension tests were performed on each fly, spaced 1 minute apart. During each feeding, the fly was only allowed to eat for two seconds. To eliminate sluggish animals, a 1% sucrose solution was provided 3-5 minutes after the test. Fruit flies that did not exhibit a proboscis excursion were excluded from the dataset. The PERI (Percussion Index) for each experiment was calculated as follows: PERI = Sum of scores / Number of fruit flies tested.
[0053] Calculate LC using the kissing index formula. 80 LC 65 LC 50 LC 35 LC 20 The corresponding perpetual kiss index (PERI) were 0.625, 0.286, 0.500, 0.550, and 0.650, respectively, while the 1% sucrose positive control was 0.857 (three significant figures are retained for ease of representation in the figure) (see...). Figure 6 As shown in the figure, the kissing index increases with decreasing LC value, indicating that in the kissing experiment, fruit flies exhibit a higher kissing frequency and greater preference as the concentration of Evodia rutaecarpa extract decreases; lower concentrations are closer to the positive control group. Using a kissing index greater than 0.5 as the standard for fruit fly preference, the LC value can be determined... 35 LC 20 The corresponding concentrations of Evodia rutaecarpa extract attracted Drosophila melanogaster, with the number of sneezing attempts increasing as the concentration decreased. LC 80 LC 65 At the corresponding concentration of Evodia rutaecarpa extract solution, fruit flies exhibited fewer sneezing attempts and lower scores, showing aversion to Evodia rutaecarpa extract at other concentrations.
[0054] (3) Construction and analysis of cascaded experimental regression model
[0055] Based on the PPI, OI, and PERI scores, with 0, 0.5, and 0.5 respectively as the boundaries for preference and aversion, the POP index was constructed by averaging the scores and comprehensively evaluating the olfactory and gustatory behavioral responses of Evodia rutaecarpa to fruit flies. A POPI of 0.3 was used as the cutoff standard to assess the aversion and preference behavior of fruit flies towards toxic drugs; a POPI greater than 0.3 indicated preference, a POPI less than 0.3 indicated aversion, and a POPI of 0.3 indicated neither aversion nor preference. Linear regression was performed using SPSS Statistics 27, yielding the linear regression equation for the POPI index: y = 0.666 - 0.935x, R = 0.977. 2 =0.955, where x is the concentration of the Evodia rutaecarpa extract solution. R, R 2 A value closer to 1 indicates a better fit, with R > 0.5 or R < 1. 2 A value >0.7 indicates a good model. To verify the reliability of the equation: a reverse validation test was performed. Using the random number generator in SPSS Statistics 27, the function Rv.Uniform was applied to generate random numbers in the range of 0-1, which were used as the concentrations of the Evodia rutaecarpa extract solution in the validation experiment. The following concentrations were obtained: 0.24 g raw drug / ml, 0.40 g raw drug / ml, 0.53 g raw drug / ml, 0.70 g raw drug / ml, 0.96 g raw drug / ml, 1.09 g raw drug / ml, and 1.26 g raw drug / ml (retaining two significant figures after conversion). These concentrations were substituted into the equation y = 0.666 - 0.935x to calculate the corresponding POPI. Then, these concentrations were used to conduct tandem experiments to calculate PI, OI, and PERI, obtaining the corresponding POPI in each experiment. Finally, a t-test was performed using GraghPad Prism 9 to check for significant differences (see...). Figure 7 Experimental results showed that the POPI obtained from the equation was not significantly different from the experimentally verified POPI, indicating that the equation was reliable. To facilitate the assessment of the toxicity of a certain concentration of Evodia rutaecarpa extract solution, LC-L... 50 As a reference indicator, the median lethal concentration (LD50) is used in LC50. 50 Substituting 0.861 into the POP regression equation, we obtain LC. 50 The POP (Potential Opposites Oral Content) is approximately -0.14, which is less than 0.3. At this concentration, fruit flies exhibit non-preference (repellent) behavior. This concentration can be used as a reference indicator to assess the toxicity of a certain concentration of Evodia rutaecarpa extract solution. To determine the trace toxicity of a certain concentration of Evodia rutaecarpa extract solution, simply substitute this concentration into the equation and compare the resulting POP with the LC50. 50The POPI can be compared. For example, to evaluate the toxicity of an Evodia rutaecarpa extract solution with a concentration of 0.034 g / ml, substituting this concentration into the equation yields y = 0.666 - 0.935 × 0.034 ≈ 0.634, which is greater than the POPI. LC50 = -0.14, toxic concentration less than LC 50 The experimental method may result in some fluctuations in the yield of Evodia rutaecarpa extract and the acute toxicity results in Drosophila, but the results of tandem experiments will not be affected by this.
[0056] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.
