Targeted delivery of ursolic acid and oleanolic acid to lungs in the form of an inhaler for the management of tuberculosis: Pharmacokinetic and toxicity assessment.

INTRODUCTION: Ursolic acid (UA) and oleanolic acid (OA) are triterpenoids. They are used to treat numerous diseases, including tuberculosis. Combinations of these drugs provide new insight into the management of tuberculosis. The major obstacle is the effective delivery of these drugs to the lungs, which are mainly affected due to M. tuberculosis. A metered-dose inhaler (MDI) was developed to address this issue containing UA and OA, followed by in-vitro and in-vivo evaluation. METHODS: In the present study, MDI formulation was prepared by incorporating UA and OA at the dose level of 120 µg/ml in each actuation. In-vitro evaluation of this MDI formulation was performed to ensure its suitability to deliver UA and OA preciously. With prior approval of IAEC, a pharmacokinetic and acute inhalation toxicity study was conducted using MDI on Wistar rats. RESULTS: The pharmacokinetic study showed an increased biological half-life of UA (9.23±0.104 h) and OA (8.93±0.166 h) in combination therapy. In-vivo toxicity study demonstrated no adverse effects on body weight and vital organs in the treatment group compared with the control group. Histopathology examination of these essential organs showed no abnormalities. Mild alternation in the biochemical and hematological parameters was observed. However, these alterations did not affect the overall health of the animals. CONCLUSION: The present study documents a detailed study for the safety and pharmacokinetics of UA and OA in-vivo for their advanced application in tuberculosis disease.

Novel Oleanane-Type Triterpene Glycosides from the Saponaria officinalis L. Seeds and Apoptosis-Inducing Activity via Mitochondria.

Saponaria officinalis L., commonly known as "Soapwort", is a rich source of triterpene glycosides; however, the chemical constituents of S. officinalis seeds have not been fully identified. In this study, we conducted a systematic phytochemical investigation of the seeds of S. officinalis and obtained 17 oleanane-type triterpene glycosides (1-17), including seven new glycosides (1-7). The structures of 1-7 were determined based on a detailed analysis of NMR spectroscopic data and chromatographic and spectroscopic analyses following specific chemical transformation. The cytotoxicities of the isolated compounds were evaluated against HL-60 human promyelocytic leukemia cells, A549 human adenocarcinoma lung cancer cells, and SBC-3 human small-cell lung cancer cells. The cytotoxicities of 1, 4, and 10 toward HL-60 cells and SBC-3 cells were nearly as potent as that of cisplatin. Compound 1, a bisdesmosidic triterpene glycoside obtained in good yield, arrested the cell cycle of SBC-3 cells at the G2/M phase, and induced apoptosis through an intrinsic pathway, accompanied by ROS generation. As a result of the mitochondrial dysfunction induced by 1, mitochondria selective autophagy, termed mitophagy, occurred in SBC-3 cells.

Farnesoid X receptor contributes to oleanolic acid-induced cholestatic liver injury in mice.

Farnesoid X receptor (FXR) is a nuclear receptor involved in the metabolism of bile acid. However, the molecular signaling of FXR in bile acid homeostasis in cholestatic drug-induced liver injury remains unclear. Oleanolic acid (OA), a natural triterpenoid, has been reported to produce evident cholestatic liver injury in mice after a long-term use. The present study aimed to investigate the role of FXR in OA-induced cholestatic liver injury in mice using C57BL/6J (WT) mice and FXR knockout (FXR-/- ) mice. The results showed that a significant alleviation in OA-induced cholestatic liver injury was observed in FXR-/- mice as evidenced by decreases in serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase as well as reduced hepatocyte necrosis. UPLC-MS analysis of bile acids revealed that the contents of bile acids decreased significantly in liver and serum, while increased in the bile in FXR-/- mice compared with in WT mice. In addition, the mRNA expressions of hepatic transporter Bsep, bile acid synthesis enzymes Bacs and Baat, and bile acids detoxifying enzymes Cyp3a11, Cyp2b10, Ephx1, Ugt1a1, and Ugt2b5 were increased in liver tissues of FXR-/- mice treated with OA. Furthermore, the expression of membrane protein BSEP was significantly higher in livers of FXR-/- mice compared with WT mice treated with OA. These results demonstrate that knockout of FXR may alleviate OA-induced cholestatic liver injury in mice by decreasing accumulation of bile acids both in the liver and serum, increasing the export of bile acids via the bile, and by upregulation of bile acids detoxification enzymes.

