Method for detecting safety and developmental toxicity of rhizoma atractylodis chinensis fibrous roots by using zebra fish

Abstract

The invention discloses a method for detecting safety and developmental toxicity of rhizoma atractylodis chinensis fibrous roots by using zebra fish, and belongs to the technical field of medicines. Comprising the following steps: preparing a rhizoma atractylodis chinensis fibrous root solution; the method comprises the following steps: preparing six types of extract diluents with different concentrations, setting a control group and a treatment group for safety detection, then preparing three types of extract diluents with different concentrations, and setting the control group and the treatment group for developmental toxicity detection. According to the method, the rhizoma atractylodis chinensis fibrous root aqueous extract is selected as a toxicity research object, zebra fish treatment is performed to obtain toxicity evaluation data, the safety of the rhizoma atractylodis chinensis fibrous roots is judged and evaluated based on analysis, the detection accuracy is high, the speed is high, and the detection method is scientific and reasonable.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more specifically to a method for detecting the safety and developmental toxicity of Atractylodes macrocephala fibrous roots by using zebrafish. Background Art

[0002] Atractylodes lancea is the dried rhizome of Atractylodes lancea (Thunb.) DC. or Atractylodes chinensis (DC.) Koidz. of the Asteraceae family. It is rich in volatile oils, aromatic glycosides, flavonoids and other chemical components, and has multiple functions such as regulating gastrointestinal motility, antibacterial, anti-inflammatory, and protecting cardiovascular. However, in traditional use, Atractylodes chinensis is only used as medicine with its rhizome, while other parts are often discarded, resulting in a waste of resources. The rational development and utilization of non-medicinal parts and processing by-products of medicinal plants can not only avoid waste of resources, but also promote the healthy development of the Chinese medicine planting industry. At present, the medicinal value of the fibrous roots of Atractylodes chinensis has been confirmed, but its toxic effects still need further study.

[0003] As a vertebrate with 85% homology to human genes, zebrafish has biological structure and physiological functions that are highly similar to those of mammals, making it very suitable for pharmacodynamic and toxicity screening of complex chemical systems such as traditional Chinese medicine. Zebrafish fertilized eggs or fry can easily absorb hydrophilic or lipophilic substances, which greatly facilitates the detection of the activity of traditional Chinese medicine.

[0004] Therefore, how to more accurately simulate the drug metabolism and pharmacodynamics of Atractylodes lancea fibrous roots in the human body, and thus develop a method for detecting the safety and developmental toxicity of Atractylodes lancea fibrous roots using zebrafish is an issue that technicians in this field urgently need to solve. Summary of the invention

[0005] In view of this, the present invention provides a method for detecting the safety and developmental toxicity of Atractylodes lancea fibrous roots using zebrafish.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for detecting the safety and developmental toxicity of Atractylodes macrocephala fibrous roots using zebrafish comprises the following steps:

[0008] (i) mixing the Atractylodes lancea fibrous root sample to be tested with triple distilled water at a mass ratio of 1:20, soaking at room temperature for 24 hours, filtering with filter paper to obtain a filtrate and a residue, mixing the residue with triple distilled water at a mass ratio of 1:10, soaking at room temperature for 10 hours, filtering with filter paper to obtain a filtrate, combining the two filtrates, evaporating and concentrating, and drying to obtain an Atractylodes lancea fibrous root extract, adding the Atractylodes lancea fibrous root extract to triple distilled water and mixing to obtain an Atractylodes lancea fibrous root solution;

[0009] (II) Safety testing:

[0010] (1) diluting the Atractylodes lancea root solution obtained in step (i) with zebrafish embryo culture medium to a concentration of 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1 mg / mL, 2.5 mg / mL, and 5 mg / mL of the Atractylodes lancea root extract, respectively, to obtain extract dilutions, and dividing zebrafish embryos into seven groups, one of which is a control group, and the other six groups are treatment groups, each group having 30 zebrafish embryos, the control group transferring the zebrafish embryos to a zebrafish embryo culture medium without extract dilution, and the treatment group transferring the zebrafish embryos to 6 different concentrations of extract dilutions, respectively, and exposing them to a constant temperature light-controlled incubator;

[0011] (2) After 24 hours of exposure, 21 embryos were randomly selected from each group and the number of spontaneous tail curling of zebrafish embryos in each group was observed;

[0012] (3) After exposure for 72 h, the hatching rate of 30 zebrafish embryos in each group was calculated;

[0013] (4) After 96 h of exposure, the mortality rate of 30 zebrafish embryos in each group was calculated and a mortality curve was drawn;

[0014] (III) Developmental toxicity testing:

[0015] 1) according to the mortality curve obtained in step (4), three concentration points are taken below 20% mortality, the Atractylodes lancea root solution obtained in step (i) is diluted with zebrafish embryo culture medium until the concentration of the Atractylodes lancea root extract is respectively the above three concentration points, to obtain extract dilutions, and zebrafish embryos are divided into four groups, one of which is a control group, and the other three groups are treatment groups, each group has 30 embryos, the control group transfers the zebrafish embryos to the zebrafish embryo culture medium without the extract dilution, and the treatment groups transfer the zebrafish embryos to the extract dilutions at the above three concentration points and expose them to a constant temperature light-controlled incubator;

[0016] After exposure to 72 hours, the hatching rate of zebrafish embryos in each group was calculated. After exposure to 96 hours, 6 fry were randomly selected from each group to observe the body length and 20-second heart rate of the fry.

