Use of active ingredients that activate the tas1r3 receptor for the preparation of a medicament for improving anemia

Abstract

The application provides a method for screening active ingredients capable of activating TAS1R3 receptors, and belongs to the technical field of screening of active ingredients of traditional Chinese medicines. The application establishes a biosensor, and the biosensor detects the current signal after each concentration of compound solution reacts with the TAS1R3 receptor; the dissociation constant K D of the interaction between the compound and the TAS1R3 receptor is calculated according to the linear fitting equation between the common logarithm of the concentration of the compound and the current change, and the compound with the K D value in the range of 10 ‑13 -10 ‑8 M is screened as the active ingredient capable of activating the TAS1R3 receptor. In the application, the interaction dissociation constant K D of the Atractylodes lactone II and the glycyrrhizic acid with the TAS1R3 protein is 3.759*10 ‑8 M and 4.435*10 ‑9 M respectively, and the binding force with the TAS1R3 protein is very strong. Moreover, the Atractylodes lactone II and the glycyrrhizic acid have the function of improving anemia within a certain concentration, and provide a new idea for the screening of candidate compounds with the sweet characteristic and the treatment of anemia or blood deficiency syndrome in traditional Chinese medicines and the research and development of new drugs.

Description

Technical Field

[0001] This invention relates to the field of screening active ingredients in traditional Chinese medicine preparations, and particularly to the application of active ingredients that activate the TAS1R3 receptor in the preparation of drugs to improve anemia. Background Technology

[0002] Sweet-tasting Chinese herbs play an indispensable role in the compatibility and efficacy of compound prescriptions. They possess unique compatibility effects in these prescriptions and are widely used to treat various clinical diseases, including spleen and stomach disorders, diabetes, gynecological diseases, eye diseases, coronary heart disease, hypertension, and perimenopausal syndrome primarily characterized by imbalances in Yin, Yang, Qi, and Blood. Treatment with sweet-tasting herbs aligns with the principle of "regulating with sweet herbs," which is the essence of TCM's syndrome differentiation and treatment. Sweet-tasting Chinese herbs mainly contain sugars, glycosides, amino acids, and proteins, and their taste expression is closely related to the TAS1R3 receptor. However, no one has yet evaluated the anti-anemic efficacy of active ingredients specifically recognized by the TAS1R3 receptor. Summary of the Invention

[0003] The purpose of this invention is to provide the application of an active ingredient that activates the TAS1R3 receptor in the preparation of a drug for improving anemia.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a method for screening active ingredients that can activate the TAS1R3 receptor, comprising the following steps:

[0006] S1. Construct a biosensor, wherein the substrate of the biosensor is a semiconductor material with biomodification function, and the biorecognition element of the biosensor is a TAS1R3 receptor;

[0007] S2. Add compound solutions of different concentration gradients in order of increasing concentration, and use a biosensor to detect the current signal after the compound solutions of each concentration react with the TAS1R3 receptor.

[0008] S3. Within a certain concentration range, the common logarithm of the compound concentration shows a linear relationship with the relative value of the current change. Determine the linear fitting equation.

[0009] S4. Calculate the dissociation constant K for the interaction between the compound and the TAS1R3 receptor based on the linear fitting equation. D value;

[0010] S5. Filter out K D Value at 10 -13 ~10 -8 Compounds within the M range are the active ingredients that can activate the TAS1R3 receptor.

[0011] Preferably, the biosensor includes the TAS1R3-HEMT biosensor and the TAS1R3@AuNPs / SPCE biosensor.

[0012] Preferably, the concentration of the compound solution is 10. -4 ~10 -14 mol / L.

[0013] This invention also provides the application of atractylodes lactone II, paeonol, or glycyrrhizic acid in the preparation of TAS1R3 receptor activating agents.

[0014] The present invention also provides the use of atractylodes lactone II, paeonol, or glycyrrhizic acid in the preparation of drugs for improving anemia.

[0015] By adopting the above technical solution, the present invention has the following beneficial effects:

[0016] This invention establishes a biosensor that detects the current signal after the reaction of compound solutions of various concentrations with the TAS1R3 receptor; and calculates the dissociation constant K of the interaction between the compound and the TAS1R3 receptor based on a linear fitting equation between the common logarithm of the compound concentration and the current change. D Value, filter out K D Value at 10 -13 ~10 -8 Compounds within the M range are the active ingredients that can activate the TAS1R3 receptor.

