Contamination of the traditional medicine Radix Dipsaci with aflatoxin B1 impairs hippocampal neurogenesis and cognitive function in a mouse model of osteoporosis.

BACKGROUND: Aflatoxin B1, which can penetrate the blood-brain barrier and kill neural cells, can contaminate traditional herbal medicines, posing a significant risk to human health. The present study examined cellular, cognitive and behavioral consequences of aflatoxin B1 contamination of the anti-osteoporotic medicine Radix Dipsaci. METHODS: A mouse model of osteoporosis was created by treating the animals with all-trans-retinoic acid. Then the animals were treated intragastically with water decoctions of Radix Dipsaci that contained detectable aflatoxin B1 or not. The animals were compared in terms of mineral density and mineral salt content of bone, production of pro-inflammatory factors, neurogenesis and microglial activation in hippocampus, as well as behavior and cognitive function. RESULTS: Contamination of Radix Dipsaci with aflatoxin B1 significantly reduced the medicine's content of bioactive saponins. It destroyed the ability of the herbal decoction to improve mineral density and mineral salt content in the bones of diseased mice, and it induced the production of the oxidative stress marker malondialdehyde as well as the pro-inflammatory cytokines interleukin-1ß and tumor necrosis factor-α. Aflatoxin B1 contamination inhibited formation of new neurons and increased the proportion of activated microglia in the hippocampus. These neurological changes were associated with anhedonia, behavioral despair, and deficits in short-term memory and social memory. CONCLUSION: Contamination of Radix Dipsaci with aflatoxin B1 not only eliminates the herbal decoction's anti-osteoporotic effects, but it also induces neurotoxicity that can lead to cognitive decline and behavioral abnormalities. Such contamination should be avoided through tightly regulated production and quality control of medicinal herbs.

[Material basis of kidney Yang deficiency rats before and after salt processing of Dipsacus asper based on spectrum-effect relationship].

This paper aims to study the spectrum-effect relationship between the fingerprints before and after salt processing of Dipsacus asper and the efficacy of warming and tonifying kidney Yang and find the main active components against kidney Yang deficiency before and after salt processing of D. asper, so as to provide the basis for clarifying the effect of salt processing on kidney Yang deficiency. The HPLC fingerprint before and after salt processing of D. asper was established by the HPLC-DAD. 15 common peaks were obtained, and 11 components were identified. The content changes of various components in rat serum were detected, and the difference in efficacy before and after salt processing was compared. The results of pharmacological experiments showed that salt processing of D. asper could enhance the kidney index. At the same dose, there was a significant difference between the raw D. asper and D. asper after salt processing groups. Compared with the model group, the contents of ACTH, cAMP, CORT, E_2, GH, Na~+-K~+-ATPase, T, and T4 in the serum of rats in the administration group increased to a certain extent, and the contents of cGMP and TNF-α decreased to a certain extent. Among them, there were significant differences in the above indexes in the serum of rats in the high-dose group of raw D. asper, middle-dose group of D. asper after salt processing, high-dose group of D. asper after salt processing, and the positive drug group. The overall results showed that D. asper after salt processing was more effective than raw D. asper in preventing kidney yang deficiency. The efficacy of D. asper was evaluated by grey correlation analysis, entropy method, and Pearson correlation analysis, and the components of D. asper after salt processing against kidney yang deficiency were screened out. According to the results of correlation degree ranking, the components with increased ranking before and after salt processing of D. asper were loganin, chlorogenic acid, dipsacoside A, asperosaponin Ⅵ, caffeic acid, and isochlorogenic acid B. It was preliminarily speculated that these compounds may be the potential pharmacodynamic components for the treatment of kidney yang deficiency before and after salt processing of D. asper. The changing components before and after the salt processing of D. asper were determined, which proved that D. asper after salt processing was superior to D. asper in the treatment of kidney yang deficiency. The spectrum-effect relationship between the efficacy of D. asper before and after salt processing and the treatment of kidney yang deficiency was established, which laid a foundation for the subsequent study on the pharmacodynamic components and molecular mechanism of salt processing of D. asper.

An exploratory metabolomic study reveals the Dipsacus asper-Achyranthes bidentate herb pair against osteoarthritis by modulating imbalance in polyunsaturated fatty acids and energy metabolism.

