Ameliorative effects of Wikstroemia trichotoma 95% EtOH extract on a mouse model of DNCB-induced atopic dermatitis.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Wikstroemia has been extensively utilized in traditional Chinese medicine (TCM) for the management of conditions such as coughs, edema, arthritis, and bronchitis. Studies have indicated that the crude extracts of Wikstroemia exhibit anti-inflammatory, anti-allergy, anti-aging, skin psoriasis, anti-cancer, and antiviral properties. In addition, these extracts are known to contain bioactive substances, including flavonoids, coumarins, and lignans. However, few studies have investigated the anti-inflammatory or anti-allergic activities of Wikstroemia trichotoma (Thunb.) Makino against atopic dermatitis (AD). AIM OF THE STUDY: The study aimed to explore the potential of a 95% ethanol extract of W. trichotoma (WTE) on the dysfunction of skin barrier and immune system, which are primary symptoms of AD, in 2,4-dinitrochlorobenzene (DNCB)-induced SKH-1 hairless mice and phorbol 12-myristate 13-acetate (PMA)/ionomycin or immunoglobulin E (IgE) + 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) stimulated rat basophilic leukemia cell line (RBL-2H3). Furthermore, we sought to identify the chemical contents of WTE using high-performance liquid chromatography equipped with a photodiode array detector (HPLC-PDA). MATERIALS AND METHODS: An in vitro study was conducted using RBL-2H3 cells stimulated with PMA/ionomycin or IgE + DNP-BSA to assess the inhibitory effects of WTE on mast cell degranulation and interleukin-4 (IL-4) mRNA expression levels. For the in vivo study, AD was induced in SKH-1 hairless mice by applying 1% DNCB to the dorsal skin daily for 7 days. Subsequently, 0.1% DNCB solution was applied on alternate days, and mice were orally administered WTE (at 30 or 100 mg/kg/day) dissolved in 0.5% carboxymethyl cellulose (CMC) daily for 2 weeks. Transepidermal water loss (TEWL), skin hydration, skin pH, and total serum IgE levels were measured. RESULTS: In DNCB-stimulated SKH-1 hairless mice, WTE administration significantly improved AD symptoms and ameliorated dorsal skin inflammation. Oral administration of WTE led to a significant decrease in skin thickness, infiltration of mast cells, and level of total serum IgE, thus restoring skin barrier function in the DNCB-induced skin lesions. In addition, WTE inhibited ß-hexosaminidase release and reduced IL-4 mRNA levels in RBL-2H3 cells. Chemical profile analysis of WTE confirmed the presence of three phenolic compounds, viz. chlorogenic acid, miconioside B, and matteucinol-7-O-ß-apiofuranosyl (1 â†’ 6)-ß-glucopyranoside. CONCLUSIONS: WTE ameliorates AD symptoms by modulating in the skin barrier and immune system dysfunction. This suggests that W. trichotoma extract may offer therapeutic benefits for managing AD.

Chemometric approach for an application of Atlantic salmons (Oncorhynchus keta) by-product for potential food sources.

This study identified the aroma profile of salmon by-product for high utilization of by-products, including hydrolysates of head, frame, and skin were treated with reducing sugars and thermal processing. Electronic nose (E-nose) and gas chromatography-mass spectrometry (GC-MS) coupled with gas chromatography-olfactometry (GC-O) were used to analyzed the aroma profile. A total of 140 and 90 volatile compounds were detected through E-nose and GC-MS respectively, and the main volatile compounds were aldehydes. A total of 23 odor active compounds were recognized using GC-O, and 3-methyl-butanal, heptanal, benzaldehyde, octanal, furfural, and methoxy-phenyl-oxime were identified as the aroma of salmon. Using multivariate analysis, the pattern between the pretreated samples and aroma profiles was confirmed, and there were clear separations among the samples. The results of this study provide the aroma profile of salmon by-products and are expected salmon by-products to be used as a potential food source.

Tumor-specific cytolysis by peptide-conjugated echogenic polymer micelles.