[0017] In this invention, the dissociation constant K of the interaction between atractylodes lactone II, glycyrrhizic acid, and TAS1R3 protein is... D The values ​​are 3.759 × 10 -8 M, 4.435×10 -9 M; has a very strong binding affinity to the TAS1R3 protein. Furthermore, atractylodes lactone II and glycyrrhizic acid, within certain concentrations, have the function of improving anemia, providing new ideas for the screening of candidate compounds in traditional Chinese medicine with sweet properties that treat anemia or blood deficiency, and for the development of new drugs. Attached Figure Description

[0018] Figure 1 The interaction analysis between TAS1R3 and atractylodes lactone II is shown in (a) IT curves of atractylodes lactone II solutions with different concentration gradients, and (b) the linear relationship between the common logarithm of atractylodes lactone II concentration and the relative value of current change.

[0019] Figure 2 The survival of zebrafish after treatment with different concentrations of active ingredients for different durations; the active ingredient in (a) is atractylodes lactone II; the active ingredient in (b) is glycyrrhizic acid.

[0020] Figure 3The effects of different phenylhydrazine concentrations on zebrafish embryonic development and erythrocytes are shown in Figure 1. (A) shows the relationship between phenylhydrazine concentration and zebrafish mortality rate; (B) shows the relationship between phenylhydrazine concentration and the staining area of ​​the zebrafish heart; and (C) shows a schematic diagram of the relationship between phenylhydrazine concentration and zebrafish heart staining.

[0021] Figure 4 Statistical analysis of the staining area of ​​the heart region in zebrafish of each drug intervention group (compared with the blank control group, #P<0.001; compared with the model control group, *P<0.001). Detailed Implementation

[0022] This invention provides a method for screening active ingredients that can activate the TAS1R3 receptor, comprising the following steps:

[0023] S1. Construct a biosensor, wherein the substrate of the biosensor is a semiconductor material with biomodification function, and the biorecognition element of the biosensor is a TAS1R3 receptor;

[0024] S2. Add compound solutions of different concentration gradients in order of increasing concentration, and use a biosensor to detect the current signal after the compound solutions of each concentration react with the TAS1R3 receptor.

[0025] S3. Within a certain concentration range, the common logarithm of the compound concentration shows a linear relationship with the relative value of the current change. Determine the linear fitting equation.

[0026] S4. Calculate the dissociation constant K for the interaction between the compound and the TAS1R3 receptor based on the linear fitting equation. D value;

[0027] S5. Filter out K D Value at 10 -13 ~10 -8 Compounds within the M range are the active ingredients that can activate the TAS1R3 receptor.

[0028] In this invention, atractylodes lactone II and glycyrrhizic acid standards are accurately weighed, and the standards are prepared to a concentration of 10 μL using PBS or deionized water. -3 A mol / L standard solution was diluted 11 times in a 10-fold gradient to obtain concentrations of 10 mol / L. -4 ~10 -14 mol / L standard solution, for later use.

[0029] This invention employs a biosensor to sequentially inject standard solutions of the active ingredient in ascending order of concentration, with a reaction time of 5–10 min. The IT curve or I curve of the reaction between the sample and TAS1R3 protein at room temperature is detected using either chronoamperometry or linear sweep voltammetry. DS -VDS Curves were generated, with each concentration measured three times. The linear relationship between the common logarithm of concentration (Lg[C]) and the relative change in current ((I-I0) / I0), as well as the linear relationship between sample concentration and concentration / current change, were observed. The biosensor described in this invention is preferably the TAS1R3-HEMT biosensor or the TAS1R3@AuNPs / SPCE biosensor.

[0030] This invention determines a linear fitting equation based on the linear relationship between concentration and concentration / current change, and calculates the dissociation constant K of the interaction between the compound and the TAS1R3 receptor based on the linear fitting equation. D Value, filter out K D Value at 10 -13 ~10 -8 Compounds within the M range are the active ingredients that can activate the TAS1R3 receptor.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1: Characterization method of interaction between active ingredient and TAS1R3 receptor

[0033] (1) Preparation of the sample solution to be tested

[0034] Accurately weigh 0.23 mg of atractylodes lactone II and 0.82 mg of glycyrrhizic acid standards into separate 2 mL centrifuge tubes. Prepare 10 mL solutions of each standard using PBS or deionized water. -3 A mol / L standard solution was diluted 11 times in a 10-fold gradient to obtain concentrations of 10 mol / L. -4 ~10 -14 mol / L standard solution, for later use.