Osteoarthritis (OA) is a degenerative joint disease primarily affecting the cartilage. The therapeutic potential of the Dipsacus asper-Achyranthes bidentate herb pair for OA has been acknowledged, yet its precise mechanism remains elusive. In this study, we conducted a comprehensive analysis of metabolomic changes and therapeutic outcomes in osteoarthritic rats, employing a gas chromatography-mass spectrometry-based metabolomics approach in conjunction with histopathological and biochemical assessments. The rats were divided into six groups: control, model, positive control, Dipsacus asper treated, Achyranthes bidentata treated, and herb pair treated groups. Compared to the model group, significant reductions in levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and iNOS were observed in the treated groups. Multivariate statistical analyses were employed to investigate metabolite profile changes in serum samples and identify potential biomarkers, revealing 45 differential biomarkers, with eighteen validated using standard substances. These analytes exhibited excellent linearity across a wide concentration range (R2>0.9990), with intra- and inter-day precision RSD values below 4.69% and 4.83%, respectively. Recoveries of the eighteen analytes ranged from 93.97% to 106.59%, with RSD values under 5.72%, underscoring the method's reliability. Treatment with the herbal pair effectively restored levels of unsaturated fatty acids such as linoleic acid and arachidonic acid, along with glucogenic amino acids. Additionally, levels of phosphoric acid and citric acid were reversed, indicating restoration of energy metabolism. Collectively, these findings highlight the utility of metabolomic analysis in evaluating therapeutic efficacy and elucidating the underlying molecular mechanisms of herb pairs in OA treatment.

Dipsacus Asperoides-Derived Exosomes-Like Nanoparticles Inhibit the Progression of Osteosarcoma via Activating P38/JNK Signaling Pathway.

Introduction: Osteosarcoma is a prevalent and highly malignant primary bone tumor. However, current clinical therapeutic drugs for osteosarcoma are not suitable for long-term use due to significant side effects. Therefore, there is an urgent need to develop new drugs with fewer side effects. Dipsacus asperoides C. Y. Cheng et T. M. Ai, a traditional Chinese medicine, is commonly used for its anti-inflammatory, anti-pain, bone fracture healing, and anti-tumor effects. In this study, we investigated the effects of exosome-like nanoparticles derived from Dipsacus asperoides (DAELNs) on osteosarcoma cells in vitro and in vivo. Methods: DAELNs were isolated and purified from Dipsacus asperoides and their physical and chemical properties were characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The cellular uptake of DAELNs in osteosarcoma cells was analyzed by PKH26 staining. The proliferation, invasion, migration, and apoptosis of osteosarcoma cells were assessed using CCK8 assay, EdU assay, colony-formation assay, transwell assay, wound healing assay, and mitochondrial membrane potential measurement, respectively. The regulatory mechanism of DAELNs inhibiting the progression of osteosarcoma via activating P38/JNK signaling pathway was investigated using Western blotting and immunohistochemistry. Moreover, the therapeutic effects of DAELNs were evaluated using in vivo small animal imaging assay, HE staining, and immunohistochemistry. Results: Our results showed that DAELNs inhibited the proliferation, invasion, migration, and fostered the apoptosis of osteosarcoma cells in vitro and suppressed the tumor growth of osteosarcoma cells in a xenograft nude mouse model. Furthermore, the bio-distribution of DiD-labeled DAELNs showed preferential targeting of osteosarcoma tumors and excellent biosafety in histological analysis of the liver and kidney. Mechanistically, DAELNs activated the P38/JNK signaling pathway-induced apoptosis. Conclusion: Taken together, DAELNs are novel, natural, and osteosarcoma-targeted agents that can serve as safe and effective therapeutic approaches for the treatment of osteosarcoma.

Investigation of Phytochemical, Antioxidant and Antidiabetic Potentials of Scabiosa L. (Caprifoliaceae) Species with Chemometric Methods.

In this research, the total phenolic and flavonoid amounts, phenolic compositions, in vitro antioxidant, antibacterial and antidiabetic properties of the methanol extracts obtained from Scabiosa L. (Caprifoliaceae) species distributed in the flora of Türkiye were investigated using chemometric methods. For this purpose, principal component (PCA) and agglomerative hierarchical clustering analysis were performed as chemometric methods. Chlorogenic acid, quinic acid and cyranoside were determined in the extracts. According to chemometric analysis, S. columbaria subsp. ochroleuca var. ochroleuca and S. triniifolia species were found to be valuable in terms of methanol extract yields, total phenolic and flavonoid contents, antioxidant and antidiabetic activities while S. columbaria subsp. ochroleuca var. webbiana species were found to be valuable in terms of phenolic composition. The methanol extracts of Scabiosa species showed high antioxidant activity, with high phenolic and flavonoid contents. Among the tested 13 bacteria, Scabiosa extracts showed only low activity against Klebsiella pneumoniae, Streptococcus pneumoniae and Pseudomonas aeruginosa. The extracts showed high α-amylase and α-glucosidase inhibitory activity. The results show that Scabiosa methanol extracts may be a source of alternative antioxidants that may be beneficial in slowing or preventing the progression of various oxidative stress-related diseases.

Evaluation of acute toxicity of Scabiosa artropurperea var.maritima aqueous extracts in Swiss mice.

