Screening of growth inhibitors for epithelial-mesenchymal transition-induced cells by TGF-ß from plant-based sources identified the active compound hydroxychavicol from Piper bitle.

Epithelial-mesenchymal transition (EMT) has recently been associated with cancer invasion, metastasis, and resistance. In our previous study, we discovered nanaomycin K, a natural growth inhibitor for EMT-induced Madin Darby canine kidney (MDCK) cells, from the cultured broth of actinomycetes. However, the screening method was undeveloped, because the activity of nanaomycin K was discovered accidentally. In this study, we established a screening method by analyzing the characteristics of nanaomycin K in MDCK cells. Nanaomycin K showed the characteristic growth inhibitory activity on MDCK cells cultured under four conditions: medium containing dimethyl sulfoxide, SB431542, TGF-ß, and a mixture of SB431542 and TGF-ß. The activity was stronger in TGF-ß-treated cells than in DMSO-treated cells. In the mixture of SB431542 and TGF-ß-treated cells, the activity of nanaomycin K was suppressed. The anti-cancer agents, mitomycin C, cisplatin, and staurosporine, lacked the characteristics as that of nanaomycin K for these four treatment conditions. Since these four conditions distinguish between the effects of nanaomycin K and other anti-cancer agents in EMT-induced cells, the screening method was established. Among the 13,427 plant extracts tested, Piper betle leaf extract displayed growth inhibitory activity against EMT-induced cells. Through the purification of the extract via bio-guided fractionation, hydroxychavicol was isolated as an active compound. The cytotoxic activity of hydroxychavicol was stronger in EMT-induced MDCK cells than in control cells. However, its cytotoxic activity was suppressed in EMT-inhibited cells. Furthermore, hydroxychavicol exhibited same activity against SAS cells (human squamous cell carcinoma of the tongue). Thus, we have successfully established a screening method for growth inhibitors of EMT-induced cells and have discovered an inhibitor from plant-based sources.

Inhibition of G protein-coupled receptor 68 using homoharringtonine attenuates chronic kidney disease-associated cardiac impairment.

Chronic kidney disease (CKD) induces cardiac inflammation and fibrosis and reduces survival. We previously demonstrated that G protein-coupled receptor 68 (GPR68) promotes cardiac inflammation and fibrosis in mice with 5/6 nephrectomy (5/6Nx) and patients with CKD. However, no method of GPR68 inhibition has been found that has potential for therapeutic application. Here, we report that Cephalotaxus harringtonia var. nana extract and homoharringtonine ameliorate cardiac inflammation and fibrosis under CKD by suppressing GPR68 function. Reagents that inhibit the function of GPR68 were explored by high-throughput screening using a medicinal plant extract library (8,008 species), and we identified an extract from Cephalotaxus harringtonia var. nana as a GPR68 inhibitor that suppresses inflammatory cytokine production in a GPR68 expression-dependent manner. Consumption of the extract inhibited inflammatory cytokine expression and cardiac fibrosis and improved the decreased survival attributable to 5/6Nx. Additionally, homoharringtonine, a cephalotaxane compound characteristic of C. harringtonia, inhibited inflammatory cytokine production. Homoharringtonine administration in drinking water alleviated cardiac fibrosis and improved heart failure and survival in 5/6Nx mice. A previously unknown effect of C. harringtonia extract and homoharringtonine was revealed in which GPR68-dependent inflammation and cardiac dysfunction were suppressed. Utilizing these compounds could represent a new strategy for treating GPR68-associated diseases, including CKD.

Improved quantitative analysis of tenuifolin using hydrolytic continuous-flow system to build prediction models for its content based on near-infrared spectroscopy.

This study used two types of analyses and statistical calculations on powdered samples of Polygala root (PR) and Senega root (SR): (1) determination of saponin content by an independently developed quantitative analysis of tenuifolin content using a flow reactor, and (2) near-infrared spectroscopy (NIR) using crude drug powders as direct samples for metabolic profiling. Furthermore, a prediction model for tenuifolin content was developed and validated using multivariate analysis based on the results of (1) and (2). The goal of this study was to develop a rapid analytical method utilizing the saponin content and explore the possibility of quality control through a wide-area survey of crude drugs using NIR spectroscopy. Consequently, various parameters and appropriate wavelengths were examined in the regression analysis, and a model with a reasonable contribution rate and prediction accuracy was successfully developed. In this case, the wavenumber contributing to the model was consistent with that of tenuifolin, confirming that this model was based on saponin content. In this series of analyses, we have succeeded in developing a model that can quickly estimate saponin content without post-processing and have demonstrated a brief way to perform quality control of crude drugs in the clinical field and on the market.