Fluorescent tagging of endogenous Heme oxygenase-1 in human induced pluripotent stem cells for high content imaging of oxidative stress in various differentiated lineages.

Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62-1000 nM) or diethyl maleate (5.62-1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.

Anticancer effects of oleanolic acid on human melanoma cells.

Owing to the ineffectiveness of the currently used therapies against melanoma, there has been a shift in focus toward alternative therapies involving the use of natural compounds. This study assessed the anticancer effects of oleanolic acid (OA) and its ability to induce apoptosis in A375SM and A375P melanoma cells in vivo. Compared to the control group, viability of A375P and A375SM cells decreased following OA treatment. In OA-treated A375SM and A375P cells, 4',6-diamidino-2-phenylindole staining showed an increase in the apoptotic body, and flow cytometry revealed increased number of apoptotic cells compared to that in the control group. OA-treated A375SM cells exhibited an increased expression of the apoptotic proteins, cleaved poly (ADP-ribose) polymerase (PARP) and B-cell lymphoma (Bcl)-2-associated X protein (Bax) as well as decreased expression of the antiapoptotic protein Bcl-2 compared to that in the control group. In OA-treated A375P cells, expression patterns of cleaved PARP and Bcl-2 were similar to those in OA-treated A375SM cells; however, no difference was reported in the expression of Bax compared to that in the control group. Additionally, OA-treated melanoma cells showed decreased expression of phospho-nuclear factor-κB (p-NF-κB), phospho-inhibitor of nuclear factor-κBα (p-IκBα), and phospho-IκB kinase αß than that in the control group. Moreover, immunohistochemistry showed a comparatively decreased level of p-NF-κB in the OA-treated group than that in the control group. Xenograft analysis confirmed the in vivo anticancer effects of OA against A375SM cells. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay revealed an increased number of TUNEL-positive cells in the OA-treated group compared to that in the control group. In conclusion, the study results suggest that OA induces apoptosis of A375SM and A375P cells in vitro and apoptosis of A375SM cells in vivo. Furthermore, the in vitro and in vivo anticancer effects were mediated by the NF-κB pathway.

Integrating non-targeted metabolomics and toxicology networks to study the mechanism of Esculentoside A-induced hepatotoxicity in rats.

Esculentoside A (EsA) is a kind of triterpenoid saponins from the root tuber of Phytolacca acinosa Roxb. It has extensive medicinal activity, such as antibacterial, anti-inflammatory, immune regulation, and cell proliferation inhibition. However, some researches suggested that EsA can cause hepatotoxicity, whose mechanism is not precise. To ensure the safety and reliability in the clinical use of Phytolacca acinosa Roxb., it is necessary to establish a rapid and accurate method to evaluate the toxicity, analyze and verify the toxicity mechanism of EsA. Therefore, this research explored the mechanism of hepatotoxicity induced by EsA in rats and analyzed endogenous metabolites' changes in rat plasma by combining network toxicology with non-targeted metabolomics. We obtained 58 critical targets of EsA induced hepatotoxicity in rats based on the strategy of network toxicology, including albumin, mitogen-activated protein kinase 1, Caspase-3, etc. Many important pathways were obtained by Kyoto Encyclopedia of Genes and Genomes enrichment analysis, such as HIF-1 signaling pathway, TNF signaling pathway, IL-17 signaling pathway, and other concerning pathways. Sixteen biomarkers, including 5-hydroxykynurenamine, N-acetylserotonin, palmitic acid, etc., were screened from rat plasma using Ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS), mainly involve Glycerophospholipid metabolism, Tryptophan metabolism, and other metabolic pathways. Further analysis showed that EsA may induce liver injury by activating oxidative stress and energy metabolism disorders, triggering inflammation and apoptosis.

New Perspectives of S-Adenosylmethionine (SAMe) Applications to Attenuate Fatty Acid-Induced Steatosis and Oxidative Stress in Hepatic and Endothelial Cells.