Interest in multifunctional polymer nanoparticles for targeted delivery of anti-cancer drugs has grown significantly in recent years. In this study, tumor-targeting echogenic polymer micelles were prepared from poly(ethylene glycol) methyl ether-alkyl carbonate (mPEG-AC) derivatives, and their potential in cancer therapy was assessed. Various mPEG derivatives with carbonate linkages were synthesized via an alkyl halide reaction between mPEG and alkyl chloroformate. Micelle formation using polymer amphiphiles in aqueous media and the subsequent carbon dioxide (CO2) gas generation from the micelles was confirmed. Their ability to target neuroblastoma was substantially enhanced by incorporating the rabies virus glycoprotein (RVG) peptide. RVG-modified gas-generating micelles significantly inhibited tumor growth in a tumor-bearing mouse model owing to CO2 gas generation within tumor cells and resultant cytolytic effects, showing minimal side effects. The development of multifunctional polymer micelles may offer a promising therapeutic approach for various diseases, including cancer.

Chemical Constituents of Halophyte Suaeda glauca and Their Therapeutic Potential for Hair Loss.

Suaeda glauca, a halophyte in the Amaranthaceae family, exhibits remarkable resilience to high salt and alkali stresses despite the absence of salt glands or vesicles in its leaves. While there is growing pharmacological interest in S. glauca, research on its secondary metabolites remains limited. In this study, chemical constituents of the aerial parts of S. glauca were identified using 1D- and 2D-NMR experiments, and its biological activity concerning hair loss was newly reported. Eight compounds, including alkaloids (1~3), flavonoids (4~6), and phenolics (7 and 8), were isolated. The compounds, except the flavonoids, were isolated for the first time from S. glauca. In the HPLC chromatogram, quercetin-3-O-ß-d-glucoside, kaempferol-3-O-ß-d-glucoside, and kaempferol were identified as major constituents in the extract of S. glauca. Additionally, the therapeutic potential of the extract of S. glauca and the isolated compounds 1~8 on the expressions of VEGF and IGF-1, as well as the regulation of Wnt/ß-catenin signaling, were evaluated in human follicle dermal papilla cells (HFDPCs) and human umbilical vein endothelial cells (HUVECs). Among the eight compounds, compound 4 was the most potent in terms of increasing the expression of VEGF and IGF-1 and the regulation of Wnt/ß-catenin. These findings suggest that S. glauca extract and its compounds are potential new candidates for preventing or treating hair loss.

Two New Components from an Association of Marine Sponges Poecillastra sp. and Jaspis sp. and Their Inhibitory Effects on Biomarkers for Benign Prostatic Hyperplasia.

Benign prostatic hyperplasia (BPH), characterized by the enlargement of the prostate gland and subsequent lower urinary tract symptoms, poses a significant health concern for aging men with increasing prevalence. Extensive efforts encompassing in vitro and in vivo models are underway to identify novel and effective agents for the management and treatment of BPH. Research endeavors are primarily channeled toward assessing the potential of compounds to inhibit cell proliferation, curb inflammation, and display anti-androgenic activity. Notably, through screening aimed at inhibiting 5-alpha reductase type 2 (5αR2) in human prostatic cells, two acyl compounds (1 and 2) were isolated from a bioactive fraction sourced from an association of marine sponges Poecillastra sp. and Jaspis sp. The complete structure of 1 was determined as (Z)-dec-3-enony (2S, 3S)-capreomycidine, ascertained by JBCA and ECD comparison. While the absolute configurations of 2 remained unassigned, it was identified as a linkage of a 2, 7S*-dihydoxy-9R*-methyloctadecanoyl group with the 2-amino position of a tramiprosate moiety referred to as homotaurine. Evaluation of both compounds encompassed the assessment of their inhibitory effects on key biomarkers (5αR2, AR, PSA, and PCNA) associated with BPH in testosterone propionate (TP)-activated LNCap and RWPE-1 cells.

Evaluation of the Chemosensoric Properties of Commercially Available Dog Foods Using Electronic Sensors and GC-MS/O Analysis.

Pet owners think of their animals as part of their family, which further promotes the growth of the pet food market, encouraging pet owners to select nutritious, palatable, and high-quality foods for pets. Therefore, the evaluation of taste and volatile compounds in pet foods is essential to improve palatability. In this study, the sensory characteristics of taste and odor compounds in 10 commercially available dry dog foods were investigated using electronic tongue (E-tongue), electronic nose (E-nose), gas chromatography-mass spectrometry (GC-MS), and gas chromatography-olfactometry (GC-O). Dry dog foods were separated based on the sensory properties of taste and volatile compounds through the multivariate analysis of integrated results of the E-tongue and E-nose. A total of 67 odor active compounds were detected through GC-MS and GC-O, and octanal, nonanal, 2-pentyl furan, heptanal, and benzaldehyde were identified as key odor compounds which may have positive effects on food intake. The multivariate analysis was used to classify samples based on key odor compounds. Volatile compounds responsible for aroma properties of samples were evaluated using GC-O and multivariate analysis in this present study for the first time. These results are expected to provide fundamental data for sensory evaluation in producing new dog foods with improved palatability.