Identification of an Oligostilbene, Vaticanol B, from Dryobalanops aromatica Leaves as an Antiviral Compound against the Hepatitis C Virus.

Chronic hepatitis C virus (HCV) infection can lead to liver cirrhosis and hepatocellular carcinoma. Although current medications using direct-acting antivirals (DAAs) are highly effective and well-tolerated for treating patients with chronic HCV, high prices and the existence of DAA-resistant variants hamper treatment. There is thus a need for easily accessible antivirals with different mechanisms of action. During the screening of Indonesian medicinal plants for anti-HCV activity, we found that a crude extract of Dryobalanops aromatica leaves possessed strong antiviral activity against HCV. Bioassay-guided purification identified an oligostilbene, vaticanol B, as an active compound responsible for the anti-HCV activity. Vaticanol B inhibited HCV infection in a dose-dependent manner with 50% effective and cytotoxic concentrations of 3.6 and 559.5 µg/mL, respectively (Selectivity Index: 155.4). A time-of-addition study revealed that the infectivity of HCV virions was largely lost upon vaticanol B pretreatment. Also, the addition of vaticanol B following viral entry slightly but significantly suppressed HCV replication and HCV protein expression in HCV-infected and a subgenomic HCV replicon cells. Thus, the results clearly demonstrated that vaticanol B acted mainly on the viral entry step, while acting weakly on the post-entry step as well. Furthermore, co-treatment of the HCV NS5A inhibitor daclatasvir with vaticanol B increased the anti-HCV effect. Collectively, the present study has identified a plant-derived oligostilbene, vaticanol B, as a novel anti-HCV compound.

Prenylated-coumarins from Gmelina arborea and evaluation for neurotrophic activity.

A MeOH extract of the stem of Gmelina arborea Roxb. ex Sm. (Lamiaceae) exhibited neurite outgrowth-promoting activity in NGF-mediated PC12 cells. Bioassay-guided fractionation resulted in the isolation of eight previously undescribed prenylated coumarin compounds along with nine known compounds. Structural elucidation of these compounds was accomplished by analysis of extensive spectroscopic data, comparison with the literature, and chemical reactions. It was the first time to find prenylated coumarin compounds from G. arborea. Among the isolated compounds, N-methylflindersine and artanin showed neurite outgrowth-promoting activity in NGF-mediated PC12 cells.

[Determination of 11 Cannabinoids in Cannabis sativa L. by Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-Q-TOF-MS)].

Eleven major cannabinoids from each subdivided tissue of drug-type and fiber-type cannabis plants were determined by means of a liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). The cannabinoids analyzed in this study were tetrahydrocannabinol acid (THCA), Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol acid (CBDA), cannabidiol (CBD), Δ8-tetrahydrocannabinol (Δ8-THC), cannabinol (CBN), cannabichromene (CBC), cannabidivarin (CBDV), cannabigerolic acid (CBGA), cannabigerol (CBG) and tetrahydrocannabivarin (THCV). As a result, THCA was detected in the bracts at 28.4 µg/mg, in the buds at 24.8 µg/mg, and in the leaves at 5.1 to 10.5 µg/mg in the drug-type cannabis plant. In addition, Δ9-THC, CBGA, CBN, CBG, CBC, and THCV were mainly detected in bracts, buds, and leaves. On the other hand, as for the fiber-type cannabis plant, CBDA was detected in the bracts at 27.5 µg/mg, in the buds at 10.6 µg/mg, and in the leaves at 1.5-3.3 µg/mg. In addition, Δ9-THCA, CBD, Δ9-THC, CBC, and CBG were mainly detected in bracts, buds, and leaves.

Essential oil composition of Curcuma species and drugs from Asia analyzed by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry.