[0035] (2) Sample testing

[0036] Using the TAS1R3-HEMT biosensor or the TAS1R3@AuNPs / SPCE biosensor, standard solutions of the active ingredient were sequentially injected from low to high concentrations, with a reaction time of 5–10 min. The IT curve or I curve of the sample reacting with TAS1R3 protein at room temperature was detected using chronoamperometry or linear sweep voltammetry. DS -V DS Curves were generated, with each concentration measured three times. The linear relationship between the common logarithm of concentration (Lg[C]) and the relative change in current ((I-I0) / I0), as well as the linear relationship between sample concentration and concentration / current change, were observed.

[0037] TAS1R3-HEMT biosensor construction method:

[0038] A three-step immobilization method was used to modify the saccharin TAS1R3 onto an AlGaAs / GaAs HEMT sensor chip (provided by the Institute of Semiconductors, Chinese Academy of Sciences). The immobilization steps are as follows: ① Using the HEMT chip as a substrate, a transparent ultrathin quartz glass tube was adhered to the gate region of the chip as a sample cell; 100 μL of 3-mercaptopropionic acid (containing sulfur) solution was added to the sample cell, and the mixture was soaked at room temperature for 17–24 h to form gold-sulfur bonds (Au-S) in the gate region of the HEMT chip. ② The residue in the sample cell was washed with deionized water, and an equal volume of 20 mM carbodiimide hydrochloride and 50 mM hydroxysuccinimide was added to form a mixed aqueous solution. The reaction was carried out at room temperature for 15 min to generate a stable amine activation product for activating the carboxyl group. ③ Clean the HEMT chip sample cell with PBS solution, add 30 μL of TAS1R3 protein, and refrigerate at 4℃ for 2-5 hours to obtain the TAS1R3-HEMT biosensor chip. Integrate the chip with the probe platform (U-Test Core Electronics Technology (Shenzhen) Co., Ltd.), CHI660E electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd.), and data processing system (Shanghai Chenhua Instrument Co., Ltd.) to obtain the TAS1R3-HEMT biosensor.

[0039] TAS1R3@AuNPs / SPCE biosensor construction method:

[0040] The screen-printed electrode (SPCE) (from Glass Carbon Technology (Weihai) Co., Ltd.) was exposed to a saturated Na₂CO₃ solution and pretreated for 5 min at 1.2 V using chronoamperometry (CA) to remove organic binders from the electrode surface. 100 μL of a 2.5 mol / L HAuCl₄ aqueous solution was then added to the SPCE electrode surface, and cyclic voltammetry was used to scan for 90–100 cycles at a voltage of -1.5–0 V. Gold ions (AuCl₄) were deposited via electrodeposition. 3+Gold nanoparticles (AuNPs) were reduced to completely cover the surface of the SPCE working electrode to obtain the AuNPs / SPCE electrode, and its morphology was observed. 100 μL of 3-mercaptopropionic acid was added dropwise to completely cover the AuNPs / SPCE working electrode, and the reaction was carried out at room temperature for 17–24 h to generate Au-S bonds on the working electrode surface, forming a self-assembled monolayer. Excess 3-mercaptopropionic acid was washed away with deionized water, and then 100 μL of a mixture of equal volumes of 20 mM carbodiimide hydrochloride and 50 mM hydroxysuccinimide was added as a crosslinking agent to completely immerse the AuNPs / SPCE working electrode surface. The reaction was carried out at room temperature for 15 min to activate the carboxyl groups. Residual reagents were washed away with deionized water, and 30 μL of... The TAS1R3 protein was incubated at 4°C for 2–5 hours and then immobilized onto the surface of gold nanoparticles via Au-S bonds, thus obtaining the TAS1R3@AuNPs / SPCE biosensor chip. Finally, the prepared TAS1R3@AuNPs / SPCE biosensor chip was placed in an adapter (BoCarbon Technology (Weihai) Co., Ltd.) and integrated with the CHI660E electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd.) and data processing system (Shanghai Chenhua Instrument Co., Ltd.) to obtain the TAS1R3@AuNPs / SPCE biosensor.