Physiological and biochemical responses of Limonium tetragonum to NaCl concentrations in hydroponic solution.

Introduction: Limonium (L.) tetragonum (Thunb.) A. A. Bullock, a halophyte that grows all over the southwest coast of Korea, is a medicinal plant with various pharmacological effects. The salt defense mechanism stimulates the biosynthesis of various secondary metabolites and improves functional substances. In this study, we investigated the optimal NaCl concentration for the growth and enhancement of secondary metabolites in hydroponically grown L. tetragonum. Methods: The seedlings grown for 3 weeks in a hydroponic cultivation system were treated with 0-, 25-, 50-, 75-, and 100-mM NaCl in Hoagland's nutrient solution for 8 weeks. No significant effect on the growth and chlorophyll fluorescence was observed for the NaCl concentrations below 100-mM. Results and discussions: The increase in the NaCl concentration resulted in the decrease in the water potential of the L. tetragonum leaves. The Na+ content accumulated in the aerial part increased rapidly and the content of K+, which acts as an antagonist, decreased with the increase in NaCl concentrations in hydroponics. The total amino acid content of L. tetragonum decreased compared to the 0-mM NaCl, and most of the amino acid content decreased as the NaCl concentration increased. In contrast, the content of urea, proline (Pro), ß-alanine, ornithine, and arginine was increased with an increase in NaCl concentration. The Pro content at 100-mM NaCl accounted for 60% of the total amino acids and was found to be a major osmoregulator as an important component of the salt defense mechanisms. The top five compounds identified in the L. tetragonum were classified as flavonoids while the flavanone compound was detected only in the NaCl treatments. A total of four myricetin glycosides were increased in comparison to the 0-mM NaCl. Among the differentially expressed genes, a significantly large change in Gene ontology was seen in the circadian rhythm. NaCl treatment enhanced the flavonoid-based substances of L. tetragonum. The optimum NaCl concentration for the enhancement of secondary metabolites of the L. tetragonum in the vertical farm-hydroponic cultivation system was 75-mM NaCl.

Sesquiterpene Lactones with Anti-Inflammatory Activity from the Halophyte Sonchus brachyotus DC.

There were five sesquiterpene lactones, belonging to the eudesmanolide class, isolated from the halophyte Sonchus brachyotus DC. The structures of the compounds were determined using spectroscopic methods, including 1D and 2D NMR spectra, MS data, and optical rotation values. Compounds 4 and 5 were characterized by the position of p-hydroxyphenylacetyl group in the sugar moiety. In the evaluation of anti-inflammatory effects on LPS-activated RAW264.7 macrophages, compound 1, 5α,6ßH-eudesma-3,11(13)-dien-12,6α-olide, potently suppressed the expression of iNOS and COS-2, as well as the production of TNF-α, IL-6, and IL-10. Treatment of 1 regulates the Nrf2/HO-1 pathway.

Adipocytolytic Polymer Nanoparticles for Localized Fat Reduction.

The demand for body fat reduction is increasing. However, conventional lipolytic approaches fail to control adipose tissue reduction and cause severe side effects in adjacent nonadipose tissues. A strategy to specifically reduce subcutaneous fat using adipocytolytic polymer nanoparticles in a minimally invasive manner is reported here. The polymer nanoparticles are designed to generate carbon dioxide gas when selectively absorbed by adipocytes. The carbon dioxide gas generated within late endosomes/lysosomes induces adipocytolysis, thereby reducing the number of cells. Localized injection of the adipocytolytic nanoparticles substantially reduces subcutaneous fat in a high-fat diet-induced obese mouse model, without significant changes in hematological or serum biochemical parameters. The adipocytolytic efficacy of the nanoparticles is also evaluated in a porcine model. This strategy addresses the need to develop safe and effective adipocytolytic agents using functional polymer nanoparticles.

Ovataline: A Polyketide Isolated from the Benthic Dinoflagellate Ostreopsis cf. ovata with 5α-Reductase Inhibitory Activity in RWPE-1 Prostatic Cells.

