Use of radix cyathulae in preparation of medicine for relieving toxicity of chlorpyrifos

Abstract

The application discloses application of Millettia pinnata in preparation of a medicine for relieving toxicity of chlorpyrifos, and belongs to the technical field of biotechnology. It is found through animal model experiments that Millettia pinnata can improve the problem of decreased reproductive performance of pregnant animals caused by chlorpyrifos, significantly increases the number of litter size, fetal weight, fetal length, fetal width and placental weight, improves intrauterine growth restriction of fetuses by promoting placental development, relieving placental inflammation, promoting placental angiogenesis and nutrient transport, and the effect can even be continued to offspring, so that the reproductive performance of the offspring is also significantly enhanced. Millettia pinnata can also improve the intestinal development of offspring, reduce intestinal inflammation caused by chlorpyrifos, restore intestinal barrier, improve intestinal flora and ensure the healthy growth of offspring. The application provides a new medicine raw material for relieving the toxicity of chlorpyrifos, and Millettia pinnata belongs to Chinese herbal medicine, is widely sourced, has natural and safe components and meets the concept of green health.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of *Smilax glabra* in the preparation of drugs to alleviate the toxicity of chlorpyrifos. Background Technology

[0002] In livestock and poultry farming, reproductive performance directly impacts production efficiency and economic benefits. Clean environmental conditions are crucial for reproductive performance; some environmental pollutants can directly reduce reproductive performance, thus affecting the economic benefits of the industry. Increased pesticide use has led to serious pesticide residues in water, soil, and crops. Chlorpyrifos, an organophosphorus pesticide, can impair intestinal development by enhancing apoptosis, oxidative stress, inflammation, and damaging the intestinal barrier, and can also harm early embryonic development and cause maternal reproductive toxicity.

[0003] Current treatments for fetal growth restriction (FGR) caused by chlorpyrifos have systemic flaws. First, clinical acute antidotes (such as atropine) only target cholinergic crises and cannot address the core pathological mechanisms leading to FGR, such as placental oxidative stress and vascular dysfunction. Second, conventional antioxidants lack placental targeting, making it difficult to achieve effective concentrations at the maternal-fetal interface and potentially interfering with normal maternal metabolism. More critically, the safety requirements for medication use during pregnancy are extremely high, and most chemical drugs are strictly restricted due to potential developmental toxicity, resulting in a clinical "no available drugs" dilemma. Furthermore, although there are numerous studies on reproductive performance, most focus only on the reproductive performance of the first generation of mothers, neglecting the adverse effects of intrauterine fetal growth restriction on offspring reproductive performance. Therefore, developing drugs that can effectively block the toxicity of chlorpyrifos to both the mother and offspring is crucial. Summary of the Invention

[0004] The purpose of this invention is to provide the application of *Smilax glabra* in the preparation of drugs to alleviate the toxicity of chlorpyrifos, thereby solving the problems existing in the prior art. This invention has found that administering *Smilax glabra* to pregnant animals can significantly improve the reproductive performance of pregnant animals exposed to chlorpyrifos, improve offspring intestinal development and intestinal microbiota structure, enhance placental function, reduce intrauterine fetal growth restriction, and improve offspring reproductive performance.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of *Smilax glabra* in the preparation of drugs to alleviate the toxicity of chlorpyrifos.

[0006] The present invention also provides the application of *Smilax glabra* in the preparation of a drug to improve the decline in reproductive performance caused by chlorpyrifos, wherein the reproductive performance includes maternal weight, litter size, fetal weight, fetal length, fetal width and placental weight.

[0007] Further, the medicament has effects of increasing litter size, fetal weight, fetal body length, fetal body width, placental weight, and increasing offspring litter size, offspring fetal weight, offspring fetal body length, offspring fetal body width, and offspring placental weight.

[0008] The present invention also provides the use of Millettia speciosa Champ. in the preparation of a medicament for treating intrauterine growth restriction caused by chlorpyrifos, wherein the medicament treats intrauterine growth restriction by promoting placental development, alleviating placental inflammation, and promoting placental angiogenesis and nutrient transport.

