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Pulmonary fibrosis (PF) is a progressive and fatal interstitial lung disease with limited therapeutic options at present, and epithelial–mesenchymal transition (EMT) is recognized as a major cause of lung fibrosis. Our previous work has confirmed that total extract of Anemarrhena asphodeloides Bunge [Asparagaceae] exerted the effect of anti‐PF. As a main constituent of Anemarrhena asphodeloides Bunge [Asparagaceae], the effect of timosaponin BII (TS BII) on drug‐induced EMT process in PF animals and alveolar epithelial cells remains unknown. In this study, we evaluated the effect of TS BII on bleomycin (BLM)‐induced PF. The results showed that TS BII could restore the structure of lung architecture and MMP‐9/TIMP‐1 balance in fibrotic rat lung and inhibit collagen deposition. Moreover, we found that TS BII could reverse the abnormal expression of TGF‐β1 and EMT‐related marker proteins including E‐cadherin, vimentin, and α‐SMA. Besides, aberrant TGF‐β1 expression and phosphorylation of Smad2 and Smad3 in BLM‐induced animal model and TGF‐β1‐induced cell model were downregulated by TS BII treatment, indicating that EMT in fibrosis was suppressed by inhibition of TGF‐β/Smad pathway both in vivo and in vitro. In summary, our study suggested that TS BII could be a promising candidate for PF treatment. [ABSTRACT FROM AUTHOR]

The safety evaluation of timosaponin BII (TBII) in beagle dogs with toxicokinetic study was performed. For the acute oral toxicity study, the minimum lethal dose (MLD) of TBII was more than 2000 mg/kg and suggested the characteristics of absorption saturation. For the 28-day repeated dose oral toxicity and toxicokinetic studies, there was no significant effect on all test parameters except for prolonged APTT in the 60 and 180 mg/kg groups, which recovered after withdrawal. The increase of drug exposure of 180 mg/kg group was not proportional to the increase of administration dose, showing the characteristics of absorption saturation. [ABSTRACT FROM AUTHOR]

Objective: To study the preventive effect of Timosaponin BII (T-BII) -loaded temperature/ion-sensitive nasal in situ hydrogels (ISGs) on Alzheimer's disease (AD), its preparation technology, characteristics and in vivo effects were evaluated. Methods: The morphological and rheological properties were evaluated. The preventive effects of T-BII ISG on scopolamine-induced AD in mice were determined with the index of muscarinicreceptor 1 (M1) expression and pathological changes. Results: Results revealed that T-BII ISG significantly increased the content of M1 choline receptors in the hippocampus of mice and ameliorated the damage incurred to the hippocampal cornu ammonis 1 (CA1) area. Conclusion: T-BII ISGs is a reasonable and convenient method of exerting an obvious preventive effect on mice with AD induced by scopolamine. This, thereby, lays forth a new treatment option for preventing AD.

Abstract Background Modern pharmacological studies have demonstrated that Baihe–Zhimu decoction (BZD) has antidepressant effects. However, the complex composition and lack of clear evaluation standards for BZD make it less likely to be understood and accepted than evidence-based active natural compounds. Methods In this study, an effective method for the identification of antidepressant components was demonstrated and applied to BZD. The first step was to evaluate the efficacy of BZD by the forced swimming test (FST) and the tail suspension test (TST), followed by successive quantitative analyses of the absorbed constituents at different stages, such as before hepatic disposition, liver distribution, after hepatic disposition and brain distribution after the oral administration of BZD. Finally, the compounds detected in the brain were confirmed by activity testing. Results Our investigation observed that timosaponin BII and timosaponin BIII were accurately determined in the brain after oral administration of BZD, and they were further confirmed to reduce the immobility time in the FST and TST. As described above, timosaponin BII and timosaponin BIII were used to scientifically and reasonably explain the effective chemical basis of the effect of BZD on depression. Conclusions This research affords an effective method to discover lead molecules for antidepressants from traditional Chinese medicine.