Ovataline (1), which is a polar metabolite containing a hexahydroquinoline moiety, was isolated from cultures of the marine dinoflagellate Ostreopsis cf. ovata. 1 was characterized as a zwitterionic compound with hexahydroquinoline and tetrahydropyran rings. The configurations of the chiral centers in 1 were established using ROESY correlations, J-based configurational and Mosher reaction analyses, and density functional theory calculations. 1 exhibited a 78% (1 µM) inhibition of type II 5α-reductase in testosterone propionate-induced RWPE-1 human prostatic cells.

Voratins A-C: Pyridinium Alkaloids from the Marine Dinoflagellate Effrenium voratum with Inhibitory Effects on Biomarkers for Benign Prostatic Hyperplasia.

Three voratin compounds (1-3) were isolated from the symbiotic marine dinoflagellate Effrenium voratum. The planar structures of 1-3 were determined by 1D and 2D NMR spectroscopy and HRESIMS, and the relative and absolute configurations were established using ROESY correlations, Mosher's method, and quantum calculations. All of the compounds are zwitterionic and contain a dihydroindolizinium ring and a spiroketal moiety. Compounds 1-3 were found to exhibit therapeutic effects against benign prostatic hyperplasia (BPH), as evaluated using testosterone propionate-treated LNCap and RWPE-1 human prostate cells. This excellent activity suggests that 1-3 are promising for the development of BPH treatments.

The efficacy of a 2,4-diaminoquinazoline compound as an intranasal vaccine adjuvant to protect against influenza A virus infection in vivo.

Adjuvants are substances added to vaccines to enhance antigen-specific immune responses or to protect antigens from rapid elimination. As pattern recognition receptors, Toll-like receptors 7 (TLR7) and 8 (TLR8) activate the innate immune system by sensing endosomal single-stranded RNA of RNA viruses. Here, we investigated if a 2,4-diaminoquinazoline-based TLR7/8 agonist, (S)-3-((2-amino-8-fluoroquinazolin-4-yl)amino)hexan-1-ol (named compound 31), could be used as an adjuvant to enhance the serological and mucosal immunity of an inactivated influenza A virus vaccine. The compound induced the production of proinflammatory cytokines in macrophages. In a dose-response analysis, intranasal administration of 1 µg compound 31 together with an inactivated vaccine (0.5 µg) to mice not only enhanced virus-specific IgG and IgA production but also neutralized influenza A virus with statistical significance. Notably, in a virus-challenge model, the combination of the vaccine and compound 31 alleviated viral infection-mediated loss of body weight and increased survival rates by 40% compared with vaccine only-treated mice. We suggest that compound 31 is a promising lead compound for developing mucosal vaccine adjuvants to protect against respiratory RNA viruses such as influenza viruses and potentially coronaviruses.

Discovery and synthesis of 1,2,4-oxadiazole derivatives as novel inhibitors of Zika, dengue, Japanese encephalitis, and classical swine fever virus infections.

Zika virus (ZIKV), an arbovirus of the Flaviviridae family, has emerged as a significant public health concern owing to its association with congenital abnormalities and severe neurological sequelae. Thus, there is an urgent need to develop effective therapeutic approaches to efficiently treat ZIKV infections. This study used phenotypic screening to identify a series of 1,2,4-oxadiazole derivatives that possess antiviral activity against ZIKV infection. Subsequently, 28 new derivatives were designed, synthesized, and evaluated for this purpose. Among these compounds, 4-(5-phenyl-1,2,4-oxadiazol-3-yl)-N-(pyridin-3-ylmethyl)aniline (5d) had potent antiviral activity against ZIKV infections. Furthermore, a structure-activity relationship analysis indicated that a benzyl substitution on the aniline nitrogen of this compound improved potency while augmenting its drug-like properties. In addition, 5d exhibited antiviral activity against various viruses of Flaviviridae family of worldwide public health importance, such as dengue, Japanese encephalitis and classical swine fever viruses, indicating its potential as a lead compound for generating 1,2,4-oxadiazole derivatives with broad-spectrum anti-flaviviral properties.

Ras-related proteins (Rab) play significant roles in sperm motility and capacitation status.