[0017] After 96 h of exposure, 6 larvae were randomly selected from each group, and the eye area, heart area, and yolk sac area of ​​the zebrafish larvae in each group were calculated;

[0018] 2) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 30 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without extract diluent, and the zebrafish embryos in the treatment groups were transferred to the extract diluents at the above three concentration points and exposed to a constant temperature light-controlled incubator;

[0019] After exposure for 120 hours, 6 fry were randomly selected from each group to observe the reactive oxygen fluorescence of the zebrafish fry and calculate the reactive oxygen fluorescence intensity of the zebrafish fry in each group;

[0020] 3) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 70 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without extract diluent, and the zebrafish embryos in the treatment groups were transferred to the extract diluents at the above three concentration points and exposed to a constant temperature light-controlled incubator;

[0021] After exposure to 120 h, 50 juveniles were randomly selected from each group, and the protein of zebrafish juveniles at each concentration was extracted. The superoxide dismutase activity and malondialdehyde content were determined based on the protein.

[0022] 4) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 30 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without extract diluent, and the zebrafish embryos in the treatment groups were transferred to the extract diluents at the above three concentration points and exposed to a constant temperature light-controlled incubator;

[0023] After exposure for 120 hours, 6 larvae were randomly selected from each group, and acridine orange fluorescence staining was used to observe the apoptosis of zebrafish larvae in each group.

[0024] 5) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 70 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without extract diluent, and the zebrafish embryos in the treatment group were transferred to the extract diluents at the above three concentration points and exposed to a constant temperature light-controlled incubator;

[0025] After exposure for 120 hours, 50 fry were randomly selected from each group, and the protein of zebrafish fry in each group was extracted. The content of apoptosis proteins Bax and BCL-2 was determined based on the extracted protein.

[0026] Further, steps (2)-(4) specifically include the following steps:

[0027] (2) After 24 hours of exposure, the number of spontaneous tail curling of zebrafish embryos in each treatment group was observed within 60 seconds. 21 embryos were randomly selected from each group, and the number of spontaneous tail curling of zebrafish embryos in the control group was used as a reference;

[0028] (3) After exposure for 72 hours, observe 30 zebrafish embryos and count the hatching status of zebrafish embryos in the control group as a reference for the quality of this batch of zebrafish. The hatching rate of the control group must be ≥90%. Count the hatching status of zebrafish embryos in each treatment group and calculate the hatching rate according to hatching rate = number of successfully hatched embryos / total number of embryos. The hatching rate of zebrafish embryos in the control group is used as a reference, and dead embryos are counted as unhatched embryos.

[0029] (4) After 96 h of exposure, the death of 30 zebrafish embryos was observed, the embryonic mortality rate of zebrafish in each group was calculated, and a mortality curve was drawn, with the embryonic mortality rate of zebrafish in the control group as a reference.

[0030] Beneficial effects of adopting the above further technical scheme: 24h spontaneous tail curling is an important indicator of developmental neurotoxicity, which can be used to preliminarily determine the concentration range of the Atractylodes lancea root solution that has potential developmental neurotoxicity to zebrafish. The 72h hatching rate is the end point of zebrafish hatching, which can determine the effect of the Atractylodes lancea root solution on zebrafish hatching; the 96h mortality rate can be used to draw a curve based on the mortality rate, which can provide a concentration range for subsequent developmental toxicity.

[0031] Further, steps 1)-4) specifically include the following steps:

[0032] 1) according to the mortality curve obtained in step (4), three concentration points are taken below 20% mortality, the Atractylodes lancea root solution obtained in step (i) is diluted with zebrafish embryo culture medium until the concentration of the Atractylodes lancea root extract is respectively the above three concentration points, to obtain extract dilutions, and zebrafish embryos are divided into four groups, one of which is a control group, and the other three groups are treatment groups, each group has 30 embryos, the control group transfers the zebrafish embryos to the zebrafish embryo culture medium without the extract dilution, and the treatment groups transfer the zebrafish embryos to the extract dilutions at the above three concentration points and expose them to a constant temperature light-controlled incubator;

[0033] After exposure to 72 hours, the hatching rate of zebrafish embryos in each group was calculated, and the hatching rate of zebrafish embryos in the control group was used as a reference; after exposure to 96 hours, 6 fry were randomly selected from each group to observe the body length and 20s heart rate of the zebrafish fry in each group, and the body length and 20s heart rate of the zebrafish fry in the control group were used as a reference;

[0034] After 96 hours of exposure, 6 juveniles were randomly selected from each group, and the eye area, heart area, and yolk sac area of ​​the zebrafish juveniles in each group were calculated, with the morphological changes of the zebrafish juveniles in the control group as a reference;

[0035] 2) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 30 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without extract diluent, and the zebrafish embryos in the treatment groups were transferred to the extract diluents at the above three concentration points and exposed to a constant temperature light-controlled incubator;

[0036] After exposure for 120 hours, DCFH-DA and triple distilled water were mixed at a volume ratio of 1:3000 to obtain an active oxygen fluorescence dye solution, namely, mixture one. The zebrafish fry were taken out and placed in mixture one for incubation at room temperature and away from light for 30 minutes. The zebrafish fry were then taken out and washed with PBS buffer at pH = 7.2 for 3 times, each time for 5 minutes. Six fry were randomly selected from each group, and the active oxygen fluorescence of the zebrafish fry was observed and photographed. ImageJ was used to calculate the active oxygen fluorescence intensity of each group, and the active oxygen fluorescence intensity of the zebrafish fry in the control group was used as a reference.