S-adenosylmethionine (SAMe) is an endogenous methyl donor derived from ATP and methionine that has pleiotropic functions. Most SAMe is synthetized and consumed in the liver, where it acts as the main methylating agent and in protection against the free radical toxicity. Previous studies have shown that the administration of SAMe as a supernutrient exerted many beneficial effects in various tissues, mainly in the liver. In the present study, we aimed to clarify the direct effects of SAMe on fatty acid-induced steatosis and oxidative stress in hepatic and endothelial cells. Hepatoma FaO cells and endothelial HECV cells exposed to a mixture of oleate/palmitate are reliable models for hepatic steatosis and endothelium dysfunction, respectively. Our findings indicate that SAMe was able to significantly ameliorate lipid accumulation and oxidative stress in hepatic cells, mainly through promoting mitochondrial fatty acid entry for ß-oxidation and external triglyceride release. SAMe also reverted both lipid accumulation and oxidant production (i.e., ROS and NO) in endothelial cells. In conclusion, these outcomes suggest promising beneficial applications of SAMe as a nutraceutical for metabolic disorders occurring in fatty liver and endothelium dysfunction.

Reparative and toxicity-reducing effects of liposome-encapsulated saikosaponin in mice with liver fibrosis.

Saikosaponin d (SSd), a primary active component of the Chinese herb Bupleurum falcatum, has antitumor and antiliver fibrosis effects. However, the toxicity of SSd at high doses can induce conditions such as metabolic disorders and hemolysis in vivo, thus hampering its clinical use. The present study investigated the toxicity-reducing effects of liposome encapsulation of pure SSd and the therapeutic action of SSd-loaded liposomes (Lipo-SSd) in liver fibrosis in vitro and in vivo. Lipo-SSd (diameter, 31.7 ± 7.8 nm) was prepared at an entrapment efficiency of 94.1%. After 10-day incubation, a slow release profile of 56% SSd from Lipo-SSd was observed. The IC50 of SSd on hepatic stellate cells was approximately 2.9 µM. Lipo-SSd exhibited much lower cytotoxicity than did pure SSd. In the in vivo toxicity assay, Lipo-SSd significantly increased mice survival rate and duration compared with pure SSd at the same dose. These in vitro and in vivo data indicate that liposomal encapsulation can reduce the cytotoxicity of SSd. The histopathological analysis results demonstrated that in mice with thioacetamide-induced liver fibrosis, Lipo-SSd exerted more obvious fibrosis- and inflammation-alleviating and liver tissue-reparative effects than did pure SSd; these effects are potentially attributable to the sustained release of SSd. In conclusion, Lipo-SSd fabricated here have antiliver fibrosis effects and lower toxicity compared with that of pure SSd.

Genotoxic and Cytotoxic Activities of Lantadene A-Loaded Gold Nanoparticles (LA-AuNPS) in MCF-7 Cell Line: An in vitro Assessment.

Gold nanoparticles (AuNPs) have been widely used in many applications. Their usage as drug delivery vehicles has also gained considerable attention due to their chemical and optical properties as well as their good biocompatibility. The present study was conducted to evaluate the efficiency of AuNPs in enhancing the cytotoxic and apoptotic induction activity of lantadene A (LA), separated from Lantana camara leaves, on the breast tumor cell line MCF-7 in vitro. By utilizing plant-mediated synthesis method of nanostructures, LA-loaded AuNPs (LA-AuNPs) were prepared and their formation was confirmed by means of ultraviolet-visible spectroscope, atomic force microscope, scanning electron microscope, and zeta potential. The cytotoxic effect of LA-AuNPs was analyzed using a methylthiazol tetrazolium assay and compared to free AuNPs and LA. The results indicated a significant increase in the reduction of MCF-7 cells viability after incubation with LA-AuNPs. As determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, LA-AuNPs induced a greater ratio of DNA-fragmented cells compared to LA-treated and untreated cells. Also, by operating real-time polymerase chain reaction, LA-AuNPs-treated cells displayed an increased upregulation of p53 expression and downregulation of BCL-2 expression in addition to a significant reduction in the level of BCL-2-BAX ratio. No significant effect was shown on the expression of BAX. Collectively, our results indicate that LA-AuNPs showed promising cytotoxicity to MCF-7 cells as a novel nanoscale preparation, likely via induction of apoptotic genes and stimulation of DNA fragmentation.

[Study on liver protection and hepatotoxicity of saikosaponin a based on zebrafish model].