Defective autophagy occurred in osteoblasts under stress induced by high glucose and played an essential role in the development of diabetic osteoporosis. Timosaponin BII, a steroidal saponin isolated from the rhizomes of Anemarrhena asphodeloides Bunge, possessed anti-osteoporosis properties. In this study, we investigated the efficacy and mechanism of timosaponin BII on diabetic osteoporosis. Timosaponin BII attenuated the deterioration in the microarchitecture of the tibias in diabetic rats. Furthermore, treatment with timosaponin BII dose-dependently reduced hyperglycemia-induced cell apoptosis in primary osteoblasts from rat calvaria. High glucose-exposed osteoblasts exhibited increased mitochondrial superoxide level, decreased mitochondrial membrane potential and impaired autophagic flux, which was attenuated by timosaponin BII, as evidenced by the upregulation of autophagosome numbers, LC3B puncta formation and Beclin1 expression. The antiapoptotic and antioxidative effect of timosaponin BII were repressed by the autophagy inhibitor 3-methyladenine and enhanced by the autophagy inducer rapamycin. Further studies showed that timosaponin BII suppressed the phosphorylation of mTOR and S6K, as well as the downstream factors NFκB and IκB, consequently activating autophagy and decreasing apoptosis. Of note, coincubation of timosaponin BII with MHY1485, a pharmacological activator of mTOR, diminished the protein expression of Bcl2 induced by timosaponin BII, which was in parallel with decreased autophagy and increased phosphorylation of NFκB and IκB. Overexpression of NFκB reduced timosaponin BII-evoked autophagy and promoted apoptosis. The in vivo results showed that oral administration of timosaponin BII downregulated the phosphorylation of mTOR and NFκB and upregulated Beclin1 expression in the proximal tibias of diabetic rats. These results suggested that timosaponin BII attenuated high glucose-induced oxidative stress and apoptosis through activating autophagy by inhibiting mTOR/NFκB signalling in osteoblasts. [Display omitted] • TBII alleviated hyperglycemia-induced osteoporosis in the diabetic rats. • TBII reduced ROS levels via activating autophagy in osteoblast under hyperglycemia. • TBII activated autophagy via inhibiting mTOR/NFκB in in vivo and in vitro models. [ABSTRACT FROM AUTHOR]

The Timosaponin BⅡ (TBⅡ) is one of the main active components of the traditional Chinese medicine Anemarrhena asphodeloides , and it is a steroidal saponin with various pharmacological activities such as anti-oxidation, anti-inflammatory and anti-apoptosis. However, its role in acute ulcerative colitis remains unexplored thus far. This study aims to investigate the protective effect of TBⅡ against dextran sulfate sodium (DSS)-induced ulcerative colitis in mice and elucidate its underlying mechanisms. Wild-type (WT) and NLRP3 knockout (NLRP3−/−) mice were applied to evaluate the protective effect of TBⅡ in DSS-induced mice colitis. Pharmacological inhibition of NLRP3 or adenovirus-mediated NLRP3 overexpression in bone marrow-derived macrophages (BMDM) from WT mice and colonic epithelial HCoEpiC cells was used to assess the role of TBⅡ in LPS + ATP-induced cell model. RNA-seq, ELISA, western blots, immunofluorescence staining, and expression analysis by qPCR were performed to examine the alterations of colonic NLRP3 expression in DSS-induced colon tissues and LPS + ATP-induced cells, respectively. In mice with DSS-induced ulcerative colitis, TBⅡ treatment attenuated clinical symptoms, repaired the intestinal mucosal barrier, reduced inflammatory infiltration, and alleviated colonic inflammation. RNA-seq analysis and protein expression levels demonstrated that TBⅡ could prominently inhibit NLRP3 signaling. TBⅡ-mediated NLRP3 inhibition was associated with alleviating intestinal permeability and inflammatory response via the blockage of communication between epithelial cells and macrophages, probably in an NLRP3 inhibition mechanism. However, pharmacological inhibition of NLRP3 by MCC950 or Ad-NLRP3 mediated NLRP3 overexpression significantly impaired the TBⅡ-mediated anti-inflammatory effect. Mechanistically, TBⅡ-mediated NLRP3 inhibition may be partly associated with the suppression of NF-κB, a master pro-inflammatory factor for transcriptional regulation of NLRP3 expression in the priming step. Moreover, co-treatment TBⅡ with NF-κB inhibitor BAY11-7082 partly impaired TBⅡ-mediated NLRP3 inhibition, and consequently affected the IL-1β mature and secretion. Importantly, TBⅡ-mediated amelioration was not further enhanced in NLPR3−/− mice. TBⅡ exerted a prominent protective effect against DSS-induced colitis via regulation of alleviation of intestinal permeability and inflammatory response via the blockage of crosstalk between epithelial cells and macrophages in an NLRP3-mediated inhibitory mechanism. These beneficial effects could make TBⅡ a promising drug for relieving colitis. [Display omitted] • TBⅡ alleviated DSS-induced colitis by alleviating the damaged intestinal barrier and inflammatory response. • TBII inhibits NLRP3 inflammasoma-mediated inflammatory responses in intestinal epithelial cells and macrophages. • The mechanism may be through the inhibition of the communication between cells mediated by NLRP3 inflammasomes. [ABSTRACT FROM AUTHOR]