[0037] 3) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 70 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without extract diluent, and the zebrafish embryos in the treatment groups were transferred to the extract diluents at the above three concentration points and exposed to a constant temperature light-controlled incubator;

[0038] After exposure to 120 h, 50 juveniles at each concentration at 120 h were taken and lysed with RIPA lysis buffer. The samples were then centrifuged at 5000 rpm and 4 °C for 15 min to obtain the supernatant. The total protein concentration of the supernatant was determined using a BCA (enhanced) protein concentration assay kit. In addition, superoxide dismutase kit and malondialdehyde kit were used for detection. The SOD activity and MDA content of zebrafish juveniles in the control group were used as a reference.

[0039] 4) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 30 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without extract diluent, and the zebrafish embryos in the treatment groups were transferred to the extract diluents at the above three concentration points and exposed to a constant temperature light-controlled incubator;

[0040] After exposure for 120 hours, acridine orange with a mass concentration of 0.2% was dissolved in a PBS buffer solution with a pH of 7.2 to obtain a second mixture. The zebrafish embryos were taken out and placed in the second mixture for incubation at room temperature for 10 minutes. The zebrafish fry were taken out and washed with PBS buffer for 3 times, each time for 5 minutes. Six fry were randomly selected from each group, and then the apoptosis of the zebrafish fry was observed. The apoptosis of the zebrafish fry in the control group was used as a reference.

[0041] 5) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 70 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without extract diluent, and the zebrafish embryos in the treatment group were transferred to the extract diluents at the above three concentration points and exposed to a constant temperature light-controlled incubator;

[0042] After exposure to 120 h, 50 larvae of each concentration at 120 h were taken and lysed with RIPA lysis buffer. The samples were then centrifuged at 5000 rpm and 4°C for 15 min to obtain the supernatant. The total protein concentration of the supernatant was determined using a BCA (enhanced) protein concentration determination kit, and the fish Bcl-2 associated X protein BAX ELISA kit and BCL-2 ELISA kit were used for protein determination. The zebrafish larvae in the control group were used as a reference.

[0043] The beneficial effects of adopting the above further technical scheme are as follows: according to the mortality curve, three concentrations with a mortality rate below 20% are selected. These three concentrations will not cause significant changes in the 24h spontaneous movement and 72h hatching rate of zebrafish in the experiment, providing a reference for the development of Atractylodes macrocephala root fibrous root drugs. The above three concentrations are not statistically significant in morphology, so subsequent developmental toxicity experiments are carried out with these three concentrations; whether these three concentrations have significant changes in the 72h hatching rate, 96h body length and 20s heart rate of zebrafish embryos, and the 96h morphological eye area, heart area and yolk sac area compared with the control group, and which of oxidative stress and apoptosis is the main cause of the developmental toxicity mechanism of zebrafish.

[0044] Furthermore, in step (i), the concentration of the Atractylodes lancea fibrous root solution is 500 mg / mL.

[0045] Further, in step (1), the solvent of the zebrafish embryo culture medium is deionized water, the concentration of anhydrous sodium chloride in the zebrafish embryo culture medium is 294.0 mg / L, the concentration of magnesium sulfate heptahydrate is 123.3 mg / L, the concentration of sodium bicarbonate is 63.0 mg / L, and the concentration of potassium chloride is 5.5 mg / L.

[0046] Furthermore, in step (1), the method for obtaining zebrafish embryos comprises the following steps:

[0047] On the afternoon before the collection of zebrafish embryos, healthy adult zebrafish were selected and placed in a breeding tank in a 1:1 ratio of male to female. The male and female zebrafish were separated by a partition. The partition was removed at 8:30 am the next day, and embryos were collected after free mating for 2 hours.

[0048] The beneficial effects of the present invention are as follows: the method of the present invention is simple to operate and has strong practicability. It can preliminarily determine the toxicity and developmental toxicity mechanism of the fibrous roots of Atractylodes lancea. It is fast, and a single test only needs 10 days to complete, with high accuracy. The present invention first obtains the safety range concentration of the fibrous root solution of Atractylodes lancea for zebrafish from the 24h spontaneous movement, 72h hatching rate and 96h mortality curve, and then takes a point at a low concentration to study the developmental toxicity and mechanism. The mechanism study uses protein extraction to measure the activity of SOD and the content of MDA, and apoptosis is performed to determine the content of protein bax and bcl-2. The present invention obtains toxicity evaluation data by selecting the fibrous root water extract of Atractylodes lancea as the research object of toxicity, and zebrafish treatment, and judges and evaluates the safety of the fibrous root of Atractylodes lancea based on analysis. The detection accuracy is high, the speed is fast, and the detection method is scientific and reasonable. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Effects of different concentrations of Atractylodes lancea fibrous root water extract on 24h spontaneous movement of zebrafish (ns: no significant difference; *: p<0.05);

[0050] Figure 2 Effects of different concentrations of Atractylodes lancea fibrous root water extract on the 72h hatching rate of zebrafish (ns: no significant difference; *: p<0.05);

[0051] Figure 3 96h concentration-mortality curve of zebrafish with different concentrations of water extract of Atractylodes lancea fibrous roots;

[0052] Figure 4 A: 72h hatching rate of zebrafish exposed to 0.1, 0.2 and 0.3mg / mL; B: 96h body length of zebrafish; C: 96h heart rate of zebrafish (ns: no significant difference; *: p<0.05)

[0053] Figure 5 A: 96h zebrafish morphology B: 96h heart area C: 96h eye area D: 96h yolk sac area (ns: no significant difference; *: p<0.05)