Claims
1. A method for constructing a model for evaluating the trace toxicity of Evodia rutaecarpa extract based on Drosophila behavior, characterized in that, Includes the following steps: S1. Evodia rutaecarpa extract solutions of different concentrations were prepared using 1% sucrose solvent. The mortality rate of fruit flies at different concentrations was detected, and LC-weighted averages were obtained. 80 LC 65 LC 50 LC 35 LC 20 Corresponding concentration; S2. Drosophila subjected to wet starvation treatment underwent a series of experiments consisting of a two-way selection feeding experiment, a Y-shaped experiment, and a proboscis test, in that order. The preference index PI, olfactory index OI, and proboscis test PERI were obtained, respectively. The concentration of the Evodia rutaecarpa extract solution used in the series experiments was the same as the LC50 concentration in S1. 80 LC 65 LC 50 LC 35 LC 20 Corresponding concentration; The method of the bidirectional selection feeding experiment is to put 3-4 days of adult female fruit flies that have been subjected to 24 h of wet starvation into a bidirectional selection medium of Evodia rutaecarpa extract solution of corresponding concentration, with 40 flies in each group. Under dark room temperature conditions, bidirectional selection feeding is carried out for 90 min. After feeding, the flies are anesthetized with carbon dioxide, and the abdominal color of each group of fruit flies is observed under a stereomicroscope. The results are statistically analyzed, and the preference index PI is calculated. PI = (number of red flies - number of blue flies) / (number of red flies + number of blue flies + number of purple flies). The Y-type experiment method involves placing cotton balls in the stimulation tube and adding Evodia rutaecarpa extract solution, and placing cotton balls in the control tube and adding an equal volume of 1% sucrose solution. Fruit flies that have undergone bidirectional selective feeding experiments at different concentrations are placed into the corresponding initial tubes. The Y-type experimental apparatus is then assembled, and the experiment is started with a timer. The number of fruit flies in the stimulation and control tubes is counted every 30 minutes for a total of 4-5 times. The number of fruit flies at each concentration and time period is combined, and the average number of fruit flies in the stimulation and control tubes at each concentration is calculated. Finally, the olfactory index OI is calculated as: OI = number of fruit flies in the stimulation tube / (number of fruit flies in the stimulation tube + number of fruit flies in the control tube). The method of the proboscis test was as follows: Drosophila that had undergone the Y-type test were anesthetized with CO2, and then the head and proboscis of the Drosophila were exposed, while the body and tarsi were restricted. After an acclimatization period of 60 minutes in a humidified chamber, the Drosophila were provided with water and allowed to drink freely until satisfied. Drosophila that did not stop responding to water within 5 minutes were discarded. A cotton swab was soaked in a solution of Evodia rutaecarpa extract of the corresponding concentration, and then the cotton swab containing the Evodia rutaecarpa extract solution was applied to the tip of the proboscis of the Drosophila for 1-2 seconds, and the proboscis extension reflex was monitored. The proboscis test was scored using three values: 1.0 points indicates that the Drosophila extended its proboscis and ingested the food for 1.0 second after smelling the soaked cotton swab; if the time it took for the fly to extend its proboscis and consume the food was less than 1.0 second, the score was 0.5; if the Drosophila failed to extend its proboscis, the score was 0. The proboscis test was performed three times consecutively for each Drosophila, with an interval of 1 minute. The proboscis index for each test was calculated using the formula: PERI = Sum of scores / Number of Drosophila tested. S3. Use SPSS Statistics 27 to perform linear regression and find the linear regression equation of the POPI (Polystic Opinion Index) for the comprehensive evaluation of olfactory and gustatory behaviors. S4. Calculate LC based on the linear regression equation described in S3. 50 The comprehensive evaluation index (POPI) of the Evodia rutaecarpa extract solution at the corresponding concentration and the concentration to be tested is used to compare the two comprehensive evaluation indices to assess whether the toxicity of the Evodia rutaecarpa extract solution at the test concentration reaches the intermediate lethal concentration.
2. The construction method according to claim 1, characterized in that, The method for detecting the mortality rate of fruit flies at different concentrations using S1 involves an acute toxicity test. Female fruit flies subjected to 24 hours of wet starvation were placed in containers containing different concentrations of Evodia rutaecarpa extract solution, and the mortality rate was calculated after 24 hours.
3. The construction method according to claim 1, characterized in that, The method for preparing the culture medium used in the bidirectional selective feeding experiment described in S2 is to take a two-compartment cell culture dish and set up sides A and B respectively. Side A consists of Evodia rutaecarpa extract solution, agarose, and Brilliant Blue FCF; side B is the control and consists of an equal amount of 1% sucrose solution of Evodia rutaecarpa extract solution of side A, an equal amount of agarose of side A, and Sulforhodamine B.
4. The construction method according to claim 1, characterized in that, The Y-type experiment described in S2 uses a glass U-shaped connecting tube and a rubber stopper to connect three flat-bottomed test tubes to form a Y-type experimental device. The Y-type experimental device has only one test tube at one end, which is the initial tube, and the other two test tubes at the other end are the stimulation tube and the control tube, respectively.
5. The construction method according to claim 1, characterized in that, S4 assesses whether the toxicity of the tested concentration of Evodia rutaecarpa extract solution reaches the median lethal concentration. If the POPI of the tested concentration of Evodia rutaecarpa extract solution is greater than the LC50 value... 50 If the POPI of the test concentration of Evodia rutaecarpa extract solution is lower than the lethal median concentration, then the POPI of the test concentration of Evodia rutaecarpa extract solution is less than the LC50 concentration. 50 For the corresponding concentration of POPI, the concentration of Evodia rutaecarpa extract solution to be tested is higher than the lethal concentration.
6. The application of the model for evaluating the trace toxicity of Evodia rutaecarpa extract obtained by any of the construction methods described in claims 1-5 in the evaluation of the trace toxicity of Evodia rutaecarpa extract.