CN112293599A

CN112293599A - Food additive for preventing pet threatened abortion and application of atractylenolide I

Info

Publication number: CN112293599A
Application number: CN202011189170.0A
Authority: CN
Inventors: 刘成功; 刘俊平; 郭羽丽; 王鹏; 王宇
Original assignee: 66399 Unit Of Chinese Pla
Current assignee: 66399 Unit Of Chinese Pla
Priority date: 2020-10-30
Filing date: 2020-10-30
Publication date: 2021-02-02

Abstract

The invention relates to the technical field of pet feeding management, in particular to a food additive for preventing pet threatened abortion and application of atractylenolide I. The food additive for preventing the pet threatened abortion contains atractylenolide I. The research of the invention discovers that the atractylenolide I can effectively prevent the occurrence of threatened abortion and has high safety, and the food additive can be freely matched with food, thereby effectively preventing the threatened abortion of pets, and being beneficial to the safe passing of the pregnancy of the pets and the delivery of healthy cubs.

Description

Food additive for preventing pet threatened abortion and application of atractylenolide I

Technical Field

The invention relates to the technical field of pet feeding management, in particular to a food additive for preventing pet threatened abortion and application of atractylenolide I.

Background

With the increase of the feeding quantity of pets, the gestational diseases and gestational health care of the pets are more and more valued. The development of the fetus protection product has important clinical application value in the aspects of reducing the abortion rate and guaranteeing the health of a parent, and plays a key role in breeding pets. In the research field, hormone therapy and antibiotic therapy are mainly used for treatment at home and abroad, no effective prevention method exists, and side effects caused by the hormone and the antibiotic are not small.

Factors causing abortion of pets are mainly classified into infectious factors and non-infectious factors, wherein abortion caused by infectious factors has been effectively controlled with improvement of breeding environment and increasing popularization of immune anthelmintic, and threatened abortion or infertility caused by luteal insufficiency is more and more common. Luteal insufficiency is characterized by delayed endometrial development in secretory phase and asynchronous endometrial development and pregnant egg development, and is clinically mainly manifested by infertility or repeated early abortion, and the reduction of endometrial receptivity is the main reason. The atractylenolide I is one of the effective components of the traditional Chinese medicine atractylenolide, has high safety, and can promote progesterone secretion so as to promote the development of endometrium. The additive can be used as a food additive, and can simply and effectively prevent the threatened abortion of pets.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims at providing a food additive for preventing the threatened abortion of pets.

The second invention aims to provide application of atractylenolide I.

In order to achieve the purpose of the invention, the technical scheme is as follows:

the invention relates to a food additive for preventing pet threatened abortion, which contains atractylenolide I.

Optionally, the food additive further comprises baicalin.

Optionally, the food additive further comprises a calcium source selected from at least one of calcium carbonate, calcium chloride, calcium phosphate calcium lactate, calcium gluconate, and calcium citrate.

Optionally, the food additive further comprises a diluent, wherein the diluent is selected from at least one of starch, powdered sugar, lactose and mannitol.

Optionally, the content of atractylenolide I in the food additive is 0.5-1.5% by mass, preferably 0.8-1.2%.

Optionally, the food additive contains baicalin 0.1-1 wt%, preferably 0.2-0.8 wt%.

Optionally, the calcium source in the food additive is 1-5% by mass.

Optionally, the baicalin is prepared by the following method: putting scutellaria baicalensis into 8-12 times of water, boiling for 30 minutes, combining the two decoction liquids, adjusting the pH value to be l-2 by using hydrochloric acid, preserving the heat at 80 ℃ for 30 minutes, standing, filtering, collecting precipitates, adding 95% ethanol for washing for 2-3 times, then carrying out vacuum filtration, and carrying out reduced pressure evaporation concentration on the filtrate at 50-60 ℃ until the filtrate is subjected to dry filtration to obtain the baicalin extract.

The invention also relates to application of the atractylenolide I in preparing food or medicine for preventing the threatened abortion of pets.

Alternatively, the pets include dogs and cats.

The invention has at least the following beneficial effects:

the research of the invention finds that the atractylenolide I can effectively prevent the occurrence of threatened abortion and has high safety, and the food additive can be matched with food of pets, effectively prevent the threatened abortion of the pets, and is beneficial to the healthy pregnancy and young baby laying.

Drawings

FIG. 1 is a photograph showing pregnant mice positive for occult blood;

FIG. 2 shows the uterine embryo morphology of different groups of mice;

FIG. 3 shows the effect of atractylenolide I on the change in body mass of pregnant mice;

FIG. 4 shows the effect of atractylenolide I on the variation of progesterone serum levels in pregnant mice;

FIG. 5 shows the effect of atractylenolide I on the variation of serum prostaglandin 2 alpha content in pregnant mice;

FIG. 6 is a schematic representation of HE staining of feline ovarian granulosa cells;

FIG. 7 is a schematic representation of staining of feline ovarian granulosa cells Gittia;

FIG. 8 is a graph showing identification of immunofluorescence staining of feline ovarian granulosa cells;

FIG. 9 is a growth curve of primary feline ovarian granulosa cells;

FIG. 10 is a graph showing the change in cell viability after treatment with varying time concentrations of atractylenolide I;

FIG. 11 is a graph showing the change in progesterone secretion from feline ovarian granulosa cells after treatment with atractylenolide I at different times;

FIG. 12 is a graph of changes in feline ovarian granulosa cells following treatment with atractylenolide I;