[0054] Figure 6 A: Schematic diagram of ROS fluorescence in zebrafish exposed for 120 hours. B: ROS fluorescence intensity diagram (ns: no significant difference; *: p<0.05)

[0055] Figure 7 Detection of MDA content in zebrafish after 120h exposure (ns: no significant difference; *: p<0.05)

[0056] Figure 8A: 120h cell apoptosis B: Bax protein content C: BCL-2 protein content D: Bax / BCL-2 (ns: no significant difference; *: p<0.05). DETAILED DESCRIPTION

[0057] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] Example 1

[0059] (I) A method for preparing an extract dilution, comprising the following steps:

[0060] 50 g of the Atractylodes lancea fibrous root sample (about 2 cm in length) produced in Heilongjiang Province to be tested was mixed with 1000 mL of triple distilled water, and after soaking at room temperature for 24 h, filtered with filter paper to obtain a filtrate and a residue, the residue and triple distilled water were mixed at a mass ratio of 1:10, and after soaking at room temperature for 10 h, filtered with filter paper to obtain a filtrate, the two filtrates were combined, evaporated and concentrated to 50 mL, and dried for 3 days at a drying temperature of 60° C. to obtain an Atractylodes lancea fibrous root extract, and 5 g of the Atractylodes lancea fibrous root extract was added to 10 mL of triple distilled water and mixed to obtain an Atractylodes lancea fibrous root solution having a concentration of 500 mg / mL of the Atractylodes lancea fibrous root extract;

[0061] (ii) A method for obtaining zebrafish embryos, comprising the following steps:

[0062] On the afternoon before the collection of zebrafish embryos, healthy adult zebrafish were selected and placed in a breeding tank in a 1:1 ratio of male to female. The male and female zebrafish were separated by a partition. The partition was removed at 8:30 am the next day, and embryos were collected after free mating for 2 hours.

[0063] (III) Safety testing:

[0064] (1) diluting the Atractylodes lancea root solution obtained in step (i) with a zebrafish embryo culture medium until the concentrations of the Atractylodes lancea root extract are 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1 mg / mL, 2.5 mg / mL, and 5 mg / mL, respectively, to obtain an extract dilution; the solvent of the zebrafish embryo culture medium is deionized water, the concentration of anhydrous sodium chloride in the zebrafish embryo culture medium is 294.0 mg / L, the concentration of magnesium sulfate heptahydrate is 123.3 mg / L, the concentration of sodium bicarbonate is 63.0 mg / L, and the concentration of potassium chloride is 5.5 mg / L.

[0065] Select wild zebrafish AB strain embryos that have just been successfully fertilized, and culture them in zebrafish embryo culture medium for 7 hours. During this period, dead embryos are removed and the remaining embryos are divided into seven groups, one of which is the control group and the other six are treatment groups, with 30 embryos in each group.

[0066] In the control group, zebrafish embryos were transferred to zebrafish embryo culture medium without extract diluent, and in the treatment group, zebrafish embryos were transferred to 6 different concentrations of extract diluent and exposed in a constant temperature and light-controlled incubator;

[0067] (2) After 24 hours of exposure, 21 embryos were randomly selected from each group to observe the number of spontaneous tail curling in each treatment group within 60 seconds. The number of spontaneous tail curling in the control group was used as a reference.

[0068] Test results such as Figure 1 As shown, the spontaneous movement of the 0.2 mg / mL group increased compared with the control group, but there was no significant difference. There was no obvious change in the 0.4 and 0.8 mg / mL groups. The number of spontaneous movements in the 1 and 2.5 mg / mL groups decreased and there was a significant difference. The 5 mg / mL group caused severe developmental retardation of the embryos and no spontaneous movement occurred. Spontaneous tail curling is the first spontaneous movement behavior of zebrafish after fertilization. It is regulated by spinal neurons. Spontaneous tail curling is an important indicator of developmental neurotoxicity, indicating that the concentration of Atractylodes lancea fibrous roots is higher than the 1 mg / mL group and has the potential to cause developmental neurotoxicity in zebrafish.

[0069] (3) After exposure for 72 hours, observe 30 zebrafish embryos and count the hatching status of zebrafish embryos in the control group as a reference for the quality of this batch of zebrafish. The hatching rate of the control group must be ≥90%. Count the hatching status of zebrafish embryos in each treatment group and calculate the hatching rate according to hatching rate = number of successfully hatched embryos / total number of embryos. The hatching rate of zebrafish embryos in the control group is used as a reference, and dead embryos are counted as unhatched embryos.

[0070] Test results such as Figure 2 As shown, after 72 hours of incubation, there was no significant difference between the 0.2 and 0.4 mg / mL groups and the control group when zebrafish embryos were exposed to different concentrations of Atractylodes macrocephala fibrous root solutions; the hatching rate of the 0.8 and 1 mg / mL groups decreased significantly, and the hatching rate of zebrafish in the 2.5 and 5 mg / mL groups was 0. The 5 mg / mL group was removed in subsequent experiments. This shows that the concentration of Atractylodes macrocephala fibrous root higher than the 0.8 mg / mL group will affect the hatching of zebrafish.

[0071] (4) After 96 h of exposure, the death of 30 zebrafish embryos was observed, the embryonic mortality rate of zebrafish in each group was calculated, and a mortality curve was drawn, with the embryonic mortality rate of zebrafish in the control group as a reference.