Timosaponin BII is one of the most abundant Anemarrhena saponins and is in a phase II clinical trial for the treatment of dementia. However, the pharmacological activity of timosaponin BII does not match its low bioavailability. In this study, we aimed to determine the effects of gut microbiota on timosaponin BII metabolism. We found that intestinal flora had a strong metabolic effect on timosaponin BII by HPLC-MS/MS. At the same time, seven potential metabolites (M1-M7) produced by rat intestinal flora were identified using HPLC/MS-Q-TOF. Among them, three structures identified are reported in gut microbiota for the first time. A comparison of rat liver homogenate and a rat liver microsome incubation system revealed that the metabolic behavior of timosaponin BII was unique to the gut microbiota system. Finally, a quantitative method for the three representative metabolites was established by HPLC-MS/MS, and the temporal relationship among the metabolites was initially clarified. In summary, it is suggested that the metabolic characteristics of gut microbiota may be an important indicator of the pharmacological activity of timosaponin BII, which can be applied to guide its application and clinical use in the future.

Abstract: Peroxide and oxygen free radicals are some of the causes of oxidative stress in brain tissue, and could lead to the change of brain structure and function. In addition, oxidative damage is one of the most important causes of the aging of the vast majority of tissues. The aim of this study is to investigate the protective effect of timosaponin BII on oxidative stress damage of PC12 induced by H2O2 using metabolomics based on the UHPLC‐Q‐TOF‐MS technique. Partial least‐squares discriminant analysis method was used to identify 35 metabolites as decisive marker compounds in a preliminary interpretation of the mechanism of the antioxidative effect of timosaponin BII. The majority of these metabolites are involved in the glutathione metabolism, amino acid metabolism, sphingolipid and glycerophospholipid metabolism. Our results suggest that timosaponin BII demonstrates systematic antioxidant effects in the PC12 oxidative damage cell model via the regulation of multiple metabolic pathways. These findings provide insight into the pathophysiological mechanisms underlying oxidative stress damage and suggest innovative and effective treatments for this disorder, providing a reliable basis for the development of novel therapeutic target in timosaponin BII treatment of oxidative stress. [ABSTRACT FROM AUTHOR]

Timosaponin BII (TBII), a major steroidal saponin isolated from Anemarrhena asphodeloides Bge., displays a variety of promising pharmacological activities, such as neuroprotection, enhancement of learning and memory, vascular protection and inhibition of platelet aggregation; therefore, it has been developed as a pharmaceutical for prevention or treatment of dementia. Given the safety concerns surrounding timosaponins and the absence of studies on the safety of TBII, the potential toxicity of TBII was evaluated in toxicity and toxicokinetic studies in rats. In the acute oral toxicity study, loose stools were observed in rats receiving 4000 mg/kg, and the symptoms recovered within 1 day. In the 28-day repeated-dose oral toxicity and toxicokinetic study, rats receiving 540 mg/kg showed loose stools and a slight deceleration of body weight growth in both sexes, and the females also showed a slight decrease in food consumption. Moreover, urinalysis indicated reversible treatment-related toxicity in rats receiving 540 mg/kg. The toxicokinetic study demonstrated a dose-dependent increase in systematic exposure to TBII after 28 successive days of oral treatment with TBII. The accumulation coefficients of TBII were 4.35, 1.70 and 1.81, respectively, in rats that received 60, 180 and 540 mg/kg. The no-observed-adverse-effect level (NOAEL) is proposed to be 180 mg/kg. [ABSTRACT FROM AUTHOR]