[0009] The present invention also provides the use of Millettia speciosa Champ. in the preparation of a medicament for treating intestinal development abnormality in offspring caused by chlorpyrifos, wherein the intestinal development abnormality refers to impaired intestinal barrier, intestinal inflammation, shortened total intestine length and colon length.

[0010] The present invention also provides the use of Millettia speciosa Champ. in the preparation of a feed additive for improving decreased reproductive performance of pregnant animals caused by chlorpyrifos, wherein the reproductive performance includes maternal body weight, litter size, fetal weight, fetal body length, fetal body width and placental weight.

[0011] The present invention also provides the use of Millettia speciosa Champ. in the preparation of a feed additive for improving fetal intrauterine growth restriction caused by chlorpyrifos, wherein the medicament improves fetal intrauterine growth restriction by promoting placental development, alleviating placental inflammation, and promoting placental angiogenesis and nutrient transport.

[0012] The present invention also provides the use of Millettia speciosa Champ. in the preparation of a feed additive for improving intestinal development abnormality in offspring caused by chlorpyrifos, wherein the intestinal development abnormality refers to impaired intestinal barrier, intestinal inflammation, shortened total intestine length and colon length.

[0013] The present invention discloses the following technical effects: Through animal model experiments, the present invention finds that Millettia speciosa Champ. can improve the problem of decreased reproductive performance of pregnant animals caused by chlorpyrifos, significantly increase litter size, fetal weight, fetal length, fetal width and placental weight, and improve fetal intrauterine growth restriction by promoting placental development, alleviating placental inflammation, promoting placental angiogenesis and nutrient transport. This effect can even be extended to the offspring, so that the reproductive performance of the offspring is also significantly enhanced. Millettia speciosa Champ. can also improve the intestinal development of offspring, reduce intestinal inflammation caused by chlorpyrifos, restore intestinal barrier, improve intestinal flora, and ensure the healthy growth of offspring. The present invention provides a new pharmaceutical raw material for alleviating the toxicity of chlorpyrifos. Millettia speciosa Champ. belongs to Chinese herbal medicine, which has wide sources, natural and safe components, and conforms to the concept of green health. Description of Drawings

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 The TIC graph of *Niu Dali* powder; Figure 2 The effect of *Smilax glabra* on the reproductive performance of first-generation pregnant female mice; where A is the weight change curve of pregnant female mice; B is the litter size, number of fetal resorptions, and fetal growth restriction rate at 15.5 days of gestation; C is the fetal weight, placental weight, and fetal body length and width at 15.5 days of gestation; D is the litter size, newborn fetal weight, and newborn fetal body length and width on the day of delivery. P<0.05 P<0.01 P < 0.001, n = 5; Figure 3 The effect of *Smilax glabra* on the reproductive performance of pregnant female mice; where A is the weight change curve of pregnant female mice; B is the litter size, number of fetal resorptions, and fetal growth restriction rate at 15.5 days of gestation; C is the fetal weight, placental weight, and fetal body length and width at 15.5 days of gestation; D is the litter size, newborn fetal weight, and newborn fetal body length and width on the day of delivery. P<0.05 P<0.01 P < 0.001, n = 5; Figure 4 The effects of *Smilax glabra* on the intestinal barrier and antioxidant capacity of offspring female mice; where A is a comparison of the normal intestine and colon of offspring pregnant female mice; B is the protein expression of the colonic barrier in offspring pregnant female mice; and C is the protein expression of antioxidants in the colon of offspring pregnant female mice. P<0.05 P<0.01 P < 0.001, n = 5; Figure 5 The study investigated the effect of *Smilax glabra* on chlorpyrifos-induced intestinal inflammation. A represents the protein expression and relative expression levels in progeny pregnant mice with colitis; B represents the expression level of the colitis gene in progeny pregnant mice. P<0.05 P<0.01 P < 0.001, n = 5; Figure 6The effects of *Smilax glabra* on placental barrier, angiogenesis, and nutrient transport are as follows: A represents the protein expression and relative expression levels of placental barrier in offspring pregnant mice; B represents the protein expression of placental angiogenesis in offspring pregnant mice; and C represents the expression level of placental nutrient transport genes in offspring pregnant mice. P<0.05 P<0.01 P < 0.001, n = 5; Figure 7 The study investigated the effect of *Smilax glabra* on placental inflammation induced by chlorpyrifos; where A represents the protein expression and relative expression levels of placental inflammation in offspring pregnant mice; and B represents the expression levels of placental inflammation genes in offspring pregnant mice. P<0.05 P<0.01 P < 0.001, n = 5; Figure 8 The effects of *Achyranthes bidentata* on gut microbiota are shown in Figure A, where A is the VENN diagram of gut microbiota abundance differences, B is the Chao and ace indices of microbial α-diversity analysis, and C is the PCoA analysis results of microbial β-diversity analysis. Figure 9 The relative abundance of the top ten microbial phyla (A) and the ratio of Firmicutes to Bacteroidetes among the four experimental groups (B). Figure 10 The relative abundance of the top ten microbial genera; Figure 11 Between the four experimental groups Lactobacillus , Staphylococcus , Mailhella , Rikenella , Ligilactobacillus Abundance comparison results; Figure 12 for Lactobacillus , Staphylococcus , Mailhella , Rikenella , Ligilactobacillus Results of correlation analysis with mouse reproductive performance (A) and inflammatory factors (B). Detailed Implementation