Bupleuri Radix has both liver protection and hepatotoxicity. Saponins are the main pharmacodynamic and toxic components of Bupleuri Radix. Based on zebrafish physical model and the model of alcoholic fatty liver( AFL) pathology,the liver toxic and protective effect of saikosaponin a( SSa) were assessed. The results indicated that 1. 77 µmol·L-1 SSa showed protective effect to AFL zebrafish. 5. 30 µmol·L-1 SSa was hepatotoxic to healthy zebrafish,but it showed protective effect to AFL zebrafish. 5. 62 µmol·L-1 SSa was hepatotoxic to healthy and AFL zebrafish. This study is benefit for clinical safety of saikosaponin a.

Modification of GSK3ß/ß-catenin signaling on saikosaponins-d-induced inhibition of neural progenitor cell proliferation and adult neurogenesis.

Saikosaponins-d (SSd) is a major bioactive compound isolated from Radix Bupleuri, an herb widely used in traditional Chinese medicine. Emerging studies demonstrate that SSd adversely affects adult neurogenesis and impairs learning and memory. However, the molecular mechanisms remain to be determined. The current study investigated the potential regulatory of GSK3ß/ß-catenin signaling on SSd-induced neurotoxicity. We demonstrated that SSd exposure inhibited the cell viability and proliferation of primary neuronal stem/progenitor cells (NPCs) from hippocampus in a concentration-dependent manner. Significantly, SSd exposure induced activation of GSK3ß and reduced the cellular ß-catenin in NPCs. Treatment with SB216763, a specific inhibitor for GSK3ß activation, we showed that inactivation GSK3ß improved the ß-catenin signaling by inhibiting degradation complex comprising Axin and APC and attenuated SSd-induced toxicity in NPCs. In addition, administration of SB216763 ameliorated SSd-induced inhibition of NPCs proliferation and enhanced radial glial cells in the hippocampus of mice. Moreover, inactivation GSK3ß promoted dendritic arborization and the survival of newborn neurons together with alleviated the impairment of SSd-induced cognitive function in mice. Overall, these data demonstrated that the significant inhibitory effects of SSd on NPCs proliferation and adult neurogenesis via GSK3ß/ß-catenin signaling pathway. Our results contribute to a better understanding of the molecular mechanisms of SSd-induced neurotoxicity.

Saikosaponin d causes apoptotic death of cultured neocortical neurons by increasing membrane permeability and elevating intracellular Ca2+ concentration.

Saikosaponins (SSs) are a class of naturally occurring oleanane-type triterpenoid saponins found in Radix bupleuri that has been widely used in traditional Chinese medicine. As the main active principals of Radix bupleuri, SSs have been shown to suppress mouse motor activity, impair learning and memory, and decrease hippocampal neurogenesis. In the present study, we investigated the effect of five SSs (SSa, SSb1, SSb2, SSc, and SSd) on neuronal viability and the underlying mechanisms in cultured murine neocortical neurons. We demonstrate that SSa, SSb1 and SSd produce concentration-dependent apoptotic neuronal death and induce robust increase in intracellular Ca2+ concentration ([Ca2+]i) at low micromolar concentrations with a rank order of SSd > SSa > SSb1, whereas SSb2 and SSc have no detectable effect on both neuronal survival and [Ca2+]i. Mechanistically, SSd-induced elevation in [Ca2+]i is the primary result of enhanced extracellular Ca2+ influx, which likely triggers Ca2+-induced Ca2+ release through ryanodine receptor activation, but not SERCA inhibition. SSd-induced Ca2+ entry occurs through a non-selective mechanism since blockers of major neuronal Ca2+ entry pathways, including L-type Ca2+ channel, NMDA receptor, AMPA receptor, Na+-Ca2+ exchanger, and TRPV1, all failed to attenuate the Ca2+ response to SSd. Further studies demonstrate that SSd increases calcein efflux and induces an inward current in neocortical neurons. Together, these data demonstrate that SSd elevates [Ca2+]i due to its ability to increase membrane permeability, likely by forming pores in the surface of membrane, which leads to massive Ca2+ influx and apoptotic neuronal death in neocortical neurons.

A comprehensive review and perspectives on pharmacology and toxicology of saikosaponins.