Timosaponin BII (TBII) is an active constituent of some Chinese medicinal herbs, and is abundant in Anemarrhena asphodeloides Bunge. To better understand the pharmaceutical mechanisms of TBII, we prepared its deglycosylated derivatives through biotransformation with fungi Colletotrichum gloeosporioides , Acremonium alternatum and Aspergillus niger. In the presence of 10 g/L glucose, transformation with C. gloeosporioides yielded 4 products: timosaponin AIII (P3) and AI (P5) and their beta isoforms (P2 and P4 respectively). However, P4 and P5 could not be detected when 0–5 g/L glucose was applied; P2 and P3 disappeared at 20 g/L glucose. P4 is a newly discovered compound. We firstly report that fungi in whole cell model can hydrolyze C3- O -glucopyranosyl residues of steroidal saponins after removing the C26 sugar. Therefore, the transformation efficiency depends on glucose. Transformation of TBII, and maybe other steroidal saponins, with C. gloeosporioides is an ideal method to prepare the metabolites in vitro . [ABSTRACT FROM AUTHOR]

Timosaponin BII (1), a steroidal saponin showing potential anti-dementia activity, was regioselectively hydrolyzed into its deglycosyl derivatives by the crude enzyme from Aspergillus niger AS 3.0739. Three biotransformation products, timosaponin BII-a (2), timosaponin BII-b (3), and timosaponin BII-c (4), were purified and their structures were elucidated on the basis of 1D NMR, 2D NMR, FAB-MS, and HR-ESI-MS spectral data. Compounds 2 and 3 are new compounds. [ABSTRACT FROM AUTHOR]

Abstract: Timosaponin BII (BII), a steroidal saponin showing potential anti-dementia activity, was converted into its glucosylation derivatives by Toruzyme 3.0L. Nine products with different degrees of glucosylation were purified and their structures were elucidated on the basis of 13C NMR, HR-ESI-MS, and FAB-MS spectra data. The active enzyme in Toruzyme 3.0L was purified to electrophoretic homogeneity by tracking BII-glycosylase activity and was identified as Cyclodextrin-glycosyltransferase (CGTase, EC 2.4.1.19) by ESI-Q-TOF MS/MS. In this work, we found that the active enzyme catalyzed the synthesis of alpha-(1→4)-linked glucosyl-BII when dextrin instead of an expensive activated sugar was used as the donor and showed a high thermal tolerance with the most favorable enzymatic activity at 100°C. In addition, we also found that the α-amylases and CGTase, that is, GH13 family enzymes, all exhibited similar activities, which were able to catalyze glucosylation in steroidal saponins. But other kinds of amylases, such as γ-amylase (GH15 family), had no such activity under the same reaction conditions. [ABSTRACT FROM AUTHOR]

An aqueous extract of Anemarrhena asphodeloides B unge (Anemarrhenae Rhizoma) showed inhibitory effects on α-glucosidase and aldose reductase (AR). We obtained two active compounds, mangiferin (α-glucosidase: IC50 = 96.1 μg/ml, AR:IC50 = 0.13 μg/ml) and mangiferin 7- O-β-D-glucopyranoside (α-glucosidase: IC50 = 158 μg/ml, AR:IC50 = 65.7 μg/ml ), from Anemarrhenae Rhizoma. In addition, the amount of mangiferin in Anemarrhenae Rhizoma changed with the method of preparation (drying conditions), namely, low-temperature, long-term drying conditions and harvesting in August were most effective. [ABSTRACT FROM AUTHOR]

Timosaponin BII is one of the most abundant Anemarrhena saponins and is in a phase II clinical trial for the treatment of dementia. However, the pharmacological activity of timosaponin BII does not match its low bioavailability. In this study, we aimed to determine the effects of gut microbiota on timosaponin BII metabolism. We found that intestinal flora had a strong metabolic effect on timosaponin BII by HPLC-MS/MS. At the same time, seven potential metabolites (M1–M7) produced by rat intestinal flora were identified using HPLC/MS-Q-TOF. Among them, three structures identified are reported in gut microbiota for the first time. A comparison of rat liver homogenate and a rat liver microsome incubation system revealed that the metabolic behavior of timosaponin BII was unique to the gut microbiota system. Finally, a quantitative method for the three representative metabolites was established by HPLC-MS/MS, and the temporal relationship among the metabolites was initially clarified. In summary, it is suggested that the metabolic characteristics of gut microbiota may be an important indicator of the pharmacological activity of timosaponin BII, which can be applied to guide its application and clinical use in the future. [ABSTRACT FROM AUTHOR]