[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0017] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0019] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0022] Example 1 The preparation process of *Smilax glabra* extract is as follows: Freshly harvested *Smilax glabra* roots are placed in a flowing pool of clean water to remove surface mud and impurities. After washing, they are fed into a fully automatic high-speed slicer to process into thin slices of approximately 5 mm thickness. The slices are then evenly spread in an oven. The drying temperature is set at 55-65℃ until completely dry. The completely dried *Smilax glabra* chips are then pulverized in a high-efficiency cyclone pulverizer. The resulting fine powder is initially collected by a built-in separator under negative pressure airflow. Subsequently, to ensure uniform powder particle size, coarse powder is screened through an 80-mesh standard sieve to remove coarse fibrous particles and substandard products, ultimately yielding a uniformly colored and fine *Smilax glabra* powder product.

[0023] The obtained *Achyranthes bidentata* powder was fully scanned using chromatography-mass spectrometry (GC-MS), and the results are as follows: Figure 1 As shown in Table 1, further analysis of the components of *Niu Da Li* powder revealed that the main components are alkaloids and organic acids, such as betaine, trigonelline, and jasmonic acid. These components possess antioxidant and anti-inflammatory properties.

[0024] Table 1. List of potential compounds in *Smilax glabra* powder Example 2 This embodiment uses pregnant mice as experimental subjects. From the beginning to the end of pregnancy, mice were administered chlorpyrifos and *Achyranthes bidentata* powder (Example 1) via gavage. Their offspring were raised to pregnancy, and their reproductive performance was observed to verify the effect of *Achyranthes bidentata* on fetal growth restriction induced by chlorpyrifos. The specific experimental procedure is as follows: 1. Mouse grouping and treatment Forty-eight ICR mice were randomly divided into four groups of 12 mice each. Pregnant mice were evenly distributed according to their physical condition and received different treatments: a control group (CON), a chlorpyrifos group (CPF), a chlorpyrifos + *Euphorbia hirta* group (CNF), and a *Euphorbia hirta* group (N). The control group was administered corn oil, the chlorpyrifos group was administered corn oil plus 5 mg / kg chlorpyrifos, the chlorpyrifos + *Euphorbia hirta* group was administered 5 mg / kg chlorpyrifos, corn oil, and 120 mg / kg *Euphorbia hirta* powder, and the *Euphorbia hirta* group was administered corn oil plus 120 mg / kg *Euphorbia hirta* powder.