FIG. 13 is a microscopic observation of liver tissue sections of the high dose group;

FIG. 14 is a microscopic observation of spleen tissue sections in the high dose group;

FIG. 15 is a microscopic observation of kidney tissue sections from the high dose group;

FIG. 16 is a microscopic observation of ovarian tissue sections from high dose groups;

fig. 17 is a microscopic observation result of a uterine tissue section of the high dose group.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise, and further, it is understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention provides a food additive for preventing pet threatened abortion, which contains atractylenolide I. The traditional miscarriage prevention traditional Chinese medicine has complex components, is influenced by the producing area and has large difference of effective components; and the traditional Chinese medicine has poor palatability and is difficult to feed on pets. The atractylodes macrocephala is a traditional 'fetus-quieting holy drug' and has definite curative effect. In the fetus-protecting prescription, the processing method of the white atractylodes rhizome is optimally stir-fried with soil, and the fetus-protecting effect of the white atractylodes rhizome stir-fried with soil is the best. In the embodiment of the invention, LC-MS detects raw rhizoma atractylodis macrocephalae, rhizoma atractylodis macrocephalae stir-fried with bran and rhizoma atractylodis macrocephalae stir-fried with soil, and the highest content of atractylenolide I in the rhizoma atractylodis macrocephalae stir-fried with soil is found. The atractylenolide I serving as a food additive for preventing the threatened abortion of the pet is proved to be safe and effective, controllable in quality, simple in feeding and convenient to popularize and apply.

The ovarian follicle is a basic functional unit of the ovary, and granulosa cells are attached around the oocyte in the cavity of the ovarian follicle, constitute the largest cell group of the ovarian follicle, and are the main functional cells of the ovary which secrete steroid hormones. Granulosa cells influence the growth, development and maturation of follicles and oocytes through gap links and paracrine factors. In the early embryonic development stage, progesterone is mainly derived from luteinizing granulosa cells, so that the activity of granulosa cells plays a very important role in the early gestation stage. The experimental example of the invention finds that the atractylenolide I has the effects of promoting the proliferation of ovarian granulosa cells of cats and the secretion of progesterone.

Optionally, the food additive also contains baicalin. The scutellaria baicalensis is often combined with the atractylodes macrocephala in the traditional fetus protection prescription, and the effective component baicalin in the scutellaria baicalensis is added in the embodiment of the invention, so that the curative effect of the food additive on preventing the threatened abortion can be further improved.

Optionally, the food additive further comprises a calcium source selected from at least one of calcium carbonate, calcium chloride, calcium phosphate calcium lactate, calcium gluconate, and calcium citrate, and the calcium source is added to supplement calcium requirement during pregnancy.

Optionally, the food additive further comprises a diluent selected from at least one of starch, sugar powder, lactose, and mannitol.

Specifically, the content of atractylenolide I in the food additive is 0.5-1.5% by mass, preferably 0.8-1.2%. The food additive contains 0.1-1 wt% of baicalin, preferably 0.2-0.8 wt%. The content of the calcium source in the food additive is 1-5% by mass.

The food additive provided by the embodiment of the invention can be prepared by directly subpackaging the raw materials in an aseptic subpackage mode, and is mixed with pet food when in use.

The recommended dosage of the food additive provided by the embodiment of the invention is 0.8-1.2 g, preferably 1g, per kilogram of body weight.

Specifically, the atractylenolide I is a commercially available product, and the baicalin is prepared by the following method:

putting scutellaria baicalensis into 8-12 times of water, boiling for 30 minutes, combining the two decoction liquids, adjusting the pH value to be l-2 by using hydrochloric acid, preserving the heat at 80 ℃ for 30 minutes, standing, filtering, collecting precipitates, adding 95% ethanol for washing for 2-3 times, then carrying out vacuum filtration, and carrying out reduced pressure evaporation concentration on the filtrate at 50-60 ℃ until the filtrate is subjected to dry filtration to obtain the baicalin extract.

The embodiment of the invention also relates to application of atractylenolide I in preparing food or medicine for preventing threatened abortion of pets, wherein the pets comprise cats and dogs. The recommended dosage of the atractylenolide I is 8-12 mg per kilogram of body weight, and preferably 10 mg.

Acute toxicity is an important index for safety evaluation of new drugs. Research experiments in the embodiment of the invention prove that the atractylenolide I is safe to use according to the clinical recommended dose. The subchronic toxicity evaluation is based on the reaction of rats and the change of corresponding indexes after different doses of atractylenolide I are infused continuously for 30 days. Experimental studies prove that the atractylenolide I does not produce chronic toxic effect on the tested animals when used according to the clinical recommended dose. The target animal safety test is also called clinical safety test and is an important basis for evaluating whether the drug is safe for the target animal. In the embodiment of the invention, a female cat is selected as a target animal test object. Experimental research proves that the recommended dose of atractylenolide I is safe in clinical use. The toxicity test result shows that the atractylenolide I is a safe medicine, has no toxic or side effect on pet organisms when used according to the clinical recommended dose, and is safe and reliable for preventing the threatened abortion of pets.

Example 1

A food additive contains Atractylodis rhizoma lactone 1.2g, baicalin 0.8g, and lactose 98 g.

The preparation method comprises the following steps: weighing the above materials, mixing, and packaging under sterile condition to obtain food additive powder.

Example 2

A food additive comprises Atractylodis rhizoma lactone 1.0g, baicalin 0.5g, calcium gluconate 1g, and lactose 97.5 g.