Rab proteins are widely known for their involvement in establishing Golgi apparatus and controlling Golgi trafficking in eukaryotic cells. Specifically, Rab proteins play significant roles in acrosome formation and exocytosis. Furthermore, mechanisms involved in the regulation of Rab proteins during capacitation have been identified. However, there has been no direct evaluation to assess the correlation between Rab proteins and sperm function. Consequently, this study was designed to analyze the correlation between Rab proteins and sperm functions. Individually, we analyzed the sperm motility patterns, motion kinematics, capacitation status, and Rab protein expression levels of sperm samples from 31 boars before and after capacitation. As a result, we discovered that Rab3A, Rab5, Rab11, Rab14, and Rab27A correlated with various sperm motility patterns, motion kinematics before capacitation. Rab3A, Rab5, Rab11, Rab14, and Rab34 correlated with various sperm motility patterns, motion kinematics after capacitation. Moreover, Rab4 and Rab34 were associated with capacitation status before capacitation, and Rab3A, 25, and 27A correlated with capacitation status after capacitation. This is the first study to analyze the correlation between Rab proteins and sperm functions. Collectively, our results indicate that specific sperm motility and kinematics, as well as the structural condition of the sperm head and capacitation status, regulate individual Rab protein. Therefore, we expect that the current findings will be used to identify the etiology of idiopathic male infertility patients and to diagnose male fertility and that Rab proteins will be employed as biomarkers to predict and analyze male fertility.

Adverse postnatal developmental effects in offspring from humidifier disinfectant biocide inhaled pregnant rats.

Inhalation exposure to polyhexamethylene guanidine phosphate (PHMG-P), one of the primary biocides used in humidifier disinfectants, caused a fatal pulmonary disease in Korea. Pregnant women were also exposed to PHMG-P, and subsequent studies showed that PHMG-P inhalation during pregnancy adversely affects their health and embryo-fetal development. However, the postnatal developmental effects after birth on prenatally PHMG-P-exposed offspring have not yet been investigated. Therefore, in this study, we aimed to examine the postnatal development of prenatally PHMG-P-exposed offspring. Pregnant rats (22 or 24 females per group) were exposed to PHMG-P during pregnancy in a whole-body inhalation chamber at the target concentrations of 0, 0.14, 1.60, and 3.20 mg/m3. After parturition, the prenatally exposed offspring were transferred to non-exposed surrogate mothers to minimize the secondary effects of severe maternal toxicities. Postnatal development of offspring was then examined with a modified extended one-generation reproductive toxicity study design. At 3.20 mg/m3 PHMG-P, increased perinatal death rates and decreased viability index (postnatal survival of offspring between birth and postnatal day 4) were observed. In addition, F1 offspring had lower body weight at birth that persisted throughout the study. PHMG-P-exposed pregnant rats also had severe systemic toxicities and increased gestation period. At 1.60 mg/m3 PHMG-P, a decreased viability index was also observed with systemic toxicities of PHMG-P-exposed pregnant rats. These results indicate that prenatal PHMG-P exposure adversely affects the offspring's future health and could be used for human risk assessment.

In Vitro Cellular Uptake and Transfection of Oligoarginine-Conjugated Glycol Chitosan/siRNA Nanoparticles.

Chitosan and its derivatives have been extensively utilized in gene delivery applications because of their low toxicity and positively charged characteristics. However, their low solubility under physiological conditions often limits their application. Glycol chitosan (GC) is a derivative of chitosan that exhibits excellent solubility in physiological buffer solutions. However, it lacks the positive characteristics of a gene carrier. Thus, we hypothesized that the introduction of oligoarginine peptide to GC could improve the formation of complexes with siRNA, resulting in enhanced uptake by cells and increased transfection efficiency in vitro. A peptide with nine arginine residues and 10 glycine units (R9G10) was successfully conjugated to GC, which was confirmed by infrared spectroscopy, 1H NMR spectroscopy, and elemental analysis. The physicochemical characteristics of R9G10-GC/siRNA complexes were also investigated. The size and surface charge of the R9G10-GC/siRNA nanoparticles depended on the amount of R9G10 coupled to the GC. In addition, the R9G10-GC/siRNA nanoparticles showed improved uptake in HeLa cells and enhanced in vitro transfection efficiency while maintaining low cytotoxicity determined by the MTT assay. Oligoarginine-modified glycol chitosan may be useful as a potential gene carrier in many therapeutic applications.

Preclinical assessment of thrombin-preconditioned human Wharton's jelly-derived mesenchymal stem cells for neonatal hypoxic-ischaemic brain injury.