[0072] Test results such as Figure 3As shown, the mortality statistics of zebrafish embryos after exposure to different concentrations of Atractylodes lancea root (0-2.5 mg / mL) for 96 hours, and the relationship curve between embryonic lethality and exposure concentration was drawn. The "concentration-mortality" effect curve shows that exposure to Atractylodes lancea root for 96 hours caused the mortality of zebrafish embryos to first plateau at 0-0.4 mg / mL, and then increased significantly in a concentration-dependent manner until it reached 100%. According to the mortality curve, 0.1, 0.2 and 0.3 mg / mL concentration groups were taken at a mortality rate of 20% for subsequent developmental toxicity testing.

[0073] (III) Developmental toxicity testing:

[0074] 1) According to the mortality curve obtained in step (4), three concentration points are taken below 20% mortality, wherein two points are taken below 10% and one point is taken between 10% and 20%, which are 0.1, 0.2 and 0.3 mg / mL respectively; the Atractylodes lancea fibrous root solution obtained in step (1) is diluted with zebrafish embryo culture medium until the concentration of the Atractylodes lancea fibrous root extract is 0.1, 0.2 and 0.3 mg / mL at three concentration points respectively, to obtain extract dilutions.

[0075] Select wild zebrafish AB strain embryos that have just been successfully fertilized, and culture them in zebrafish embryo culture medium for 7 hours. During this period, dead embryos are removed, and the remaining embryos are divided into four groups, one of which is a control group, and the other three groups are treatment groups, each with 30 embryos. The zebrafish embryos in the control group are transferred to a zebrafish embryo culture medium without an extract diluent, and the zebrafish embryos in the treatment group are transferred to the extract diluents at the above three concentration points and exposed to a constant temperature light-controlled incubator;

[0076] After exposure to 72 hours, the hatching rate of zebrafish embryos in each group was calculated by observation under a stereomicroscope, and the hatching rate of zebrafish embryos in the control group was used as a reference. After exposure to 96 hours, 6 fry were randomly selected from each group to measure the body length and 20-second heart rate of zebrafish embryos in each group, and the body length and 20-second heart rate of zebrafish fry in the control group were used as a reference.

[0077] Test results such as Figure 4 As shown in A, B, and C, the hatching rate at 72 h, body length at 96 h, and heart rate at 0.1, 0.2, and 0.3 mg / mL were not significantly different from those in the control group.

[0078] After 96 hours of exposure, 6 fry were randomly selected from each group. The development of various organs of the fry were observed under a stereo microscope. The eye area, heart area and yolk sac area of ​​the fry were calculated using ImageJ. The morphological changes of the fry in the control group were used as a reference.

[0079] Test results such as Figure 5As shown in A, the green dotted line is the eye part, the red dotted line is the heart part, and the yellow part is the yolk sac part. Figure 5 B is the calculated area of ​​the heart. The heart area of ​​the 0.3 mg / mL group increased significantly. Figure 5 The eye area and yolk sac area in C and D were not statistically significant compared with the control group.

[0080] 2) Freshly fertilized wild zebrafish AB strain embryos were cultured in zebrafish embryo culture medium for 7 hours, during which dead embryos were removed, and the remaining zebrafish embryos were divided into four groups, one of which was a control group, and the other three were treatment groups, with 30 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without an extract diluent, and the zebrafish embryos in the treatment groups were transferred to the extract diluents at the above three concentration points and exposed to a constant temperature light-controlled incubator;

[0081] After exposure to 120 hours, DCFH-DA (active oxygen fluorescence probe) was mixed with triple distilled water at a volume ratio of 1:3000 to obtain an active oxygen fluorescence dye solution, namely, mixture one. The zebrafish fry were taken out and placed in mixture one for incubation at room temperature and away from light for 30 minutes. The zebrafish fry were then taken out and washed with PBS buffer at pH = 7.2 for 3 times, each time for 5 minutes. Six fry were randomly selected from each group, and the active oxygen fluorescence of the zebrafish fry was observed and photographed. ImageJ was used to calculate the active oxygen fluorescence intensity of each group, and the active oxygen fluorescence intensity of the zebrafish fry in the control group was used as a reference.

[0082] Test results such as Figure 6 As shown, we first determined whether the developmental toxicity of Atractylodes macrocephala root to zebrafish was caused by oxidative stress, which is defined as an imbalance between oxidants and antioxidants. Oxidative stress plays a key role in the occurrence and development of various diseases, such as heart failure, atherosclerosis, Alzheimer's disease, and cancer. To date, oxidative stress is one of the main influencing indicators of toxic effects of aquatic organisms. There was no significant difference in reactive oxygen species between the 0.1, 0.2, and 0.3 mg / mL groups and the control group. It was preliminarily determined that the developmental toxicity of Atractylodes macrocephala root to zebrafish was not caused by oxidative stress.