Experimental example 1

1. Instruments and reagents:

test drugs: atractylenolide I (AT-I) was purchased from british biotechnology limited, product number: MB6520, quality standard: not less than 98 percent, and storing the standard product in a sealing way at 4 ℃ for later use. During gastric lavage, purified water is used for preparing suspension for use according to the gastric lavage dosage and volume designed by experiments.

BC-2800vet full-automatic blood cell analyzer (Beijing Manchu technologies, Inc.); an OLYMPUS (OLYMPUS) series of fully automated biochemical analyzers; d228-1 electronic counting scale, Shanghai Gaoshi precision instruments products; synergy H4 full-function enzyme-labeling instrument, Boteng instruments Inc. USA;

mouse ELISA kit: mouse PGF2 alpha ELISA kit (lot MB-5673A), mouse PROG ELISA kit (lot MB-3304A), mouse E2 ELISA kit (lot MB-3302A), Jiangsu enzyme-labeled Biotech Co., Ltd.

2. Test animals:

healthy Kunming mice (70 females and 35 males) are 7 weeks old, the female mice have the physical mass of 18-22g, the male mice have the physical mass of 25-30g, the physical mass is provided by the animal experiment center of the university of inner Mongolia, and the qualification license number is SCXK (Mongolia) 2016-0001. Before the test, the male and female mice are separated, the mice are normally bred for 10 days at room temperature, the mice are freely fed with food and water, the padding is replaced 3 times per week, and the health condition is observed.

3. The test method comprises the following steps:

(1) threatened abortion model establishment

Selectively mature mice nighttime 19: 2 after 00: 1, closing the cage, and 7% the next morning: the fertilization of the mice was observed before 00, and the day 0 of pregnancy was indicated as the occurrence of vaginal embolus. On the 7 th day of pregnancy, the patient is gavaged with mifepristone 4.15mg/kg and misoprostol 50 mug/kg to produce a threatened abortion model. A small cotton ball is put into the vagina of a female mouse, and the success of modeling is determined by the positive BLD (occult blood) in the cotton ball through occult blood detection, as shown in figure 1.

(2) Test grouping

Pregnant mice were randomly divided into: the normal pregnancy group, the AT-I low dose group, the AT-I medium dose group, the AT-I high dose group, the progesterone group, the threatened abortion model group and the combined action group are 10 groups, and the rat body quality among the groups has no significant difference.

Normal pregnancy group (10): the physiological saline is infused from the first day of pregnancy, and the dosage is equal to that of the administration group;

AT-I Low dose group (10): administering AT-I0.6 mg/kg/d on the first day of gestation; intragastrically administering mifepristone 4.15mg/kg and misoprostol 50 μ g/kg on the seventh day;

AT-I Medium dose group (10): AT-I was administered by drench from the first day of pregnancy AT 1.2 mg/kg/d. Intragastrically administering mifepristone 4.15mg/kg and misoprostol 50 μ g/kg on the seventh day;

AT-I high dose group (10): AT-I was administered by drench from the first day of pregnancy AT 2.4 mg/kg/d. Intragastrically administering mifepristone 4.15mg/kg and misoprostol 50 μ g/kg on the seventh day;

progesterone group (10): 1mg/kg/d of progesterone is infused from the first day of pregnancy, and 4.15mg/kg of mifepristone and 50 mu g/kg of misoprostol are infused into the stomach on the seventh day;

threatened abortion model group (10): the physiological saline is infused from the first day of pregnancy, the dosage is equal to the dosage of the administration group, and the intragastric administration is carried out by using mifepristone 4.15mg/kg and misoprostol 50 mu g/kg on the seventh day;

combined action group (10): AT-I1.2 mg/kg/d + baicalin 0.8mg/kg/d is drenched from the first day of pregnancy, and mifepristone 4.15mg/kg and misoprostol 50 mu g/kg are drenched from the seventh day.

4. Sample collection and detection:

on day 13 of pregnancy, blood sampling is carried out on eyeballs of the mice, centrifugation is carried out at 3000r/min, and upper serum is taken for testing. The neck was sacrificed, the uterus of the mice was collected, and the number of embryos and the abortion rate were calculated. Normal embryos: blood stasis is not seen in the uterus, and the embryo is in a bead shape; abortion embryo: irregular morphology, and dark brown intrauterine embryo. The serum was stored in a refrigerator at-20 ℃ and returned to room temperature before detection, and the contents of PGF2 alpha and PROG were measured by ELISA kit.

5. Data processing

The data of each test is expressed by mean value plus standard deviation, and the significance of the difference between each group of data is statistically analyzed by using SPSS 20.0. Differences between groups were compared using t-test and one-way ANOVA analysis, with P <0.05 being statistically significant. The histograms were analyzed for One-way ANOVA and Tukey's Multiple Comparison test using the statistical software GraphPad Prism 7.04 statistical program.

6. Test results

6.1 Effect of AT-I on the fetus protection Rate of mice

Typical morphology of intrauterine embryos is shown in figure 2, and the influence on body mass changes of pregnant mice is shown in figure 3, the body mass changes of mice in a normal pregnancy group are remarkably higher than those in a threatened abortion group and a low dose group (P <0.01), and the body mass changes of other dose groups are remarkably lower than those in the normal pregnancy group (P < 0.05). The combination group, the high dose group and the medium dose group did not significantly differ from the progesterone group (P > 0.05).