[0025] 2. Sample collection and testing methods Based on previous research on first-generation pregnant female mice, the offspring were raised to pregnancy. Six female mice in each group were kept until delivery, and the weight changes of each pup were recorded every other day. At weaning, six weaned female mice from each group were randomly selected for dissection, and the following samples were collected: feces, blood, maternal intestines, placenta, and fetus. Weaned pup weight, body length, body width, organ index, and uterine weight were recorded. After the pups reached adulthood, twelve female mice from each group were mated again with normal male mice. After this new mating, six female mice were randomly selected, and at 15.5 days of gestation, the following samples were collected: maternal feces, blood, intestines, placenta, and fetus. Organ index, uterine weight, and length were measured. The collected samples were frozen in liquid nitrogen for subsequent RNA and protein extraction.

[0026] Gene expression: 30 mg of tissue was placed in a 2.5 mL enzyme-free grinding tube, and 700 μL of lysis buffer was added. The mixture was incubated at room temperature for 5 min for lysis. After lysis, RNA was extracted according to the instructions provided by the EZB kit (EZB-RN001-plus, EZBioscience, Shanghai, China). The concentration and purity of the RNA were detected using a Nanodrop-2000, and cDNA was synthesized using a reverse transcription kit. The obtained cDNA was stored at -20°C for later use. β-actin was used as an internal control, and quantitative methods (2...) were employed. -ΔΔCt (Methods) The relative expression levels of target genes (TLR4, TNF-α, IL-6, IL-1β, IL-10, GLUT1, GLUT2, GLUT3, GLUT4, SLC2A5, SNAT2, SNAT3, SNAT4, SLC43A1, SLC7A8, CD36, FATP1, FATP4, FABP1, FABP3, β-actin) were determined (Livak and Schmittgen, 2001). The primers used are shown in Table 2.

[0027] Protein Expression: Preserved colon and placental tissues were collected and subjected to Western blotting. Total protein was extracted from the homogenized tissues, and the concentration and denaturation were determined. SDS-PAGE gel electrophoresis was performed based on molecular weight, and the protein was transferred to a PVDF membrane. The membrane was then blocked with 5% skim milk at room temperature for 2 hours. After washing four times, the membrane was incubated overnight at 4°C with primary antibody. After washing four times with Tween-20 buffered saline, the membrane was incubated with secondary antibody (511203, ZenBio, Chengdu, China) at room temperature for 1.5 hours. The target bands were then visualized using an Image Quant LAS 4000 mini system and an enhanced chemiluminescence kit (P1020, Applygen, Beijing, China). Finally, protein expression analysis was performed using ImageJ software.

[0028] Table 2 Primers for Real-Time Quantitative PCR Target Genes 3. Data processing and analysis ANOVA analysis was performed on the data using SPSS 26 (IBM Version 26.0, Armonk, NY, USA), and multiple comparisons were performed using the LSD test. Data are expressed as mean ± SEM. Alpha and beta diversity analyses were performed using the Kidd platform. Prism 9.5 was used for plotting. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0029] 4. Experimental Results Effect of Millettia speciosa Champ. on the reproductive performance of first-generation pregnant mice is as Figure 2 shown. After intragastric administration of Millettia speciosa Champ., the body weight of pregnant mice was weighed every day. No difference among the four groups was observed in early pregnancy. With the increase of gestational days, the body weight of pregnant mice in the chlorpyrifos group showed a significant decreasing trend compared with the other three groups, and the addition of Millettia speciosa Champ. could alleviate the weight loss of pregnant mice induced by chlorpyrifos ( Figure 2 A). On day 15.5 of gestation, the number of litter size, fetal weight, fetal length and fetal width in the chlorpyrifos group were significantly lower than those in the control group, while the Millettia speciosa Champ. treatment group significantly increased the litter size, fetal weight and fetal length, and also showed an increasing trend in fetal width ( Figure 2 B and C). In addition, the litter size, neonatal weight, body length and body width of the chlorpyrifos group in the neonatal period were significantly lower than those of the control group, while Millettia speciosa Champ. could significantly increase the litter size and fetal body length, and also showed an increasing trend in neonatal weight and body width ( Figure 2 D).