[0041] (3) Test results

[0042] Taking the interaction analysis between atractylodes lactone II and TAS1R3 protein as an example, the detection results are as follows: Figure 1 As shown. By Figure 1 It can be seen that as the concentration of atractylodes lactone II gradually increases, the current response value gradually decreases. With the continuous increase in the concentration of atractylodes lactone II solution, the small drug molecules in the solution continuously bind to the TAS1R3 protein, leading to an increase in impedance and thus a gradual decrease in the current response value. Until the concentration of atractylodes lactone II exceeds 0.1 mM, the current response value shows an upward trend, indicating that the binding of TAS1R3 protein to the small molecules of atractylodes lactone II has reached saturation at this point. The concentration of atractylodes lactone II solution at 1×10⁻⁶ mM... -13 M to 1×10 -4 Within the range of M, Figure 1 (b) shows a good linear relationship between the common logarithm of concentration (LgC) and the relative change in current response ((I-I0) / I0, where I0 represents the current response of the blank solvent). The linear fitting equation is y = -0.0133x - 0.9637 (R²). 2 =0.9754), the linear fitting equation for concentration and concentration / current change (C / ΔI) is y = 986925x + 0.0371 (R = 0.9754). 2=1.0000), calculate the dissociation constant K of the interaction between atractylodes lactone II and TAS1R3 protein. D The value is 3.759 × 10 -8 M.

[0043] Similarly, the dissociation constant K of the interaction between glycyrrhizic acid and TAS1R3 protein was calculated. D The value is 4.435 × 10 -9 M.

[0044] K D Value at 10 -13 ~10 -8 Within the M range, this indicates a very strong binding force. In summary, the K-axis of the interaction between the TAS1R3 protein and active molecules... D The values ​​are all above 10 -13 ~10 -8 Within the M range, it indicates that the TAS1R3 protein has a very strong affinity for the above-mentioned active ingredients, and the active ingredients can activate the TAS1R3 receptor.

[0045] Example 2: Characterization of the efficacy of the active ingredient in improving anemia

[0046] (1) Solution preparation

[0047] Zebrafish embryo culture medium: Weigh 1462.5 mg sodium chloride, 242.583 mg calcium chloride, 63.325 mg potassium chloride, and 198 mg magnesium sulfate into a 100 mL beaker. Add an appropriate amount of deionized water to dissolve the contents. Sonicate for 20 minutes until completely dissolved. Transfer the solution to a 500 mL volumetric flask and bring it to a final volume with deionized water. Store at 4°C for later use. Dilute 10 times with deionized water and preheat to approximately 28.5°C before use. Adjust the pH to approximately 7.2 using sodium bicarbonate solution. Ensure the salinity of the culture medium is within the range of 0.25–0.5%. Add 1–2 drops of methylene blue solution per 1 L before use.

[0048] Active ingredient sample solution: Accurately weigh 0.23 mg of atractylodes lactone II and 0.82 mg of glycyrrhizic acid standards into 2 mL centrifuge tubes, dissolve them in embryo culture medium to prepare a 1 mM solution of atractylodes lactone II and glycyrrhizic acid for later use. During the experiment, dilute to the grouped concentrations with zebrafish embryo culture medium and preheat to approximately 28.5℃.

[0049] (2) Zebrafish embryo culture

[0050] Cultured according to the relevant regulations of the National Zebrafish Resource Center, the embryonic development temperature of AB wild-type zebrafish was strictly controlled at 28.5℃, the dark / light time was 10h / 14h, and the light intensity was 54~324 lux. The fetal membranes or dead embryos were aspirated daily, and the preheated culture medium was replaced once a day. In order to reduce the interference of melanin in zebrafish on the calculation of the staining intensity of cardiac erythrocytes, PTU was added to the zebrafish culture medium to inhibit melanin production.

[0051] (3) Drug tolerance study

[0052] Zebrafish embryos that were 3 days post-fertilization and under normal development were selected and randomly transferred into 12-well plates with 10 juvenile fish per well. They were given seven different concentrations of atractylodes lactone II and glycyrrhizic acid active ingredient sample solutions at 25 μM, 50 μM, 100 μM, 200 μM, 500 μM, 1000 μM and 2000 μM respectively. Three replicates were set for each concentration group. The samples were placed in a constant temperature and light incubator under the culture conditions of (2) and cultured for 24 h. The teratogenicity and mortality of the zebrafish were observed.