The influence of atractylenolide I on the survival rate of mice is shown in Table 1, the mean survival rate of the group of the threatened abortion model is 3.82%, is significantly lower than that of other groups (P <0.01), the mean survival rate of the combined action group and the group of AT-I high dose is improved to 14.5% and 13.7%, and compared with the mean survival rate of the progesterone group of 15.27%, the difference is not significant (P > 0.05). The effect of the combined action group is equivalent to that of the progesterone group, and compared with the effect of singly using the atractylenolide I, the effect is better, the economic benefit is better, and the combination accords with the compatibility of the traditional prescription.

TABLE 1

Group of	n	Average number of live tires/piece	Average live embryo rate/%)
				Group of normal pregnancies	10	8.33±1.03^a	38.17
AT-I high dose group	10	3.0±0.63^b	13.7
				AT-I Medium dose group	10	2.5±1.05^b	11.5
AT-I Low dose group	10	0.67±0.82^c	3.05
				Progesterone group	10	3.33±1.03^b	15.27
Threatened abortion model group	10	0.84±0.75c	3.82
				Combined action group	10	3.17±0.75b	14.5

Note: the data in the same column are marked with the same shoulder mark to indicate that the difference is not significant (P >0.05), and the data in the same column are marked with the different shoulder mark to indicate that the difference is significant.

6.2 Effect of AT-I on mouse serum PGF2 alpha, PROG

The influence of atractylenolide I on the change of the serum progesterone content of pregnant mice is shown in the result of fig. 4, and the difference of the PROG content of a normal pregnancy group is very obvious (P is less than 0.01) compared with a traditional Chinese medicine low-dose group and a threatened abortion model group; compared with the progesterone group, the content of progesterone in the traditional Chinese medicine high-dose group and the traditional Chinese medicine medium-dose group has no significant difference (P >0.05), and the fact that the high-dose AT-I can promote the secretion of progesterone and has the effect similar to that of progesterone treatment is proved.

The influence of atractylenolide I on the change of serum prostaglandin 2 alpha content of pregnant mice is shown in fig. 5, the content of PGF2 alpha in the group of threatened abortion models is significantly different from that in other groups (P <0.05), while the content of PGF2 alpha in the drug-treated group is not significantly different from that in the group of progesterone and the group of normal pregnancy (P >0.05), which indicates that AT-I has certain anti-inflammatory effect.

The experimental results of the Kunming mouse model for threatened abortion are analyzed and compared, so that the atractylenolide I has the fetus protection effect on the Kunming mouse for threatened abortion modeled by mifepristone and misoprostol, has a better effect after being combined with baicalin, and has no adverse effect on the normal pregnancy of the mouse.

Experimental example 2 cell assay study

1. Primary culture of cat ovarian granulosa cells

Selecting ovary (soaked by 3% streptomycin physiological saline) taken down by healthy female cat sterilization, transporting the ovary in a vacuum cup at 37 ℃, sending the ovary to a laboratory within 3 hours after collection, cutting off mesentery and adipose tissue in an ultra-clean bench after cleaning by using 3% streptomycin physiological saline, extracting 1-3mL follicle follicular fluid by using a 5mL injector, collecting cumulus oocyte complexes (cumulus-oophores COCs) under a picking scope, reversely cleaning by using a culture medium containing 3% streptomycin and 12% fetal calf serum, blowing, culturing by using DMEM/F12 medium (Gibco) containing 1% streptomycin and 12% fetal calf serum based on 37 ℃ and 5% CO₂Culturing in the environment, and changing the culture solution every other day.

2. Ovarian granulosa cell HE staining and gipsy staining identification

When the cat ovary granular cells are subcultured to 3 rd generation, the cells are digested by 0.25% pancreatin, and the cells are cultured for 24h in a climbing film mode. The coverslips were rinsed 3 times with DPBS for 5min each. Cell slide was fixed with 4% paraformaldehyde for 20 min. Taking out the slide, washing the slide by DPBS, dyeing with hematoxylin for 2min, washing away unbound dye solution with running water, soaking in hydrochloric acid alcohol to remove excessive dye bound in cell nucleus and dye adsorbed in cytoplasm, washing with running water for 5min, dyeing the cell nucleus under microscope with hematoxylin to blue, and leaving cytoplasm uncolored. Then 5% eosin is used for dyeing for 3min, and the washing is carried out by running water, so as to wash out the unbound dye liquor. Gradient alcohol dehydration, xylene transparency and mounting. Cell morphology was observed under light microscope.

The experimental results are as follows: HE staining is shown in fig. 6 and gipsy staining is shown in fig. 7, with intact cell morphology, clear edges, triangular pyramidal shape or irregular star shape under the microscope.

3. Immunofluorescence identification of cat ovarian granulosa cells

When the cat ovary granular cells are subcultured to 3 rd generation, the cells are digested by 0.25% pancreatin and cultured in a 12-well plate for 24h in a climbing-sheet manner. Discarding the culture medium, adding 3mL of 0.01mol/L DPBS (Thermo HyClone) into each well, washing on a room temperature decolorizing shaker, and continuously hooking a climbing sheet with a self-made hook needle in the washing process to shake and wash for 3 times at 10 min/time. Cells were fixed with 4% paraformaldehyde for 30min and washed on a shaking table at room temperature. 1mL of freshly prepared 0.4% Triton X-100(pH 7.40) was added to each well and the mixture was protected from light at room temperature for 20 min. Washing on decolorizing shaking bed at room temperature for 10 min/time, 3 times. The cells were then washed 3 times for 10 min/time in a dark environment on a room temperature decolorizing shaker using 3% normal donkey serum (D9663, Sigma) blocking solution containing 0.1% Triton X-100(Sigma) for 1 h. Primary anti-AntiFSHR (FSH receptor) antibody (1: 200, bios) was incubated overnight at 4 ℃ and after washing, secondary antibody Alx647 (done-antibody) (1: 1000, Jackson) was incubated 2h away from light at room temperature. DAPI staining for 10min, and washing on a decolorization shaking bed for 10 min/time and 4 times. After the slide is naturally air-dried, the slide is transferred to a glass slide, and laser confocal direct observation is carried out.