Essential oils (EOs) comprised of various bioactive compounds have been widely detected in the Curcuma species. Due to the widespread distribution and misidentification of Curcuma species and differences in processing methods, inconsistent reports on major compounds in rhizomes of the same species from different geographical regions are not uncommon. This inconsistency leads to confusion and inaccuracy in compound detection of each species and also hinders comparative study based on EO compositions. The present study aimed to characterize EO compositions of 12 Curcuma species, as well as to detect the compositional variation among different species, and between the plant specimens and their related genetically validated crude drug samples using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. The plant specimens of the same species showed similar EO patterns, regardless of introducing from different geographical sources. Based on the similarity of EO compositions, all the specimens and samples were separated into eight main groups: C. longa; C. phaeocaulis, C. aeruginosa and C. zedoaria; C. zanthorrhiza; C. aromatica and C. wenyujin; C. kwangsiensis; C. amada and C. mangga; C. petiolata; C. comosa. From EOs of all the specimens and samples, 54 major compounds were identified, and the eight groups were chemically characterized. Most of the major compounds detected in plant specimens were also observed in crude drug samples, although a few compounds converted or degraded due to processing procedures or over time. Orthogonal partial least squares-discriminant analysis allowed the marker compounds to discriminate each group or each species to be identified.

Flavonoids from Sedum japonicum subsp. oryzifolium (Crassulaceae).

Twenty-two flavonoids were isolated from the leaves and stems of Sedum japonicum subsp. oryzifolium (Crassulaceae). Of these compounds, five flavonoids were reported in nature for the first time, and identified as herbacetin 3-O-xyloside-8-O-glucoside, herbacetin 3-O-glucoside-8-O-(2'''-acetylxyloside), gossypetin 3-O-glucoside-8-O-arabinoside, gossypetin 3-O-glucoside-8-O-(2'''-acetylxyloside) and hibiscetin 3-O-glucoside-8-O-arabinoside via UV, HR-MS, LC-MS, acid hydrolysis and NMR. Other seventeen known flavonoids were identified as herbacetin 3-O-glucoside-8-O-arabinoside, herbacetin 3-O-glucoside-8-O-xyloside, gossypetin 3-O-glucoside-8-O-xyloside, quercetin, quercetin 3-O-glucoside, quercetin 3-O-xylosyl-(1→2)-rhamnoside-7-O-rhamnoside, quercetin 3-O-rhamnoside-7-O-glucoside, kaempferol, kaempferol 3-O-glucoside, kaempferol 7-O-rhamnoside, kaempferol 3,7-di-O-rhamnoside, kaempferol 3-O-glucoside-7-O-rhamnoside, kaempferol 3-O-glucosyl-(1→2)-rhamnoside-7-O-rhamnoside, kaempferol 3-O-xylosyl-(1→2)-rhamnoside, kaempferol 3-O-xylosyl-(1→2)-rhamnoside-7-O-rhamnoside, myricetin 3-O-glucoside and cyanidin 3-O-glucoside. Some flavonol 3,8-di-O-glycosides were found in Sedum japonicum subsp. oryzifolium as major flavonoids in this survey. They were presumed to be the diagnostic flavonoids in the species. Flavonoids were reported from S. japonicum for the first time.

Effect of Cultivation Conditions on Components of Ephedra sp. Using Liquid Chromatography-Mass Spectrometry and Multivariate Analysis.

In this study, we investigated the correlation between the cultivation conditions and chemical composition of Ephedra sinica and E. sp. (denoted EP-13, which has been grown at the National Institutes of Biomedical Innovation, Health, and Nutrition for many years). The total contents of ephedrine and pseudoephedrine are specified in the Japanese Pharmacopoeia; therefore, we investigated the changes in their content under different cultivation conditions, including varying soil conditions and fertilization or the lack of fertilization. Poor growth due to low soil nutrition and lack of sunlight caused decrease of the alkaloid content. As expected, the plants accumulated proline, although the proline content varied considerably with cultivation location. The proline concentration correlated with the content of methanoproline. Moreover, a new compound, namely N,N-dimethyl-p-hydroxyphenylethylamine-O-[ß-D-glucopyranosyl-(1→3)-α-L-rhamnopyranoside], was isolated from E. sinica but was absent in EP-13. This study on the correlation between cultivation methods and the alkaloid content in Ephedra is expected to assist in the future production of quality Ephedra herb.

Near-Infrared Metabolic Profiling for Discrimination of Apricot and Peach Kernels.

Apricot and Peach Kernels are commercial crude drugs used in many formulas of traditional Japanese medicine, Kampo. Although their applications are quite different, it is difficult to distinguish them using conventional methods such as HPLC. The study aimed at near-infrared (NIR) metabolic profiling to discriminate Apricot and Peach Kernels (Armeniacae Semen and Persicae Semen) collected from Japanese markets. A fast, simple, non-destructive, and robust NIR measurement of kernel surface with no sample pre-treatment was achieved in situ. Principal component analysis and orthogonal partial least squares discriminant analysis (OPLS-DA) models showed discrimination between the two crude drugs with good fitting and prediction values. These results indicate that NIR metabolic profiling is useful for discriminating Apricot and Peach Kernels based on their chemical constituents using a simple and non-destructive procedure.