[0083] 3) Freshly fertilized wild zebrafish AB strain embryos were cultured in zebrafish embryo culture medium for 7 hours, during which dead embryos were removed, and the remaining zebrafish embryos were divided into four groups, one of which was a control group, and the other three were treatment groups, with 70 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without extract diluent, and the zebrafish embryos in the treatment groups were transferred to the extract diluents at the above three concentration points and exposed to a constant temperature light-controlled incubator;

[0084] After exposure to 120 hours, 50 juveniles of each concentration at 120 hours were taken and lysed with RIPA lysis buffer (RIPALysis Buffer) produced by Biyuntian. The samples were then centrifuged at 5000 rpm and 4°C for 15 minutes to obtain the supernatant. The total protein concentration of the supernatant was determined using the BCA protein concentration determination kit (Enhanced) (Enhanced BCA Protein Assay Kit) produced by Biyuntian. In addition, the SOD (superoxide dismutase kit) and MDA (malondialdehyde) kits of Nanjing Jiancheng Bioengineering Institute were used for detection, and the SOD activity and MDA content of the zebrafish juveniles in the control group were used as references;

[0085] Test results such as Figure 7 As shown, oxidative damage can cause chemical-induced developmental defects in zebrafish. SOD and MDA are two important biomarkers of oxidative stress and are considered to be key endogenous antioxidant enzymes that protect cells from oxidative damage. SOD is the first enzyme to scavenge oxygen free radicals and defend against oxidative stress. Malondialdehyde is a byproduct of free radical lipid peroxidation. MDA levels can be used to assess the severity of tissue oxidative stress damage. The 0.1, 0.2 and 0.3 mg / mL groups did not show activity for SOD, so there is no data graph. In the MDA (malondialdehyde) content determination, the 0.1 mg / mL group showed a significant difference compared with the control group. According to a large number of previous experiments in our laboratory, it is likely that low concentrations of Atractylodes lancea root extracts promote hatching of zebrafish and accelerate the body length of zebrafish ( Figure 4 B) and yolk sac ( Figure 5 D), therefore, the developmental toxicity of Atractylodes lancea fibrous roots at 0.1, 0.2, and 0.3 mg / mL on zebrafish was not caused by oxidative stress.

[0086] 4) Take wild zebrafish AB strain embryos that have just been successfully fertilized, and culture them in zebrafish embryo culture medium for 7 hours. During this period, remove the dead embryos, and divide the remaining zebrafish embryos into four groups, one of which is a control group, and the other three groups are treatment groups, with 30 embryos in each group. The zebrafish embryos in the control group are transferred to a zebrafish embryo culture medium without an extract diluent, and the zebrafish embryos in the treatment group are transferred to the extract diluents at the above three concentration points and exposed to a constant temperature light-controlled incubator;

[0087] After exposure to 120 hours, 0.2% AO (acridine orange) was dissolved in PBS (phosphate buffered saline) buffer at pH = 7.2 using the Acridine Orange Staining Kit produced by Beyotime to obtain mixture 2. The zebrafish embryos were taken out and placed in mixture 2 for incubation at room temperature for 10 minutes. The zebrafish larvae were taken out and washed with PBS buffer for 3 times, each time for 5 minutes. Six larvae were randomly selected from each group, and then the apoptosis of zebrafish larvae was observed under a stereo fluorescence microscope. The apoptosis of zebrafish larvae in the control group was used as a reference.

[0088] 5) Take wild zebrafish AB strain embryos that have just been successfully fertilized, and culture them in zebrafish embryo culture medium for 7 hours. During this period, remove the dead embryos, and divide the remaining zebrafish embryos into four groups, one of which is a control group, and the other three groups are treatment groups, with 70 embryos in each group. The zebrafish embryos in the control group are transferred to a zebrafish embryo culture medium without an extract diluent, and the zebrafish embryos in the treatment group are transferred to the extract diluents at the above three concentration points and exposed to a constant temperature light-controlled incubator;

[0089] After exposure to 120 h, 50 fry of each concentration at 120 h were taken and lysed with RIPA lysis buffer (RIPALysis Buffer) produced by Bio-Time. The samples were then centrifuged at 5000 rpm and 4°C for 15 min to obtain the supernatant. The total protein concentration of the supernatant was determined using the BCA protein concentration determination kit (Enhanced) (Enhanced BCA Protein Assay Kit) produced by Bio-Time. The fish Bcl-2 associated X protein (BAX) ELISA kit and (BCL-2) ELISA kit of Nanjing Boyan Biotechnology Co., Ltd. were used for protein determination. The zebrafish fry in the control group were used as a reference.

[0090] Test results such as Figure 8 As shown in Figure 2, BAX is a pro-apoptotic protein. When cells are damaged or stressed, BAX is activated and translocated to the mitochondrial membrane, promoting changes in the permeability of the mitochondrial membrane, leading to the release of pro-apoptotic factors such as cytochrome c, and ultimately inducing cell apoptosis. BCL-2 is an anti-apoptotic protein that can inhibit cell apoptosis. It competes with BAX and other pro-apoptotic proteins for binding, thereby interfering with the transmission of apoptotic signals and protecting cells from death signals. The expression levels and relative proportions of BAX and BCL-2 are closely related to the occurrence of a variety of diseases and dysregulation of cell apoptosis. Therefore, these two proteins have become an important target for research. Figure 8 In A, the apoptotic cells stained with AO appear as bright spots, such as the white area. Figure 8Each concentration group in B was significantly different from the control group, indicating that 0.1, 0.2 and 0.3 mg / mL of the main fibrous roots of A. serrata promoted cell apoptosis and caused developmental toxicity in zebrafish. Figure 8 The expression of BCL-2 protein in C also increased significantly, indicating that the body's anti-apoptotic system was activated to resist cell apoptosis. Figure 8 The ratio of BAX / BCL-2 in D increased significantly, indicating that the production of apoptotic proteins in the body was higher than that of anti-apoptotic proteins, indicating that the developmental toxicity of Atractylodes lancea fibrous roots on zebrafish was caused by apoptosis.

[0091] In summary, the safety range of Atractylodes lancea fibrous roots to zebrafish is less than 0.2 mg / mL, and the developmental toxicity is caused by cell apoptosis.