Background: Timosaponin BⅡ (TBⅡ), one of the primary bioactive compounds from Anemarrhena asphodeloides Bunge, possesses potential cardioprotective effects. However, the mechanism underlying TBⅡ-mediated cardioprotection, especially the involvement of endoplasmic reticulum stress, remains largely unknown.Purpose: This study was designed to evaluate the role of TBⅡ in myocardial injury protection and explore its possible mechanisms.Methods: In vivo models of isoproterenol-induced myocardial injury and H2O2-induced cytotoxicty were established to investigate the effect of anti-myocardial injury of TBⅡ. The potential mechanisms were investigated in vitro and in vivo using multiple detection methods like electrocardiography, histo-pathological examination, JC-1 staining, TUNEL staining, ELISA technology, and western blot analysis.Results: In vivo study revealed that TBⅡ improved electrocardiography and heart vacuolation, reduced myocyte apoptosis, and improved the antioxidant potential. In vitro investigation demonstrated that TBⅡ pretreatment inhibited ER stress-mediated apoptosis pathways. Further investigation of the underlying mechanisms revealed that TBⅡ prevented H2O2-induced H9c2 cardiomyocytes injury by the PI3K/Akt pathways, whereas the addition of LY294002, the pharmacologic antagonist of PI3K, attenuated TBⅡ-induced expression of apoptotic protein and cytoprotective effects.Conclusion: These results suggested that TBⅡ protects against myocardial injury in vitro and enhances cellular defense capacity by inhibiting ER stress-mediated apoptosis pathways in vivo by activating the PI3K/Akt pathways. [ABSTRACT FROM AUTHOR]

• A novel thermo- and ion-sensitive in-situ hydrogels (ISGs) of timosaponin BII were prepared. • Timosaponin BII ISGs significantly improved the behavior manifestation of LPS-injured mice. • Brain-targeted timosaponin BII ISGs had mucosa adhesion. Alzheimer's disease (AD) is a common and severe brain disease with a high mortality among the elders, but no highly efficient medications are currently available. For example, timosaponin BII, an efficient anti-AD agent, has low oral bioavailability. Here, timosaponin BII was formulated in a temperature/ion-sensitive in situ hydrogel (ISG) that was well transformed into gels in the nasal environment. Timosaponin BII protected the PC12 cells injured by lipopolysaccharides (LPS) by decreasing TNF-α and IL-1β and stabilizing F-actin. Timosaponin BII ISGs were intranasally administered to the mice every day for 38 days. On Day 36, LPS was injected to the mice to establish an AD model. Morris water maze experiments showed that the number of the animals that were able to cross the platform returned to normal and the total distance over which the animals moved in the open field also increased, which demonstrated that the spatial memory and spontaneous behavior were improved after treatment compared to the model. Moreover, an AD improver, inducible nitric oxide synthase (iNOS) in the brain, was reduced after treatment. High brain targeting effect of timosaponin BII ISGs was confirmed by in vivo fluorescence imaging. The nasal timosaponin BII dually sensitive ISGs can serve as a promising medication for local prevention of AD. [ABSTRACT FROM AUTHOR]

Anemarrhena asphodeloides Bunge is a traditional Chinese medicine. The timosaponin BII is one of the most abundant and widely studied active ingredients in Anemarrhena asphodeloides Bunge. Related studies have shown that timosaponin BII has potential value for development and further utilization. The protective effect of timosaponin BII on islet β cells under type 2 diabetes was investigated in the glycolipid toxic INS-1 cell model and possible biomarkers were explored by lipidomics analysis. Timosaponin BII was isolated from Anemarrhena asphodeloides Bunge by polyamide resin and Sephadex LH-20. Then, the glycolipid toxicity INS-1 cell model was established to investigate the protective effect of timosaponin BII. The results showed that timosaponin BII could significantly influence the levels of malondialdehyde (MDA) and glutathione (GSH), thereby restoring the insulin secretion ability and cell viability of model cells. Lipidomics analysis was combined with multivariate statistical analysis for marker selection. The four most common pathological and pharmacological lipid markers were phosphatidylserine (PS), suggesting that timosaponin BII had protective effects on model cells related to the reduction oxidative stress and macrophage inflammation. RAW264.7 macrophages were stimulated by LPS to establish a model of inflammation and study the effect of timosaponin BII on the nodes of NOD-like receptor P3 (NLRP3) inflammasome pathway in the model cells. In conclusion, timosaponin BII may have the effect of protecting INS-1 pancreatic β cells through reducing IL-1β (interleukin-1β) production by inhibiting the NLRP3 inflammasome in macrophage and restoring the insulin secretion ability and cell viability by reducing oxidative stress., (© 2020 Wiley-VHCA AG, Zurich, Switzerland.)