The experimental results are as follows: immunofluorescent staining of cat ovarian granulosa cells is shown in fig. 8, with the left panel being the positive group: feline ovarian granulosa cells that express an FSH receptor; the right panel is a negative control group: feline ovarian granulosa cells that do not express an FSH receptor. FSHR immunofluorescent chemical staining is specific staining of ovarian granulosa cells, with FSHR positive staining being present in the cytoplasm, with green fluorescence, and the nucleus with blue fluorescence. The positive rate of FSHR compared with the control group is more than 90 percent under the microscope. Therefore, the purity of the cat ovarian granulosa cells cultured in vitro can be shown to reach more than 90%. (Scale: 100 μm)

4. CCK-8 detection of feline ovarian granulosa cell growth curve

Taking 3 rd generation cat ovary granular cells, washing the cells by using DPBS, adding 1mL of trypsin for digestion for 3min, adding a culture medium (containing 12% serum) to stop digestion, and repeatedly blowing to ensure that the cells are separated from the wall. After digestion, cell suspension concentrations were adjusted to 5 × 10³one/mL. A96-well cell culture plate was taken, and 100. mu.L of the culture medium was added to each well, followed by culture for 12 hours. The remaining wells were loaded with 100. mu.L of DPBS to reduce evaporation. Then purifying for 6h (adding serum-free culture medium), pouring out cell liquid after purification, adding 100UL culture medium (containing 12% serum) to culture for 6h, 12h, 24h, 36h and 48h respectively, adding CCK-8 to act for 4h, and measuring the light absorption value (OD value) of each hole at the wavelength of 450nm by using a microplate reader.

The growth curve of primary cat ovarian granulosa cells is shown in fig. 9.

5. Cytotoxicity detection of different concentrations of atractylenolide I on cat ovarian granulosa cells at different action times

After the cells were purified as described above, 100. mu.L of the culture medium containing atractylenolide I at different concentrations (0. mu. mol/L, 10. mu. mol/L, 20. mu. mol/L, 40. mu. mol/L, 80. mu. mol/L, 160. mu. mol/L, 320. mu. mol/L) was added thereto, and after incubation at 37 ℃ for 12 hours, 24 hours, 36 hours, and 48 hours, CCK-8 was added thereto for 4 hours, and the absorbance (OD value) of each well was measured at a wavelength of 450nm using a microplate reader.

The change of cell viability after treatment with atractylenolide I at different time concentrations is shown in fig. 10, and according to the results of CCK-8, atractylenolide I has a proliferation effect on cat ovarian granulosa cells within a certain concentration range and has an inhibition effect on cells at a high concentration. The cell survival rate is highest and the inhibition rate is lowest when the cell is acted for 36 hours at the concentration of 10 mu mol/L. Therefore, the optimal action time of the atractylenolide I on the cat ovarian granulosa cells is 36 hours, and the optimal action concentration is 10 mu mol/L, and subsequent experiments are carried out at the optimal action concentration.

6. Effect of Atractylodes macrocephala lactone I on progesterone secretion from luteinizing granulosa cells at different treatment times

By adopting a double-antibody sandwich ELISA method, the color depth is in positive correlation with the progesterone to be detected in the sample. Adding 10 mu mol/l of atractylenolide I into the granular cells, respectively incubating at 37 ℃ (0h, 12h, 24h, 48h and 72h), collecting supernatant, and detecting.

The change of the secretion of progesterone of the cat ovarian granulosa cells treated by the atractylenolide I at different time is shown in fig. 11, and it can be seen that the atractylenolide I can effectively promote the secretion of progesterone along with the increase of the treatment time, and the increase of the secretion of progesterone is very obvious when the cat ovarian granulosa cells are treated for 24 hours.

The morphology of the cells was observed under the mirror after the treatment with atractylenolide I, and the results are shown in FIG. 12 (scale: 100 μm). As can be seen in figure 12, the cell volume of the ovarian granules of the cat is increased, the lipofuscin content is increased, and the secretion capacity is obviously improved.

Experimental example 2 toxicity test study

1. Instruments and reagents:

BC-2800vet full-automatic blood cell analyzer (Beijing Manchu technologies, Inc.); an OLYMPUS (OLYMPUS) series of fully automated biochemical analyzers; OLYMPUS (OLYMPUS) light microscope; SC-04 type low speed centrifuge (Anhui Zhongzhongjia scientific instruments, Inc.); a VetScan HM5 five-classification cytometer; bs-180vet blood biochemical analyzer (Abaxis, usa); hunan instruments centrifuge instruments Ltd. Aspartate aminotransferase (AST, lot # 140216017), alanine aminotransferase (ALT, lot # 140116022), glutamyl transferase (r-GGT, lot # 140916015), alkaline phosphatase (ALP, lot # 140316003), total cholesterol (TC, lot # 141616018), glucose (GLU, lot # 141516018), albumin (ALB, lot # 140916013), total protein (TP, lot # 140816010), UREA (UREA, lot # 141316019) creatinine (CREA, lot # 141016003) reagent cartridges were purchased from inner Mongolian first reagent supplies, Inc. Intragastric perfusion needles, disposable blood collection needles and associated vacuum blood collection tubes were purchased from Dayin Shanghai, Inc., of inner Mongolia Huo and Haote.