[0092] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting the safety and developmental toxicity of Atractylodes macrocephala fibrous roots using zebrafish, characterized in that: The steps include: (i) mixing the Atractylodes lancea fibrous root sample to be tested with triple distilled water at a mass ratio of 1:20, soaking at room temperature for 24 hours, filtering with filter paper to obtain a filtrate and a residue, mixing the residue with triple distilled water at a mass ratio of 1:10, soaking at room temperature for 10 hours, filtering with filter paper to obtain a filtrate, combining the two filtrates, evaporating and concentrating, and drying to obtain an Atractylodes lancea fibrous root extract, adding the Atractylodes lancea fibrous root extract to triple distilled water and mixing to obtain an Atractylodes lancea fibrous root solution; (II) Safety testing: (1) diluting the Atractylodes lancea root solution obtained in step (i) with zebrafish embryo culture medium to a concentration of 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1 mg / mL, 2.5 mg / mL, and 5 mg / mL of the Atractylodes lancea root extract, respectively, to obtain extract dilutions, and dividing zebrafish embryos into seven groups, one of which is a control group, and the other six groups are treatment groups, each group having 30 zebrafish embryos, the control group transferring the zebrafish embryos to a zebrafish embryo culture medium without extract dilution, and the treatment group transferring the zebrafish embryos to 6 different concentrations of extract dilutions, respectively, and exposing them to a constant temperature light-controlled incubator; (2) After 24 h of exposure, 21 embryos were randomly selected from each group, and the number of spontaneous tail curling of zebrafish embryos in each group was calculated; (3) After exposure for 72 h, the hatching rate of 30 zebrafish embryos in each group was calculated; (4) After 96 h of exposure, the mortality rate of 30 zebrafish embryos in each group was calculated and a mortality curve was drawn; (III) Developmental toxicity testing: 1) according to the mortality curve obtained in step (4), three concentration points are taken below 20% mortality, the Atractylodes lancea root solution obtained in step (i) is diluted with zebrafish embryo culture medium until the concentration of the Atractylodes lancea root extract is respectively the three concentration points, to obtain extract dilutions, and zebrafish embryos are divided into four groups, one of which is a control group, and the other three groups are treatment groups, each group having 30 embryos, the control group transfers the zebrafish embryos to the zebrafish embryo culture medium without the extract dilution, and the treatment groups transfer the zebrafish embryos to the extract dilutions at the three concentration points and expose them to a constant temperature light-controlled incubator; After exposure to 72 hours, the hatching rate of zebrafish embryos in each group was calculated. After exposure to 96 hours, 6 fry were randomly selected from each group to observe the body length and 20-second heart rate of the fry. After 96 h of exposure, 6 larvae were randomly selected from each group, and the eye area, heart area, and yolk sac area of ​​the zebrafish larvae in each group were calculated; 2) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 30 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without an extract diluent, and the zebrafish embryos in the treatment groups were transferred to the extract diluents at the three concentration points and exposed in a constant temperature light-controlled incubator; After exposure for 120 hours, 6 fry were randomly selected from each group to observe the reactive oxygen fluorescence of the zebrafish fry and calculate the reactive oxygen fluorescence intensity of the zebrafish fry in each group; 3) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 70 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without an extract diluent, and the zebrafish embryos in the treatment groups were transferred to the extract diluents at the three concentration points and exposed to a constant temperature light-controlled incubator; After exposure to 120 h, 50 juveniles were randomly selected from each group, and the protein of zebrafish juveniles at each concentration was extracted. The superoxide dismutase activity and malondialdehyde content were determined based on the protein. 4) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 30 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without an extract diluent, and the zebrafish embryos in the treatment group were transferred to the extract diluents at the three concentration points and exposed in a constant temperature light-controlled incubator; After exposure for 120 hours, 6 larvae were randomly selected from each group, and acridine orange fluorescence staining was used to observe the apoptosis of zebrafish larvae in each group. 5) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 70 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without an extract diluent, and the zebrafish embryos in the treatment groups were transferred to the extract diluents at the three concentration points and exposed to a constant temperature light-controlled incubator; After exposure for 120 hours, 50 fry were randomly selected from each group, and the protein of zebrafish fry in each group was extracted. The content of apoptosis proteins Bax and BCL-2 was determined based on the extracted protein.

2. According to claim 1, a method for detecting the safety and developmental toxicity of Atractylodes lancea fibrous roots using zebrafish, characterized in that: Steps (2) to (4) specifically include the following steps: (2) After 24 hours of exposure, the number of spontaneous tail curling of zebrafish embryos in each treatment group was observed within 60 seconds. 21 embryos were randomly selected from each group, and the number of spontaneous tail curling of zebrafish embryos in the control group was used as a reference; (3) After exposure for 72 hours, observe 30 zebrafish embryos and count the hatching status of zebrafish embryos in the control group as a reference for the quality of this batch of zebrafish. The hatching rate of the control group must be ≥90%. Count the hatching status of zebrafish embryos in each treatment group and calculate the hatching rate according to hatching rate = number of successfully hatched embryos / total number of embryos. The hatching rate of zebrafish embryos in the control group is used as a reference, and dead embryos are counted as unhatched embryos. (4) After 96 h of exposure, the death of 30 zebrafish embryos was observed, the embryonic mortality rate of zebrafish in each group was calculated, and a mortality curve was drawn, with the embryonic mortality rate of zebrafish in the control group as a reference.