Background: Modern pharmacological studies have demonstrated that Baihe–Zhimu decoction (BZD) has antidepressant effects. However, the complex composition and lack of clear evaluation standards for BZD make it less likely to be understood and accepted than evidence-based active natural compounds. Methods: In this study, an effective method for the identification of antidepressant components was demonstrated and applied to BZD. The first step was to evaluate the efficacy of BZD by the forced swimming test (FST) and the tail suspension test (TST), followed by successive quantitative analyses of the absorbed constituents at different stages, such as before hepatic disposition, liver distribution, after hepatic disposition and brain distribution after the oral administration of BZD. Finally, the compounds detected in the brain were confirmed by activity testing. Results: Our investigation observed that timosaponin BII and timosaponin BIII were accurately determined in the brain after oral administration of BZD, and they were further confirmed to reduce the immobility time in the FST and TST. As described above, timosaponin BII and timosaponin BIII were used to scientifically and reasonably explain the effective chemical basis of the effect of BZD on depression. Conclusions: This research affords an effective method to discover lead molecules for antidepressants from traditional Chinese medicine. [ABSTRACT FROM AUTHOR]

Ethnopharmacological Relevance: Oxidative damage to osteoblasts was a key factor in the development of osteoporosis. Er-Xian Decotion (EXD) is widely used in China for the treatment of osteoporosis, which has a variety of antioxidant active ingredients. EXD may be an important source of protection against oxidative damage in osteoblasts, but the anti-osteoporotic active components of EXD is currently unclear., Aim of the Study: This work established an effective and reliable drug screening method to find main active ingredients in EXD (M-EXD) that can protect osteoblasts against oxidative stress and achieve anti-osteoporosis effects., Materials and Methods: H 2 O 2 -induced osteoblast cell fishing with UHPLC-QTOF/MS was firstly used to discover the potential active components from EXD. Afterword, the EXD compound-osteoporosis target network was constructed using network pharmacology, thus potentially anti-osteoporosis ingredients were founded, and their combination were defined as the M-EXD. Finally, pharmacology effects of M-EXD was evaluated by ovariectomized rats, prednisolone induced-zebrafish and H 2 O 2 -induced osteoblasts., Results: 40 candidate active ingredients in EXD were initially screened out via pathological cell fishing. According to network pharmacology result, M-EXD consisted of 13 ingredients since they had a close relationship with 65 osteoporosis-related targets. Pharmacological evaluation showed that M-EXD significantly ameliorated oxidative stress in H 2 O 2 -induced osteoblast model, evidently reversed the activity of ALP, ROS, GSH-px, NO and MDA compared with the model group. M-EXD showed better anti-oxidative activities than individual ingredients, presenting obvious synergetic effects. In osteoporosis rat and zebrafish models, M-EXD also demonstrated good anti-osteoporotic properties by mitigating the osteoporosis bone loss and increasing serum bone morphogenetic protein 2, and reversing osteocalcin expression in bone tissue. It significantly ameliorated oxidative stress in the in-vivo models. Moreover, M-EXD and EXD showed similar anti-osteoporotic and anti-oxidative properties, while the rest components of EXD had no satisfactory anti-osteoporotic efficacy., Conclusions: Our work successfully identified the main active components in EXD, which could represent the efficacy of EXD on treating osteoporosis, and meanwhile, it also provided an effective strategy to investigate active ingredients from natural medicines, which might be helpful for drug development and application., (Copyright © 2019 Elsevier B.V. All rights reserved.)