80 healthy Kunming female mice (about 6 weeks old, body mass 18-22 g) and 24 healthy female Wistar rats (about 7 weeks old, body mass 110-130 g) are provided by the animal experiment center of the university of inner Mongolia, and the qualification license number is SCXK (Mongolia) 2016-0001. The room was kept for 10 days at room temperature for the test, and the bedding was changed 3 times per week for free food and water intake and health observation.

20 healthy adult domestic cats, about 2-3 years old, have been immunized against parasites.

2. Test method

(1) Acute toxicity test

Taking 60 healthy female Kunming mice, randomly and averagely dividing into 5 groups, taking 1-4 groups as a test group, taking the 5 th group as a control group, fasting each group of mice for 8 hours, then performing intragastric administration, re-eating after administration for 4 hours, performing intragastric administration once according to the intragroup dose ratio of 1:0.8 for the test group, respectively taking 6000, 4800, 3840 and 3072mg/kg for the 1 st-5 groups, and taking pure water with the same volume as the control group, wherein the intragastric volume of the liquid medicine of each group of mice is 0.4mL per 20g of body mass. After administration, the mice were bred for 7 days, and the mental, appetite, drinking water and activity thereof were observed daily, and the intoxication symptoms, death time and number were recorded, and the dead animals were examined immediately and the ocular pathological changes were recorded, and LD50 was calculated according to the modified kouynian method. If the mice do not die and the administration cannot be continued (no LD50 can be detected), the maximum tolerance test is required.

(2) Maximum tolerated dose test

20 female mice were taken and divided into test and control groups, each group containing 10 mice, and fasted for 8h (without water deprivation). The test group mice are drenched with 0.2g/mL liquid medicine 3 times in 12h, and the volume of each time of the drenching is 0.8mL (maximum drenching volume) per 20g of body mass; the control mice were drenched with purified water of equal volume. After the gavage, the mice are conventionally bred for 7 days, the spirit, appetite, drinking water and activity conditions of the mice are observed every day, the poisoning symptoms, death time and number of the mice are recorded, the maximum tolerance amount and the tolerance times are calculated, then the mice are killed by dislocation of cervical vertebrae and are subjected to autopsy, and whether macroscopic pathological changes exist in organs such as liver, spleen, kidney, ovary and uterus of 2 groups of mice is observed. The maximum tolerance times of the mice (mouse tolerance dose/average body mass of mice) x (average body mass of domestic cats/daily dose of domestic cats), wherein the average body mass of the domestic cats is 3kg and the daily dose of the domestic cats is 30 mg.

(3) Subchronic toxicity test

Taking 24 female rats, randomly and equally dividing into control group and atractylenolide I high, medium and low dosage groups, wherein the gavage dosage is 6000, 3000, 1500mg/kg respectively, 1 time per day, and the control group is regularly administered with equal volume of purified water every day. The gavage is continuously performed for 30d in each group (every 7d is called as the body mass for adjusting the gavage volume), and the behavior, the appearance signs, the toxic reaction and the death of the animals are observed and recorded in the morning and at the evening every day during the whole test period. After the administration period, the hearts of rats in each group are subjected to blood sampling, and blood routine and blood biochemical indexes are detected.

Measurement of body mass: the rat body mass was measured before and at 7, 14, 21, 28, 30d after drug administration.

Measurement of blood routine and blood biochemical indexes: the main physiological indicators of blood, including the Hemoglobin (HGB) content, the red blood cell number (RBC), the white blood cell number (WBC) and the proportion of various white blood cells (lymphocytes (Lym), monocytes (Mon), neutrophils (Neu), eosinophils (Eo) and basophils (Ba)) in the total number of white blood cells, were determined by a fully automatic blood cell analyzer. And (3) determination of biochemical indexes: after separating serum, an automatic blood biochemical index analyzer is adopted to measure blood biochemical indexes including aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), glutamyltransferase (r-GGT), alkaline phosphatase (ALP) activity, Total Protein (TP), Albumin (ALB), creatinine (CREA-J), UREA (UREA), Glucose (GLU) and Total Cholesterol (TC) content.

Observation of pathological changes and measurement of organ coefficients: after blood collection, 4 groups of rats were immediately examined by dissection to observe the ocular pathological changes of organs such as liver, spleen, stomach, kidney, and lung, and organs such as liver, spleen, kidney, ovary, and uterus of each group of rats were weighed, fixed with 10% formalin, dehydrated with different gradient alcohols, embedded with wax, sectioned, and examined for histopathological changes of organs under an optical microscope after HE staining, and organ coefficients of organs such as liver, spleen, kidney, ovary, and uterus were calculated (organ coefficient ═ wet mass/body mass × 100%).