Machine Learning-Assisted Screening of Herbal Medicine Extracts as Vaccine Adjuvants.

Adjuvants are important vaccine components, composed of a variety of chemical and biological materials that enhance the vaccine antigen-specific immune responses by stimulating the innate immune cells in both direct and indirect manners to produce a variety cytokines, chemokines, and growth factors. It has been developed by empirical methods for decades and considered difficult to choose a single screening method for an ideal vaccine adjuvant, due to their diverse biochemical characteristics, complex mechanisms of, and species specificity for their adjuvanticity. We therefore established a robust adjuvant screening strategy by combining multiparametric analysis of adjuvanticity in vivo and immunological profiles in vitro (such as cytokines, chemokines, and growth factor secretion) of various library compounds derived from hot-water extracts of herbal medicines, together with their diverse distribution of nano-sized physical particle properties with a machine learning algorithm. By combining multiparametric analysis with a machine learning algorithm such as rCCA, sparse-PLS, and DIABLO, we identified that human G-CSF and mouse RANTES, produced upon adjuvant stimulation in vitro, are the most robust biological parameters that can predict the adjuvanticity of various library compounds. Notably, we revealed a certain nano-sized particle population that functioned as an independent negative parameter to adjuvanticity. Finally, we proved that the two-step strategy pairing the negative and positive parameters significantly improved the efficacy of screening and a screening strategy applying principal component analysis using the identified parameters. These novel parameters we identified for adjuvant screening by machine learning with multiple biological and physical parameters may provide new insights into the future development of effective and safe adjuvants for human use.

Retusone A, a Guaiane-Type Sesquiterpene Dimer from Wikstroemia retusa and Its Inhibitory Effects on Histone Acetyltransferase HBO1 Expression.

Retusone A (1), a new sesquiterpene dimer consisting of two guaiane-type sesquiterpenoids, and oleodaphnal (2) were isolated from heartwood of Wikstroemia retusa (Thymelaeaceae). The planar structure of 1 was elucidated on the basis of HRESIMS and NMR spectroscopic data, and the relative stereochemistry was established by X-ray diffraction analysis. The absolute configuration of 1 was determined by electronic circular dichroism. Compound 1 suppressed luciferase reporter gene expression driven by the HBO1 (histone acetyltransferase binding to ORC1) gene promoter in human breast cancer MCF7 cells. Compound 1 also decreased the expression of endogenous HBO1 mRNA and protein, and inhibited proliferation of the cells. These results suggest that retusone A (1), which has a unique dimeric sesquiterpenoid structure with inhibitory activity against HBO1 expression, may contribute to the development of a novel therapeutic candidate for the treatment of breast cancer.

Development of SCAR Markers to Identify Medicinal Cultivars of Paeonia lactiflora.

Paeoniae Radix, the dried root of Paeonia lactiflora, is one of the most important ingredients in Kampo medicine. It is known that Paeoniae Radix is derived from various P. lactiflora cultivars, including medicinal and horticultural cultivars, and that cultivar identification by DNA analysis has been unsuccessful. We attempted to develop sequence characterized amplified region (SCAR) markers as useful DNA markers for the identification and herbal medicine authentication of two cultivars developed in Japan, 'Bonten' and 'Kitasaisho,' which are two superior medicinal strains of P. lactiflora. Sequence-related amplified polymorphism (SRAP) analysis was conducted on fourteen P. lactiflora cultivars, and polymorphic fragments specific to 'Bonten' or 'Kitasaisho' were detected. Then, SCAR markers for 'Bonten' and 'Kitasaisho' were developed from the sequence information of these polymorphic fragments. Thirty cultivars of P. lactiflora and five herbal medicine samples were used to validate the specificity of the developed SCAR markers. As a result, we confirmed that our SCAR markers can identify 'Bonten' or 'Kitasaisho' from the plant samples and the herbal medicine samples. Thus, we have successfully designed two highly specific DNA markers and established an easy, rapid, and cost-efficient method to identify specific cultivars of P. lactiflora. Our SCAR markers are expected to contribute to the maintenance of P. lactiflora cultivars such as 'Bonten' as superior medicinal strains, the development of more elite cultivars in the future, and the deterrence of outflow of original cultivars to foreign countries.