3. The method for detecting the safety and developmental toxicity of Atractylodes lancea fibrous roots using zebrafish according to claim 1, characterized in that: Steps 1)-4) specifically include the following steps: 1) according to the mortality curve obtained in step (4), three concentration points are taken below 20% mortality, the Atractylodes lancea root solution obtained in step (i) is diluted with zebrafish embryo culture medium until the concentration of the Atractylodes lancea root extract is respectively the three concentration points, to obtain extract dilutions, and zebrafish embryos are divided into four groups, one of which is a control group, and the other three groups are treatment groups, each group having 30 embryos, the control group transfers the zebrafish embryos to the zebrafish embryo culture medium without the extract dilution, and the treatment groups transfer the zebrafish embryos to the extract dilutions at the three concentration points and expose them to a constant temperature light-controlled incubator; After exposure to 72 hours, the hatching rate of zebrafish embryos in each group was calculated, and the hatching rate of zebrafish embryos in the control group was used as a reference; after exposure to 96 hours, 6 fry were randomly selected from each group to observe the body length and 20s heart rate of the zebrafish fry in each group, and the body length and 20s heart rate of the zebrafish fry in the control group were used as a reference; After 96 hours of exposure, 6 juveniles were randomly selected from each group, and the eye area, heart area, and yolk sac area of ​​the zebrafish juveniles in each group were calculated, with the morphological changes of the zebrafish juveniles in the control group as a reference; 2) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 30 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without an extract diluent, and the zebrafish embryos in the treatment groups were transferred to the extract diluents at the three concentration points and exposed in a constant temperature light-controlled incubator; After exposure for 120 hours, DCFH-DA and triple distilled water were mixed at a volume ratio of 1:3000 to obtain an active oxygen fluorescence dye solution, namely, mixture one. The zebrafish fry were taken out and placed in mixture one for incubation at room temperature and away from light for 30 minutes. The zebrafish fry were then taken out and washed three times with PBS buffer at pH = 7.2, each time for 5 minutes. Six fry were randomly selected from each group, and the active oxygen fluorescence of the zebrafish fry was observed and photographed. ImageJ was used to calculate the active oxygen fluorescence intensity of each group, and the active oxygen fluorescence intensity of the zebrafish fry in the control group was used as a reference. 3) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 70 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without an extract diluent, and the zebrafish embryos in the treatment groups were transferred to the extract diluents at the three concentration points and exposed to a constant temperature light-controlled incubator; After exposure to 120 h, 50 juveniles at each concentration at 120 h were taken and lysed with RIPA lysis buffer. The samples were then centrifuged at 5000 rpm and 4 °C for 15 min to obtain the supernatant. The total protein concentration of the supernatant was determined using a BCA enhanced protein concentration assay kit. In addition, superoxide dismutase kit and malondialdehyde kit were used for detection. The SOD activity and MDA content of zebrafish juveniles in the control group were used as a reference. 4) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 30 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without an extract diluent, and the zebrafish embryos in the treatment group were transferred to the extract diluents at the three concentration points and exposed in a constant temperature light-controlled incubator; After exposure for 120 hours, acridine orange with a mass concentration of 0.2% was dissolved in a PBS buffer solution with a pH of 7.2 to obtain a second mixture. The zebrafish embryos were taken out and placed in the second mixture for incubation at room temperature for 10 minutes. The zebrafish fry were taken out and washed with PBS buffer for 3 times, each time for 5 minutes. Six fry were randomly selected from each group, and then the apoptosis of the zebrafish fry was observed. The apoptosis of the zebrafish fry in the control group was used as a reference. 5) Zebrafish embryos were divided into four groups, one of which was a control group and the other three were treatment groups, with 70 embryos in each group. The zebrafish embryos in the control group were transferred to a zebrafish embryo culture medium without an extract diluent, and the zebrafish embryos in the treatment groups were transferred to the extract diluents at the three concentration points and exposed to a constant temperature light-controlled incubator; After exposure to 120 h, 50 larvae of each concentration at 120 h were taken and lysed with RIPA lysis buffer. The samples were then centrifuged at 5000 rpm and 4°C for 15 min to obtain the supernatant. The total protein concentration of the supernatant was determined using a BCA (enhanced) protein concentration determination kit, and the fish Bcl-2 associated X protein BAX ELISA kit and BCL-2 ELISA kit were used for protein determination. The zebrafish larvae in the control group were used as a reference.

4. The method for detecting the safety and developmental toxicity of Atractylodes macrocephala fibros using zebrafish according to claim 1, characterized in that: In step (a), the concentration of the Atractylodes lancea fibrous root solution is 500 mg / mL.

5. The method for detecting the safety and developmental toxicity of Atractylodes macrocephala fibros using zebrafish according to claim 1, characterized in that: In step (1), the solvent of the zebrafish embryo culture medium is deionized water, the concentration of anhydrous sodium chloride in the zebrafish embryo culture medium is 294.0 mg / L, the concentration of magnesium sulfate heptahydrate is 123.3 mg / L, the concentration of sodium bicarbonate is 63.0 mg / L, and the concentration of potassium chloride is 5.5 mg / L.

6. The method for detecting the safety and developmental toxicity of Atractylodes macrocephala fibros using zebrafish according to claim 1, characterized in that: In step (1), the method for obtaining zebrafish embryos comprises the following steps: On the afternoon before the collection of zebrafish embryos, healthy adult zebrafish were selected and placed in a breeding tank in a 1:1 ratio of male to female. The male and female zebrafish were separated by a partition. The partition was removed at 8:30 am the next day, and embryos were collected after free mating for 2 hours.

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