(4) Clinical safety test

20 healthy female domestic cats are selected, observed and raised for 7 days, and randomly divided into 4 groups of 5 cats. The domestic cat administration dosage is 1 time, 2 times and 4 times of the recommended administration dosage, and respectively comprises the following components: the low dose group is 10mg/kg, the medium dose group is 20mg/kg, the high dose group is 40mg/kg, the oral administration is carried out for 1 time per day for 7 days continuously, and the blank control group is not administered. All domestic cats eat and drink water freely; appetite, mental state and defecation were observed daily. Measurement of blood routine and blood biochemical indexes: after the administration, namely blood collection is carried out on all domestic cats at the same time point on the 7 th day of the experiment, whole blood is collected by adopting an EDTA-K2 anticoagulation tube and is analyzed by a full-automatic blood analyzer, and detection indexes comprise Hemoglobin (HGB), red blood cell count (RBC), white blood cell count (WBC), Granulocyte (GRA), Lymphocyte (LYM), Monocyte (MON) classification counting and the like. Biochemical detection of blood: after the administration is finished, namely on the 7 th day of the test, performing venous blood collection on each domestic cat, collecting the blood in a blood collection tube without anticoagulant, centrifuging for 15min at 3000r/min, taking out serum, and performing blood biochemical index detection by using a full-automatic biochemical analyzer. The detection index includes alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), urea nitrogen (BUN), Creatinine (CREA), blood Glucose (GLU), serum Albumin (ALB), total egg (TP), Total Cholesterol (TCH), Triglyceride (TG), etc.

3. Data processing

The data of each test is expressed by mean value plus standard deviation, and the significance of the difference between each group of data is statistically analyzed by using SPSS 20.0.

4. Test results

4.1 acute toxicity test

After the oral administration is carried out by drenching for 1 time, each group of mice is in a state of standing for 2-3 min due to gastric lavage stress, the mental state is gradually recovered to be normal, the movement is free, the fur is smooth, the excrement and urine are discharged normally, no abnormal secretion exists, and no adverse reaction exists. After continuous observation for 7 days, the mice in each group have no abnormal spirit, appetite, drinking water and activity, and the main organs have no abnormal change after general autopsy and pathological examination. The main measurement index of the acute toxicity test is the measurement of half lethal dose (LD50), and LD50 is measured if animals die; however, some drugs are of low toxicity, and if the number of deaths in an animal remains zero when a total dose of greater than 5000mg/kg is administered, more administrations are discontinued and the maximum tolerated dose of the drug is determined.

This test revealed that the maximum tolerance test was carried out in mice because LD50 was difficult to measure due to the limitations of the dose volume and drug concentration.

4.2 maximum tolerated dose test

After the mice are continuously drenched with the maximum volume for 3 times in 12h, the cumulative dosage of the test group reaches 24g/kg, the two groups of mice are both coiled and suspended due to the stress of drenching the stomach, the activity is reduced, and the activity and the diet condition are recovered to be normal after 10 minutes. No death and abnormal conditions are observed after 7d observation, and no ocular pathological changes appear in each organ after the autopsy. The maximum tolerated dose was calculated to be 0.48 g/mouse. The maximum tolerance multiple of the mice taking atractylenolide I is 0.48/20 multiplied by 3000/0.03 is 2400, which shows that the maximum tolerance of the mice 1d calculated according to the constitutional weight is 2400 times of the clinically recommended dose of domestic cats, and the mice do not have abnormal changes under the dose, which indicates that the atractylenolide I is safe to use according to the clinically recommended dose.

4.3 subchronic toxicity test

(1) General condition observation and measurement of physical quality

In the test period, the rats of the 4 treatment groups have gastric lavage stress in the first 3 days and recover to a normal state after 15 minutes; the mental state, the activity degree and the diet condition of the rats have no abnormal change in the whole gastric lavage period, the defecation and the urine are normal, no abnormal secretion exists, and the difference between the body quality of the rats in the 3 atractylenolide I dosage groups and the body quality of the rats in the control group is not significant (P is more than 0.05) as shown in the table 1, which indicates that the atractylenolide I has no obvious influence on the growth and development of the rats.

TABLE 1 Effect of different doses of Atractylodes macrocephala lactone I on rat body quality (g)

(2) Blood routine and blood biochemical index examination

As can be seen from tables 2 and 3, the conventional blood index and the biochemical blood index of each group of rats have no significant difference (P >0.05), which indicates that the atractylenolide I has no obvious influence on the conventional blood index, liver function and kidney function of the rats during oral administration.

TABLE 2 Effect of different doses of Atractylodes macrocephala lactone I on the blood routine of rats

TABLE 3 Effect of different doses of Atractylodes macrocephala lactone I on the biochemical indicators of rat blood

(2) Pathological anatomy change and measurement of organ coefficient

After the autopsy, the heart, liver, spleen, lung, stomach and intestine, kidney, uterus and ovary of the rat are observed, and no abnormality is found on the surface and section of other tissues except the heart which is damaged and bleeds due to blood collection. As shown in Table 4, the coefficients of organs of the 4 treated rats were not significantly different (P >0.05), indicating that atractylenolide I did not substantially damage the organs of the rat tissues.