Phenanthroindolizine alkaloids from Boehmeria sieboldiana leaves exhibit cytotoxicity against human cancer cell lines.

To explore useful natural compounds from indigenous medicinal plants, the cytotoxic properties from a methanolic extract of Boehmeria sieboldiana leaves against human cancer cell lines were isolated in the present study. After purification of the extract, seco-dehydroantofine B (1) together with two known phenanthroindolizine alkaloids, seco-dehydroantofine A (2) and septicine (3), were isolated. The structure of seco-dehydroantofine B was elucidated by performing comprehensive one- and two-dimensional nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. The cytotoxicity of these compounds against five human tumor cell lines was evaluated. Compound 3 exhibited anti-tumor activity at IC50 values of 50.0, 66.9, 50.0, and 153.7 µM against MKN1, SAS, HL-60, and THP-1 cells, respectively.

Essential oil components in the seed masses of Amomum xanthioides and its related species from Southeast Asia and China.

Previously, to develop an objective identification method for Amomi Semen (AS), the nucleotide sequences of nrDNA ITS region and two cpDNA regions of nine Amomum taxa specimens from Southeast Asia and China were determined, and the generated phylogenetic tree showed six taxa specimens were divided into four groups. In this study, 51 crude drug samples of AS in Japanese markets were classified into four groups or species based on their ITS sequences. Approximately 67% of samples were derived from A. villosum var. xanthioides or A. xanthioides, A. villosum var. villosum and A. longiligulare prescribed in Japanese Pharmacopoeia, and the rest were mixed with A. uliginosum and A. microcarpum. Subsequently, the essential oil compositions of Amomum taxa specimens and AS samples were determined by GC-MS to characterize each group or species. Group 1(A. xanthioides) samples were characterized by containing higher amount of camphor(6) than bornyl acetate(9), and a specific germacrene D-4-ol; group 2(Chinese A. villosum var. villosum and var. xanthioides) by containing higher amount of 9 than 6, a specific isobornyl acetate; group 3(Laotian A. villosum var. villosum and A. longiligulare) by containing higher amount of 6 than 9, and a characteristic neointermedeol, except for A. longiligulare specimen from Hainan, China; group 4(A. uliginosum) by containing equivalent amount of 6 and 9, and the specific (E,E)-farnesyl acetate and (E,E)-farnesol. A. microcarpum samples were discriminated from the above groups by absence of 6 and 9, and with higher amount of (E)-nerolidol. There was a good correlation between genetic classification and chemical discrimination.

Discrimination of Curcuma species from Asia using intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase.

Recently, Curcuma rhizome-related foods with claimed health benefits have been used worldwide; however, correct identification and quality assessment have not been conducted. Due to the wide distribution and morphological similarities of Curcuma species, the classification of some species is debated and nomenclature is inconsistent among countries. In this study, to elucidate specific molecular markers of medicinally used Curcuma species in Asia, and to solve the confusion on the reported botanical origin of crude drugs, molecular analysis based on the intron length polymorphism (ILP) in genes encoding diketide-CoA synthase and curcumin synthase and the trnK intron sequences was performed using 59 plant specimens and 42 crude drug samples from 13 Curcuma species, obtained from Asian countries. The ILP patterns of the respective species from both plant specimens and crude drug samples revealed high consistency in C. aromatica, C. zedoaria, C. phaeocaulis, C. aeruginosa, C. wenyujin, and C. zanthorrhiza, but showed intraspecies polymorphism in C. longa, C. kwangsiensis, C. amada, C. mangga and C. comosa. The C. longa specimens and samples were separated into three subgroups which were highly consistent with their geographical origins. Based on the ILP markers and the trnK intron sequences, the botanical origins of "Khamin oi" from Thailand were correctly determined to be C. longa or a hybrid between C. longa and other species, and "Wan narn kum" from Thailand and "Kasturi manjal" from India were correctly determined to be C. zanthorrhiza.

Diversity of Adenostemma lavenia, multi-potential herbs, and its kaurenoic acid composition between Japan and Taiwan.