[0053] Drug tolerance study results as follows Figure 2 As shown, by Figure 2 As shown in (a), treatment with 200 μM atractylodes lactone II for 24 h induced 100% (30 / 30) mortality in zebrafish. Figure 2 As shown in (b), normal zebrafish tolerate glycyrrhizic acid well. After treatment with concentrations of 25–100 μM for 24 h, 48 h, and 72 h, no significant malformations or mortality were observed in zebrafish embryos. However, at a concentration of 500 μM for 24 h, 48 h, and 72 h, the mortality rate exceeded 70%. With increasing drug concentration and prolonged treatment time, the mortality rate rose to 100%. Therefore, the IC50 of atractylodes lactone II after 24 h of administration was finally calculated. 50 The concentration was 139.75 μM; the IC50 values ​​for glycyrrhizic acid treatment at 24 h, 48 h, and 72 h were... 50 The values ​​were 437.97 μM, 388.45 μM, and 326.88 μM, respectively.

[0054] (4) Investigation on the concentration of phenylhydrazine, a modeling agent for zebrafish anemia model

[0055] Healthy wild-type AB strain zebrafish, 24 hpf post-fertilization, were used as experimental animals. After demembranes removal, they were transferred to 12-well plates, 10 zebrafish per well. The blank control group was given zebrafish culture water, while other groups were given different concentrations of phenylhydrazine. Each group had three replicate wells. The plates were incubated at 28.5℃ until 48 hpf, and the growth and development of the zebrafish were observed. The modeling agent was removed, and the zebrafish were washed three times with zebrafish culture water. All groups were then given zebrafish culture water. The zebrafish were incubated at 28.5℃ until 5 dpf. Red blood cells from each group were stained using the o-anisidine staining method. After staining, the cells were fixed with 4% neutral paraformaldehyde, photographed under a microscope, and the stained area and intensity of red blood cells were measured using Image-Pro Plus 6 software to observe the success of the zebrafish anemia model and to screen for suitable phenylhydrazine concentrations for anemia model establishment.

[0056] The effects of different concentrations of phenylhydrazine on zebrafish embryonic development are shown in Table 1:

[0057] Table 1. Effects of different concentrations of phenylhydrazine on zebrafish embryonic development.

[0058]

[0059]

[0060] Studies have found that small doses of phenylhydrazine can help zebrafish growth and development, but excessive doses can adversely affect their physiological activities. Phenylhydrazine can also cause behavioral disorders in zebrafish; large doses can lead to head-biting and cannibalism, as well as reduced swimming ability. All zebrafish in the dose group above 1E-3 μg / mL died, while the survival rate in the dose group below 1E-4 μg / mL was 100%. Figure 3 As shown in (A).

[0061] Further staining was performed on zebrafish with a 100% survival rate, by... Figure 3 As shown in (B), with increasing phenylhydrazine concentration, the staining area in the zebrafish heart region significantly decreased (p<0.001). Figure 3 As shown in Figure (C), at a concentration of 1E-6 μg / mL, the zebrafish heart showed no color change, a significant difference compared to the blank control group (p<0.001). When the concentration increased to 1E-5 μg / mL and 1E-4 μg / mL, the zebrafish turned yellow all over, and some even developed deformities or died. Therefore, based on the effects of phenylhydrazine on the growth status and staining area of ​​the heart in zebrafish, the final concentration of phenylhydrazine as the modeling agent for zebrafish anemia was determined to be 1E-6 μg / mL. The zebrafish group treated at this concentration served as the model control group.

[0062] (5) Evaluation method of zebrafish anemia model

[0063] Healthy wild-type AB strain zebrafish treated with PTU at 28 hpf post-fertilization were used as experimental animals. After demembranes were removed, they were transferred to 12-well plates with 10 zebrafish per well.

[0064] The blank control group was treated by administering zebrafish embryo culture medium.

[0065] The model control group was treated with 1E-6 μg / mL phenylhydrazine solution and incubated at 28.5℃ until 48 hpf. The modeling agent was removed, and the cells were washed three times with embryo culture medium, followed by incubation of embryo culture medium.