[0030] Effect of Millettia speciosa Champ. on the reproductive performance of offspring pregnant mice is as Figure 3 shown. First, a difference in body weight among the four groups was observed in early pregnancy. The body weight of pregnant mice in the chlorpyrifos group showed a significant decreasing trend compared with the other three groups, and the addition of Millettia speciosa Champ. could alleviate the weight loss of pregnant mice induced by chlorpyrifos ( Figure 3 A). On day 15.5 of gestation, the number of litter size, fetal weight, fetal length and placental weight in the chlorpyrifos group were significantly lower than those in the control group, while the Millettia speciosa Champ. treatment group significantly increased the litter size, fetal weight, fetal length and placental weight, and also showed an increasing trend in body width ( Figure 3 B and C). In addition, the litter size, neonatal weight and body length of the chlorpyrifos group in the neonatal period were significantly lower than those of the control group, while Millettia speciosa Champ. could significantly increase the litter size and fetal body length, and also showed an increasing trend in neonatal body width ( Figure 3 D).

[0031] Effect of Millettia speciosa Champ. on the intestinal barrier and antioxidant capacity of offspring pregnant mice is as Figure 4 shown. Compared with the other three groups, the total intestinal length and colon length of the chlorpyrifos group were significantly reduced ( Figure 4 A). The relative protein expression levels of ZO-1, Claudin-1 and Occludin in the chlorpyrifos group were significantly lower than those in the control group, and this change could be reversed in the Millettia speciosa Champ. treatment group ( Figure 4 B). Meanwhile, HO-1 and phosphorylated Nrf2 (P-Nrf2) in the chlorpyrifos group were significantly lower than those in the control group, and Millettia speciosa Champ. exerted a good alleviating effect ( Figure 4 C).

[0032] Effect of Millettia speciosa Champ. on intestinal inflammation in offspring pregnant mice is as Figure 5As shown. The chlorpyrifos group had significantly higher levels of P-NF-κB / NF-κB, P-ERK / ERK, P-JNK / JNK, and P-P38 / P38 than the control group. The addition of *Achyranthes bidentata* significantly reduced phosphorylated NF-κB and JNK. Figure 5 (A). Correspondingly, the inflammatory genes TLR4, IL-1β, TNF-α, and IL-6 in the chlorpyrifos group were significantly higher than those in the control group, while the anti-inflammatory gene IL-10 was significantly lower in the chlorpyrifos group. Meanwhile, *Smilax glabra* significantly reduced TLR4 and significantly increased IL-10. Figure 5 (B). These results indicate that chlorpyrifos causes intestinal inflammation, and the addition of *Smilax glabra* significantly reduces intestinal inflammation.

[0033] Effects of *Smilax glabra* on placental barrier, angiogenesis, and nutrient transport in offspring pregnant rats, such as Figure 6 As shown. The placenta is the link between the mother and the developing fetus, promoting nutrient and gas exchange, waste removal, and hormone production, which are crucial for optimal fetal growth and development in utero. In the chlorpyrifos group, the barrier proteins ZO-1, Claudin-1, and Occludin were significantly reduced compared to the control group. The treatment group with *Niu Dali* significantly increased the expression level of Occludin and also significantly increased the expression levels of ZO-1 and Claudin-1. Figure 6 (A). VEGF-A and P-VEGFR2, which are associated with placental development, were lowest in the chlorpyrifos group, while the addition of *Achyranthes bidentata* significantly increased VEGF-A and phosphorylated VEGFR2, indicating that *Achyranthes bidentata* enhances placental development (A). Figure 6 (B). Furthermore, the chlorpyrifos group had the lowest levels of placental glucose transport, amino acid transport, and fatty acid transport-related genes compared to the other three groups, indicating that *Smilax china* can significantly enhance placental nutrient transport (…). Figure 6 (C).