Hypoxic-ischaemic encephalopathy (HIE) is a type of brain injury affecting approximately 1 million newborn babies per year worldwide, the only treatment for which is therapeutic hypothermia. Thrombin-preconditioned mesenchymal stem cells (MSCs) exert neuroprotective effects by enriching cargo contents and boosting exosome biogenesis, thus showing promise as a new therapeutic strategy for HIE. This study was conducted to evaluate the tissue distribution and potential toxicity of thrombin-preconditioned human Wharton's jelly-derived mesenchymal stem cells (th-hWJMSCs) in animal models before the initiation of clinical trials. We investigated the biodistribution, tumorigenicity and general toxicity of th-hWJMSCs. MSCs were administered the maximum feasible dose (1 × 105 cells/10 µL/head) once, or at lower doses into the cerebral ventricle. To support the clinical use of th-hWJMSCs for treating brain injury, preclinical safety studies were conducted in newborn Sprague-Dawley rats and BALB/c nude mice. In addition, growth parameters were evaluated to assess the impact of th-hWJMSCs on the growth of newborn babies. Our results suggest that th-hWJMSCs are non-toxic and non-tumorigenic in rodent models, survive for up to 7 days in the brain and hold potential for HIE therapy.

Densazalin, a New Cytotoxic Diazatricyclic Alkaloid from the Marine Sponge Haliclona densaspicula.

Densazalin, a polycyclic alkaloid, was isolated from the marine sponge Haliclona densaspicula collected in Korea. The complete structure of the compound was determined by spectroscopic methods, including 1D and 2D nuclear magnetic resonance techniques, high-resolution mass spectrometry, and comparison of the calculated and measured electronic circular dichroism spectra. Densazalin possesses a unique 5,11-diazatricyclo[7.3.1.02,7]tridecan-2,4,6-triene moiety, which is connected by two linear carbon chains. This compound was derived from the biogenetic precursor bis-1,3-dialkylpyridnium. Densazalin exhibited cytotoxic activity on two human tumor cell lines (AGS and HepG2) in the Cell Counting Kit-8 (CCK-8) bioassay, with IC50 values ranging from 15.5 to 18.4 µM.

Stereochemical Determination of Fistularins Isolated from the Marine Sponge Ecionemia acervus and Their Regulatory Effect on Intestinal Inflammation.

By activity-guided fractionation based on inhibition of nitric oxide (NO) and prostaglandin E2 (PGE2), six fistularin compounds (1-6) were isolated from the marine sponge Ecionemia acervus (order Astrophorida). Based on stereochemical structure determination using Mosher's method, fistularin-3 was assigned as a new stereoisomer. On the basis of the stereochemistry of fistularin-3, the stereochemical homogeneity of all six compounds was established by comparing carbon and proton chemical shifts. For fistularin-1 (1) and -2 (2), quantum calculations were performed to confirm their stereochemistry. In a co-culture system of human epithelial Caco-2 cells and THP-1 macrophages, all six isolated compounds showed potent anti-inflammatory activities. These bioactive fistularins inhibited the production of NO, PGE2, TNF-α, IL-1ß, and IL-6 induced by lipopolysaccharide and interferon gamma. Inducible NO synthase and cyclooxygenase-2 expression and MAPK phosphorylation were downregulated in response to the inhibition of NF-κB nuclear translocation. Among the compounds tested, fistularin-1 (1) and 19-deoxyfistularin-3 (4) showed the highest activity. These findings suggest the potential use of the marine sponge E. acervus and its metabolites as pharmaceuticals for the treatment of inflammation-related diseases including inflammatory bowel disease.

Serial blood sampling effects in rat embryo-fetal development studies for toxicokinetics.

Serial blood sampling for toxicokinetics is generally conducted in regulatory embryo-fetal development (EFD) studies in rats. EFD studies are designed to detect the potential adverse effects of pharmaceuticals on pregnant females and their fetuses; this information is useful for understanding the relationships between systemic exposure levels and toxicity profiles. However, additional satellite pregnant females are needed for toxicokinetics because comprehensive information regarding the potential impact of serial blood sampling on pregnant females is scarce. Here, in this study, we investigated the potential impact of serial blood sampling in pregnant female rats using a typical EFD study design. Additionally, we investigated the additional endpoints (clinical pathology, organ weights, and histopathology) that were deemed likely to be sensitive to blood sampling. Results indicated that serial blood sampling in pregnant females induced physiological adaptive changes and did not affect the general endpoints in EFD studies. Nevertheless, inclusion of satellite groups in EFD studies may be a more prudent approach considering the physiological changes in pregnant females and potential off-target effects of candidate pharmaceuticals. These results provide background information on the impact of serial blood sampling in pregnant females and will be useful to design the regulatory EFD studies.