BACKGROUND: Radix Bupleuri (RB) has been widely used in Chinese Traditional Medicine for over 2000 years and is currently marketed in China as Chai-Hu-Shu-Gan tablets and Xiao-Yao-Wan tablets. Saikosaponins (SSs, especially SSa, SSc and SSd), as the major bioactive compounds in RB, represent anti-inflammatory, anti-tumor, anti-oxidant, anti-viral and hepatoprotective effects. PURPOSE: To summarize recent findings regarding to the extraction, detection, biosynthesis, metabolism, pharmacological/toxicological effects of SSs. METHODS: Online academic databases (including PubMed, Google Scholar, Web of Science and CNKI) were searched using search terms of "Saikosaponin", "Radix Bupleuri", "Bupleurum" and combinations to include published studies of SSs primarily from 2003 to 2018. Several critical previous studies beyond this period were also included. RESULTS: 354 papers were found and 165 papers were reviewed. SSs have drawn great attention for their anti-inflammation, anti-viral and anti-cancer effects and contradictory roles in the regulation of cell apoptosis, oxidative stress and liver fibrosis. Meanwhile, increased risks of overdose-induced acute or accumulation-related chronic hepatotoxicity of SSs and RB have also been reported. However, underlying mechanisms of SSs bioactivities, the metabolism of SSs and bioactivities of SSs metabolites are largely unknown. CONCLUSION: This comprehensive review of SSs provides novel insights and perspectives on the limitations of current studies and the importance of metabolism study and the dose-pharmacological/toxic relationship of SSs for the future discovery of SSs-based therapeutic strategies and clinical safe practice.

Design, Synthesis, and Cytotoxic Analysis of Novel Hederagenin⁻Pyrazine Derivatives Based on Partial Least Squares Discriminant Analysis.

Hederagenin (He) is a novel triterpene template for the development of new antitumor compounds. In this study, 26 new He⁻pyrazine derivatives were synthetized in an attempt to develop potent antitumor agents; they were screened for in vitro cytotoxicity against tumor and non-tumor cell lines. The majority of these derivatives showed much stronger cytotoxic activity than He. Remarkably, the most potent was compound 9 (half maximal inhibitory concentration (IC50) was 3.45 ± 0.59 µM), which exhibited similar antitumor activities against A549 (human non-small-cell lung cancer) as the positive drug cisplatin (DDP; IC50 was 3.85 ± 0.63 µM), while it showed lower cytotoxicity on H9c2 (murine heart myoblast; IC50 was 16.69 ± 0.12 µM) cell lines. Compound 9 could induce the early apoptosis and evoke cell-cycle arrest at the synthesis (S) phase of A549 cells. Impressively, we innovatively introduced the method of cluster analysis modeled as partial least squares discriminant analysis (PLS-DA) into the structure⁻activity relationship (SAR) evaluation, and SAR confirmed that pyrazine had a profound effect on the antitumor activity of He. The present studies highlight the importance of pyrazine derivatives of He in the discovery and development of novel antitumor agents.

Macranthoidin B Modulates Key Metabolic Pathways to Enhance ROS Generation and Induce Cytotoxicity and Apoptosis in Colorectal Cancer.

BACKGROUND/AIMS: Induction of oxidative stress and reactive oxygen species (ROS) mediated-apoptosis have been utilized as effective strategies in anticancer therapy. Macranthoidin B (MB) is a potent inducer of ROS-mediated apoptosis in cancer, but its mechanism of action is poorly understood. METHOD: Superoxide production with MB exposure in colorectal cancer (CRC) cells was measured using lucigenin chemiluminescence and real-time PCR. MB's inhibitory effect on proliferation and viability of CRC cells was determined by proliferation assays. MB's effect on apoptosis of CRC cells was determined by Western blotting and annexin V-FITC/PI staining. MB's effect on the growth of CRC xenografts in mice was assessed. An established metabolomics profiling platform combining ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS) with gas chromatography-mass spectrometry (GC-MS) was performed to determine MB's effect on total metabolite variation in CRC cells. RESULTS: We found that MB increases ROS generation via modulating key metabolic pathways. Using metabolomics profiling platform combining LC-MS with GC-MS, a total of 236 metabolites were identified in HCT-116 cells in which 31 metabolites were determined to be significantly regulated (p ≤ 0.05) after MB exposure. A number of key metabolites revealed by metabolomics analysis include glucose, fructose, citrate, arginine, phenylalanine, and S-adenosylhomocysteine (SAH), suggesting specific modulation of metabolism on carbohydrates, amino acids and peptides, lipids, nucleotide, cofactors and vitamins in HCT-116 CRC cells with MB treatment highly associated with apoptosis triggered by enhanced ROS and activated caspase-3. CONCLUSION: Our results demonstrate that MB represses CRC cell proliferation by inducing ROS-mediated apoptosis.