[0034] Effects of *Smilax glabra* on placental inflammation in offspring pregnant mice, such as Figure 7 As shown. The chlorpyrifos group showed significantly higher levels of P-P65 / P65, P-ERK / ERK, P-JNK / JNK, and P-P38 / P38 than the control group. The addition of *Achyranthes bidentata* significantly reduced phosphorylated P65, ERK, JNK, and P38. Figure 7 (A). Correspondingly, the inflammatory genes TLR4, IL-1β, TNF-α, and IL-6 in the chlorpyrifos group were significantly higher than those in the control group, while the anti-inflammatory gene IL-10 was significantly lower in the chlorpyrifos group. Meanwhile, *Smilax glabra* significantly reduced TLR4 and IL-6 and significantly increased IL-10. Figure 7 B).

[0035] The effects of *Smilax glabra* on the gut microbiota of offspring pregnant mice, such as Figures 8-12As shown in the figure. The VENN plot was used to illustrate the differences in microbial abundance among the four groups, with 136, 120, 132, and 80 unique OUTs in the four groups, respectively. Figure 8 The Chao and ace indices were used to assess α-diversity of the microorganisms, and there were no significant differences in α-diversity of the microorganisms among the four groups. Figure 8 (B). The four groups of microorganisms differed in composition at both the phylum and genus levels; the chlorpyrifos group showed a decrease in beneficial bacteria. Lactobacillus , Ligilactobacillus and Rikenella Increased harmful bacteria Mailhella and Staphylococcus Niu Dali can reverse this change ( Figures 9-11 Next, a correlation analysis was performed on the gut microbiota of offspring pregnant mice and their reproductive performance and inflammatory markers. The results showed that beneficial bacteria ( Lactobacillus , Ligilactobacillus and Rikenella It was negatively correlated with pro-inflammatory genes (IL-1β, TNF-α, IL-6, and TLR4) and positively correlated with anti-inflammatory factors (IL-10), placental weight, fetal weight, and placental efficiency. Harmful bacteria ( Staphylococcus and Mailhella It is positively correlated with pro-inflammatory genes (IL-1β, TNF-α, IL-6, and TLR4) and negatively correlated with anti-inflammatory factors (IL-10), fetal weight, and placental efficiency. Figure 12 ).

[0036] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of *Smilax glabra* in the preparation of drugs to alleviate the toxicity of chlorpyrifos, characterized in that... The toxicity of chlorpyrifos refers to the pregnancy-related decline in reproductive performance, intrauterine growth restriction in fetuses, and abnormal intestinal development in offspring caused by chlorpyrifos.

2. The application of *Smilax glabra* in the preparation of drugs to improve reproductive performance decline caused by chlorpyrifos, characterized in that... The reproductive performance includes maternal weight, litter size, fetal weight, fetal length, fetal width, and placental weight.

3. The application according to claim 2, characterized in that, The drug has the effect of increasing litter size, fetal weight, fetal length, fetal width, and placental weight, as well as increasing the litter size, fetal weight, fetal length, fetal width, and placental weight of offspring.

4. The application of *Niu Dali* in the preparation of drugs for treating intrauterine growth restriction in fetuses caused by chlorpyrifos, characterized in that... The drug treats intrauterine growth restriction by promoting placental development, relieving placental inflammation, and promoting placental angiogenesis and nutrient transport.

5. The application of *Smilax glabra* in the preparation of drugs for treating intestinal developmental abnormalities in offspring caused by chlorpyrifos, characterized in that... The intestinal developmental abnormalities refer to impaired intestinal barrier, intestinal inflammation, and shortened length of the intestine and colon.

6. The application of *Smilax glabra* in the preparation of feed additives to improve the reproductive performance of pregnant animals induced by chlorpyrifos, characterized in that... The reproductive performance includes maternal weight, litter size, fetal weight, fetal length, fetal width, and placental weight.

7. The application of *Smilax glabra* in the preparation of feed additives to improve intrauterine growth restriction in fetuses caused by chlorpyrifos, characterized in that... The feed additive improves intrauterine growth restriction by promoting placental development, alleviating placental inflammation, and promoting placental angiogenesis and nutrient transport.

8. The application of *Smilax glabra* in the preparation of feed additives to improve intestinal developmental abnormalities in offspring caused by chlorpyrifos, characterized in that... The intestinal developmental abnormalities refer to impaired intestinal barrier, intestinal inflammation, and shortened length of the intestine and colon.

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