Adenostemma lavenia (L.) Kuntze (Asteraceae) is widely distributed in tropical regions of East Asia, and both A. lavenia and A. madurense (DC) are distributed in Japan. In China and Taiwan, A. lavenia is used as a folk medicine for treating lung congestion, pneumonia, and hepatitis. However, neither phylogenic nor biochemical analysis of this plants has been performed to date. We have reported that the aqueous extract of Japanese A. lavenia contained high levels of ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (11αOH-KA; a kaurenoic acid), which is a potent anti-melanogenic compound. Comparison of chloroplast DNA sequences suggested that A. lavenia is originated from A. madurense. Analyses of kaurenoic acids revealed that Japanese A. lavenia and A. madurense contained high levels of 11αOH-KA and moderate levels of 11α,15OH-KA, while Taiwanese A. lavenia mainly contained 9,11αOH-KA. The diverse biological activities (downregulation of Tyr, tyrosinase, gene expression [anti-melanogenic] and iNOS, inducible nitric oxide synthase, gene expression [anti-inflammatory], and upregulation of HO-1, heme-oxygenase, gene expression [anti-oxidative]) were associated with 11αOH-KA and 9,11αOH-KA but not with 11α,15OH-KA. Additionally, 11αOH-KA and 9,11αOH-KA decreased Keap1 (Kelch-like ECH-associated protein 1) protein levels, which was accompanied by upregulation of protein level and transcriptional activity of Nrf2 (NF-E2-related factor-2) followed by HO-1 gene expression. 11αOH-KA and 9,11αOH-KA differ from 11α,15OH-KA in terms of the presence of a ketone (αß-unsaturated carbonyl group, a thiol modulator) at the 15th position; therefore, thiol moieties on the target proteins, including Keap1, may be important for the biological activities of 11αOH-KA and 9,11αOH-KA and A. lavenia extract.

Genetic analysis of Curcuma species from Asia based on intron regions of genes encoding diketide-CoA synthase and curcumin synthase.

Intron length polymorphism (ILP) markers in genes encoding diketide-CoA synthase (DCS) and curcumin synthase (CURS) showed high identification rates in 13 Curcuma species from Asia. However, the sequences of the intron regions have not yet been analyzed. To elucidate the sequence differences in intron regions of the DCS and CURS genes and to search for specific sequences suitable for the identification of Curcuma species, a large number of sequences were determined through subcloning coupled with sequencing analysis of six Curcuma plant specimens belonging to five species that showed distinct ILP patterns. More than 30 sequences of each region from each specimen were grouped into genes DCS1, DCS2, or CURS1-3 and subsequently the sequences of the same genes were compared. Sequences belonging to the same gene showed inter-species similarity, and thus, these intron sequences were less informative within each single-gene region. The determined sequences from each specimen showed 3-5 kinds of sequence lengths in DCS intron I region, and 5-7 kinds of sequence lengths in CURS intron region. The length of determined sequences and the fragment number in each intron region were different among species, or specimens in C. longa, which were in accordance with the fragment lengths and numbers in their corresponding ILP patterns.

Toxicity of Jegosaponins A and B from Styrax japonica Siebold et al. Zuccarini in Prostate Cancer Cells and Zebrafish Embryos Resulting from Increased Membrane Permeability.

(1) Background: Screening of medicinal herbs is one of the most powerful approaches to identifying novel therapeutic molecules against many human diseases. To avoid potential harmful effects during medicinal use, toxicity testing is necessary in the early stages of drug discovery. The objective of this study was to identify the cytotoxic mechanisms of jegosaponin A and B from Styrax japonica Siebold et al. Zuccarini; (2) Methods: We screened Japanese medicinal herb extracts using PC-3 prostate cancer cells and found that a methanol extract isolated from the unripe fruit of Styrax japonica Siebold et al. Zuccarini (SJSZ) had an inhibitory effect on cell viability. We further performed fractionation assays with PC-3 cells and identified the bioactive compounds using LC/MS and NMR analysis. We clarified the toxic mechanisms of these compounds using PC-3 cells and zebrafish embryos; (3) Results: We identified two active molecules, jegosaponin A and jegosaponin B, in the inhibitory fractions of the methanol extract. These jegosaponins are toxic to zebrafish embryos during the early developmental stage. Jegosaponin A and B showed strong haemolytic activity in sheep defibrinated blood (EC50 = 2.1 µM, and 20.2 µM, respectively) and increased the cell membrane permeability in PC-3 cells and zebrafish embryos, which were identified using a membrane non-permeable DRAQ7, a fluorescent nucleus staining dye; (4) We identified the cytotoxic compounds jegosaponin A and B from SJSZ, which we showed to exhibit cell membrane disruptive properties using cell- and zebrafish-based testing.