TABLE 4 Effect of different doses of atractylenolide I on rat organ coefficients (%)

(3) Changes in histopathology

The observation of each tissue under an optical microscope shows that: the hepatic lobules consist of hepatic plates which are radially arranged by taking a central vein as a center, hepatic cells are complete, boundaries among the hepatic lobules can be clear but not obvious, necrosis and inflammatory cell infiltration are not seen in the intralobular area and the manifold area, and bile ducts are not abnormal; the spleen essence has no abnormal change, the white marrow, the red marrow, the trabecula, the lymph nodule and the venous sinus structure are clearly visible, and no obvious abnormal change is seen; the kidney tissue has a complete morphological structure, nephrons can be clearly seen, glomeruli and renal tubules have no abnormal change, inflammatory exudates and cell infiltration are not seen in renal vesicle cavities, and the structures of proximal convoluted tubules, distal convoluted tubules and collecting vessels are complete and have no abnormal change; the uterine tissue is complete, the endometrium and the endometrial gland have no abnormal change, the myometrium has no necrosis, and the blood vessel layer has no abnormal change such as congestion; the ovary cortex and medulla are complete in shape, the nucleus, cytoplasm, corona radiata and granulosa of the oocyte are clearly visible and do not change abnormally, which shows that the traditional Chinese medicine has no toxic effect on main organs and target organs of rats. The microscopic observation results of the tissue sections of the respective organs in the high dose group are shown in FIGS. 13 to 17.

4.4 clinical safety test

(1) General observations

During the test period, the domestic cats in each group had normal diet and drinking water conditions, and the food intake of the drug-treated groups was increased, the fur was bright and soft, the behavior and activity were normal, no abnormal excitation or mental depression occurred, the defecation frequency and form were normal, and no acute emaciation, poisoning, death or the like occurred.

(2) Blood routine and blood biochemical index examination

As can be seen from tables 5 and 6, on the 7 th day of treatment, there was no significant difference in the blood routine and blood biochemical indicators (P >0.05) in each group of cats, indicating that atractylenolide I had no significant effect on the blood routine, liver function and kidney function of cats during oral administration.

TABLE 5 Effect of Atractylodes macrocephala lactone I on Haemology index of domestic cats

TABLE 6 Effect of Atractylodes macrocephala lactone I on blood biochemical indicators of domestic cats

Note: the data in the same column are marked with no mark on the shoulder, which indicates that the difference is not significant (P >0.05), and marked with a mark on the shoulder, which indicates that the difference is significant.

In this experimental example, the acute toxicity test was affected by the concentration of the drug solution and the gavage volume, and the LD50 of the drug could not be calculated. According to the research and development specifications and reporting guidelines of Chinese new veterinary drugs, the maximum dosage of the drug which can not be detected by LD50 needs to be analyzed, and the reaction of animals after taking the maximum dosage of the drug for one time or 2-3 times in one day is generally detected. The cumulative dose of the mice after being drenched with the maximum dose of the liquid medicine for 3 times reaches 24g/kg, and 2 groups of mice have no abnormal change within 1 week, and all organs have no eye pathological changes after the autopsy. The maximum tolerance of the mouse 1d calculated according to the constitutional quantity is 2400 times of the clinical recommended dose of the domestic cat every day, and the mouse does not have abnormal change under the dose, which indicates that the atractylenolide I is safe to use according to the clinical recommended dose.

The subchronic toxicity evaluation is based on the reaction of rats and the change of corresponding indexes after different doses of atractylenolide I are infused continuously for 30 days. The test results show that the body mass, blood routine, blood biochemical index and organ coefficient of the rats in the three dose groups are not significantly different (P is more than 0.05) compared with those in the control group, and each organ has no other abnormal changes except that part of myocardial fiber necrosis and interstitial vascular hemorrhage caused by heart blood collection. The medicament is shown to have no chronic toxic effect on the tested animals when used according to the clinical recommended dose.

In the embodiment of the invention, a female cat is selected as a target animal test object. Three dose groups and a control group are designed by adopting 1 time, 2 times and 4 times of recommended dose of target animals, the normal food intake of domestic cats, the normal mental state, the normal glossiness of furs and the normal excrement condition of domestic cats in different dose groups are observed, and the comparative difference between hematology indexes and blood biochemical indexes and the control group is not obvious (P is more than 0.05). Since each group of animals did not show signs of intoxication and death, the experimental cats were not necropsied and histopathologically examined according to animal welfare. Experiments show that the recommended dose of atractylenolide I is safe for clinical use.

The toxicity test result shows that the atractylenolide I is a safe medicine, has no toxic or side effect on the body of the domestic cat when used according to the clinical recommended dose, and is safe and reliable for preventing the threatened abortion of the domestic cat.

Although the present application has been described with reference to preferred embodiments, it is not intended to limit the scope of the claims, and many possible variations and modifications may be made by one skilled in the art without departing from the spirit of the application.

Claims

1. A food additive for preventing threatened abortion of pets is characterized by comprising atractylenolide I.

2. The food additive according to claim 1, wherein the food additive further comprises baicalin.

3. The dietary supplement of claim 1, further comprising a calcium source selected from at least one of calcium carbonate, calcium chloride, calcium lactate phosphate, calcium gluconate, and calcium citrate.

4. The dietary supplement of claim 1, further comprising a diluent selected from at least one of starch, powdered sugar, lactose, and mannitol.

5. The food additive according to claim 1, wherein the atractylenolide I is contained in the food additive in an amount of 0.5-1.5% by mass, preferably 0.8-1.2% by mass.

6. The food additive according to claim 2, wherein the baicalin content in the food additive is 0.1-1% by mass, preferably 0.2-0.8% by mass.

7. The food additive according to claim 3, wherein the calcium source is contained in the food additive in an amount of 1 to 5% by mass.

8. The food additive according to any one of claims 1 to 7, wherein the baicalin is prepared by the following method:

9. Application of atractylenolide I in preparing food or medicine for preventing pet threatened abortion is provided.

10. A food additive according to any one of claims 1 to 8 or the use of claim 9 wherein the companion animal comprises a dog or a domestic cat.