Synthesis of Oleanolic Acid Analogues and Their Cytotoxic Effects on 3T3 Cell Line.

BACKGROUND: Oleanolic acid (OA) is a known natural compound with many important biological activities. Thirteen oleanolic acid derivatives linked at C-3 and C-28 were synthesized and their structures were confirmed by 1H- and 13C NMR and mass spectral analyses. Among them, compounds 4, 6, 8-10, 12, 13 were synthesized for the first time. They were evaluated for their cytotoxic activity. They showed proliferative effect at low concentrations while cytotoxic effect was observed at high concentrations in a dose dependent manner. METHODS: We have first synthesized compounds 1 and 2 from the reaction of methyl iodide and OA. Compound 1 was reduced with LiAlH4 to give compound 3, and compound 2 gave compound 9 with MOMBr as a new compound. The compound 10 was then obtained from the reduction of compound 9 with LiAlH4 as a new oleanolic acid derivative. A diol derivative 11 was synthesized from OA and LiAlH4 at the room temperature. Compound 4 was obtained from the reaction of compound 3 with CBr4 as a new analogue of OA, and the reduction of compound 4 afforded compound 5 as a known product. In addition, we synthesized compounds 6-8 from compound 3 using MsCl, MeI and p-nitrobenzoyl chloride, respectively, in good yields. Compounds 6 and 8 are new analogues of OA. The new compounds 12 and 13 were also synthesized starting from OA using with MOMBr and TBDMSiCl as the reagents. The all synthesized compounds were purified by using column chromatography and/or crystallization. RESULTS: In the present study, thirteen OA derivatives linked at C-28 and (or) C-3 were synthesized and evaluated for their cytotoxic activity on 3T3 cell lines which are the standard fibroblast cell lines, derived from Swiss albino mouse embryo tissue. 3T3 cell viability was observed at low concentrations of the tested triterpenoids while they displayed anti-proliferative effect at higher concentrations. CONCLUSION: Oleanolic acid 28-methyl ester (2) showed fairly different behavior from all the other compounds tested and found to be the least cytotoxic compound. However, at 200 µM concentration, it exhibited the same cytotoxicity with compounds 3, 9 and 10 around 58-59%. Among the tested 13 compounds, 7 exhibited the most drastic decline for the viability from 12,5 µM to 25 µM concentration. Compound 6 displayed the most cytotoxic effect, almost in all concentrations, particularly at 6.25 and 25 µM concentrations while the highest cytotoxic effect at 50 µM was observed for compound 11 among all the tested triterpenoids. As a result, all the tested OA derivatives showed proliferative effect at 1,56 µM although no proliferative effect was observed for OA. Moreover, OA exhibited higher cytotoxic effect than its derivatives, particularly at higher concentrations (50, 100, 200 µM) with an exception for compound 11. Because, the latter showed highest proliferative effect at lowest concentration, and highest anti-proliferative effect at highest concentration which surpassed all the OA derivatives.

Saikosaponin-d-mediated downregulation of neurogenesis results in cognitive dysfunction by inhibiting Akt/Foxg-1 pathway in mice.

Saikosaponin-d (SSd), one of the main constituents of the total saikosaponins extracted from Bupleurum falcatum L, possesses anti-inflammatory and anti-apoptosis effect. Recently, SSd was proved to improve depressive symptoms although exhibit hepatotoxicity in animals, but the central nervous system (CNS) toxicity of SSd remains unclear. The present study investigated the SSd-induced impairment in hippocampal cognitive function and explored the possible mechanisms involved. After intragastric administration of SSd (4mg/kg, 8mg/kg) for 7days, the learning and memory abilities of mice were evaluated by behavioral experiments. In the step-down passive avoidance test, we found that the mice treated with SSd showed a significant decrease of step-down latency and increase of the frequency of errors. In the Morris water maze task, both the escape latency and swimming distance of the mice treated with SSd were increased, correspondingly, both the time of mice staying in the target zone and the frequency of crossing platform were decreased. These neurobehavioral changes were accompanied by the reduction of the expression of 5-bromo-2'-deoxyuridine (BrdU), nestin, doublecortin (Dcx) and microtubule associated protein 2 (MAP2). Moreover, SSd significantly inhibited the expression of p-Akt, Foxg-1 and fibroblast growth factor 2 (FGF2) in the hippocampus of mice. These results indicated that SSd had a toxic effect on cognitive function in mice, which was associated with inhibiting the hippocampal neurogenesis via Akt/Foxg1 pathway.