[0066] Treatment method for the drug intervention group: Administer 1E-6 μg / mL phenylhydrazine solution and incubate at 28.5℃ until 48 hpf. Remove the modeling agent, wash three times with embryo culture medium, and then administer low, medium, and high concentrations (25 μM, 50 μM, 100 μM) of atractylodes lactone II or glycyrrhizic acid, with three replicate wells for each group.

[0067] The blank control group, model control group, and drug intervention group were incubated in a constant temperature incubator at 28.5℃ until 5 days post-flop (dpf). Red blood cells of zebrafish in each group were stained using the o-anisidine staining method for 15 minutes. After staining, the cells were rinsed 2-3 times with DMSO solution. The juvenile fish were then removed using a disposable dropper, placed laterally on a biconcave slide, fixed with 4% neutral paraformaldehyde, and photographed under a microscope. The stained area and intensity of red blood cells were measured using Image-Pro Plus 6 software for quantitative analysis. The staining intensity (SI) of zebrafish heart red blood cells was analyzed and statistically analyzed to evaluate the anti-anemia (blood-tonifying) efficacy of the active ingredient.

[0068] Anti-anemia efficacy (%) = [SI (drug intervention group) - SI (model control group)] / [SI (blank control group) - SI (model control group)] × 100%.

[0069] SPSS Statistics 2.0 was used for data processing. All data were expressed as mean ± variance. Analysis of variance (ANOVA) was used, and P < 0.05 was considered statistically significant. Origin 2021 was used for image processing.

[0070] (6) Evaluation results of the anti-anemia efficacy of the active ingredients

[0071] After administering atractylodes lactone II at concentrations of 25 μM, 50 μM, and 100 μM for 24 hours to a zebrafish anemia model, zebrafish in the 50 μM and 100 μM groups lost their ability to move, and some even developed deformities or died. This indicates that excessively high concentrations of atractylodes lactone II and its strong odor can irritate zebrafish, leading to loss of activity. Subsequently, the high concentration of atractylodes lactone II was set at 25 μM, and the low and medium concentrations were diluted to 6.25 μM and 12.5 μM, respectively. After a certain period of intervention with atractylodes lactone II, compared with the model control group, the SI (staining intensity of zebrafish cardiac erythrocytes) of each concentration group was significantly increased (P < 0.001). Figure 4 The results indicate that atractylodes lactone II has a significant effect on improving anemia within the concentration range of 6.25 μM to 25 μM. The anti-anemia efficacy of atractylodes lactone II at concentrations of 6.25 μM, 12.5 μM, and 25 μM was 82.2%, 110.0%, and 52.7%, respectively. The results show that the anemia-improving effect of atractylodes lactone II did not show a significant dose-dependent relationship with the administered concentration. Within the concentration range of 6.25 μM to 25 μM, the anemia-improving effect of atractylodes lactone II first increased and then decreased. When the concentration exceeded 12.5 μM, its stimulating effect gradually increased, leading to a decrease or even disappearance of the function of restoring red blood cells in zebrafish hearts. Zebrafish embryos died due to prolonged insufficient blood supply.

[0072] After administering glycyrrhizic acid at concentrations of 50 μM, 100 μM, and 200 μM to a zebrafish anemia model for 24 hours, all zebrafish in the 200 μM group died, the mortality rate in the 100 μM group was 50%, and the survival rate in the other groups was 100%. Subsequently, the high concentration of glycyrrhizic acid was set at 50 μM, and the low and medium concentrations were diluted to 12.5 μM and 25 μM, respectively. After a certain period of glycyrrhizic acid intervention, compared with the model control group, the serum index (SI) of each concentration group was significantly increased (P < 0.001). Figure 4 The study found that glycyrrhizic acid significantly improved anemia within the concentration range of 12.5 μM to 50 μM; the anti-anemia efficacy of each concentration group was 44.8%, 46.8%, and 37.1%, respectively. The results showed that within the concentration range of 12.5 μM to 50 μM, the effect of glycyrrhizic acid in improving anemia did not show a significant dose-dependent relationship with the administered concentration, and there were no significant differences between the concentration groups. Notably, when the concentration of glycyrrhizic acid exceeded 50 μM, its irritant effect gradually increased, reducing the recovery function of hemoglobin cells in the zebrafish heart, leading to death due to long-term insufficient blood supply.

[0073] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of atractylodes lactone II in the preparation of TAS1R3 receptor activator.

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