Novel Derivative of Bardoxolone Methyl Improves Safety for the Treatment of Diabetic Nephropathy.

Currently, no effective and safe medicines are available to treat diabetic nephropathy (DN). Bardoxolone methyl (CDDO-Me) has displayed promising anti-DN activity as well as serious side effects in clinical trials, probably because the highly reactive α-cyano-α,ß-unsaturated ketone (CUK) in ring A of CDDO-Me can covalently bind to thiol functionalities in many biomacromolecules. In this study, we designed and synthesized a γ-glutamyl transpeptidase (GGT)-based and CUK-modified derivative of CDDO-Me (2) to address this issue. 2 can be specifically cleaved by GGT, which is highly expressed in the kidney, to liberate CDDO-Me in situ. It should be noted that 2 exhibited anti-DN efficacy comparable to that of CDDO-Me with much less toxicity in cells and db/db mice, suggesting that its safety is better than CDDO-Me. Our findings not only reveal the therapeutic potential of 2 but also provide a strategy to optimize other synthetic molecules or natural products bearing a pharmacophore like CUK to achieve safer pharmaceutical drugs.

Oleanolic-bioenhancer coloaded chitosan modified nanocarriers attenuate breast cancer cells by multimode mechanism and preserve female fertility.

Addressing multidrug resistant stage of breast cancer is an impediment for chemotherapy. Moreover, breast cancer chemotherapy has potential enduring confrontations i.e. related toxicity including effect on fertility of young female patients. The co-delivery of polyphenolic bio-enhancers with oleanolic acid in chitosan coated PLGA nanoparticles was designed for oral delivery with enhanced antitumor effect consecutively preserving the female fertility. The optimized oleanolic- bio-enhancer nano formulation CH-OA-B-PLGA with particle size was 342.2±3.7nm and zeta potential of 34.2±3.1mV was capable of lowering viability in MDAMB 231 cell line 16 times than OA. Further, mechanistic studies in MDAMB-231 cells revealed that CH-OA-PLGA induces apoptosis by mitochondrial membrane disruption; follows ROS mediated and caspase dependent apoptosis. The antitumor effect studied in 4-T1 induced Balb/c mice mammary tumor model displayed augmented antitumor potency by CH-OA-B-PLGA in comparison to OA. In the in vivo toxicity on Sprague-Dawley rat model, CH-OA-B-PLGA significantly displayed the safe profile and also preserves fertility in female rats. The experiment result suggests co-delivery of oleanolic acid with bio-enhancers as a breakthrough for developing safe chemotherapy for hormone independent breast cancer therapy countering the toxicity issues.

Content decline of SERCA inhibitors saikosaponin a and d attenuates cardiotoxicity and hepatotoxicity of vinegar-baked Radix bupleuri.

Improper usage of unprocessed Radix bupleuri root (chaihu) may cause cardiotoxicity and liver injury. Baking herb with vinegar is believed to attenuate the adverse responses. However, the chemical and molecular basis involved remained unclear. To this end, we investigated the in vitro toxicity of saikosaponin a, c, d, and their hydrolysates saikosaponin b1 and b2. Results showed that SSa and SSd possessed higher affinity with sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) by molecular docking, and exhibited stronger toxic responses on cardiomyocytes and hepatocytes than the other three saikosaponins in equivalent concentrations. Further, SSa and SSd induced LC3 puncta formation in U2OS-mCherry-EGFP-LC3 cells. Blockage of autophagy by 3-methyladenine did not abrogate the cytotoxicities induced by SSa and SSd. In parallel, none of SSc, SSb1, or SSb2 caused cell injury. Our study reveals how changes in chemical ingredients are connected to the toxicity of Chaihu during vinegar baking process and also provides a guidance for structure optimization to reduce drug induced toxicity.