Scabiosa artropurperea var.maritima is a plant widely distributed in the Mediterranean region and used as a traditional medicine. The present study evaluated the biochemical composition and the potential toxicity of aqueous extract of whole Scabiosa artropurperea var.maritima through acute toxicity oral administration in male mice. Phytochemical analysis of the Scabiosa artropurperea var.maritima revealed high levels of reductor sugars and significant flavonoid and total phenol content. The aqueous extract of Scabiosa artropurperea var.maritima was daily oral administered to mice at doses of 300 (group 1), 2000 (group 2) and 4000 (group 3) mg/kg body weight per day for 14 days. We observed no significant difference in the consumption of food, body weight and relative organ weights except for an increase in the seminal vesicles weight in group 3. Hematological parameters revealed the non-adverse effects of prolonged oral consumption of Scabiosa artropurperea var.maritima except for a slight increase but significant of percentage of hematocrit in group 1 and 3 and a decrease in percentage of granulocytes in group 2. The histopathologic examination did not show any differences in vital organs. We also observed non-adverse effects on the reproductive parameters including testosterone concentration, spermatozoa motility and morphologies. Based on our findings, the aqueous extract of Scabiosa artropurperea var.maritima could be considered safe for oral medication in animals.

Four new triterpene saponins from Cephalaria speciosa and their potent cytotoxic and immunomodulatory activities.

Four new triterpene saponins, namely speciosides A-D (1-4) along with six known saponins were isolated from the n-butanol extract of Cephalaria speciosa. In addition to these, three new prosapogenins (2a-4a) were obtained after alkaline hydrolysis. Elucidation of the structures of the isolated compounds was carried out by 1D, 2D NMR, HR-ESI/MS and GC-MS analyses. Cytotoxic activity was investigated on A549, CCD34-Lu, MDA-MB-231, PC-3, U-87MG, HeLa, HepG-2 cells by MTT method. Additionally, the immunomodulatory effect of compounds was evaluated for macrophage polarization with/without inactivated IBV D274 antigen treatment on THP-1 cells originated macrophage cells in terms of M1 or M2. According to the cytotoxicity results, compound 1 and prosapogenin 2a exhibit significant cytotoxicity than doxorubicin by comparison. The results demonstrated that saponin molecules treated THP-1 originated macrophages were induced M1 and/or M2 polarization. Additionally, macrophage cells treated with/without IBV D274 antigen contained saponin compounds were triggered significantly M2 polarization relative to M1. Notably, monodesmosidic saponins (1 and 2a-4a) in comparison with bisdesmosidic ones (2-4) demonstrated the most effect on M2 polarization. In conclusion, the results showed that all the isolated new saponins and their prosapogenins have immunomodulatory potential on macrophage cells increasing immune response without significant cytotoxic effect on THP-1 originated macrophages.

Oleanolic Acid Glycosides from Scabiosa caucasica and Scabiosa ochroleuca: Structural Analysis and Cytotoxicity.

In the field of research on medicinal plants from the Armenian flora, the phytochemical study of two Scabiosa L. species, S. caucasica M. Bieb. and S. ochroleuca L. (Caprifoliaceae), has led to the isolation of five previously undescribed oleanolic acid glycosides from an aqueous-ethanolic extract of the roots: 3-O-α-L-rhamnopyranosyl-(1→3)-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester, 3-O-ß-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester, 3-O-ß-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid, 3-O-ß-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-ß-D-xylopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester, 3-O-α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester. Their full structural elucidation required extensive 1D and 2D NMR experiments, as well as mass spectrometry analysis. For the biological activity of the bidesmosidic saponins and the monodesmosidic saponin, their cytotoxicity on a mouse colon cancer cell line (MC-38) was evaluated.

The Anti-Acne Potential and Chemical Composition of Knautia drymeia Heuff. and Knautia macedonica Griseb Extracts.

The treatment of acne and other seborrheic diseases has arisen as a significant clinical challenge due to the increasing appearance of multi-drug resistant pathogens and a high frequency of recurrent lesions. Taking into consideration the fact that some Knautia species are valuable curatives in skin diseases in traditional medicine, we assumed that the thus far unstudied species K. drymeia and K. macedonica may be a source of active substances used in skin diseases. The purpose of this study was to evaluate the antioxidant, anti-inflammatory, antibacterial, and cytotoxic activities of their extracts and fractions. An LC-MS analysis revealed the presence of 47 compounds belonging to flavonoids and phenolic acids in both species while the GC-MS procedure allowed for the identification mainly sugar derivatives, phytosterols, and fatty acids and their esters. The ethanol as well as methanol-acetone-water (3:1:1) extracts of K. drymeia (KDE and KDM) exhibited great ability to scavenge free radicals and good capacity to inhibit cyclooxygenase-1, cyclooxygenase-2, and lipoxygenase. Moreover, they had the most favorable low minimal inhibitory concentration values against acne strains, and importantly, they were non-toxic toward normal skin fibroblasts. In conclusion, K. drymeia extracts seem to be promising and safe agents for further biomedical applications.

Dipsacus and Scabiosa Species-The Source of Specialized Metabolites with High Biological Relevance: A Review.

The genera Dipsacus L. and Scabiosa L. of the Caprifoliaceae family are widely distributed in Europe, Asia, and Africa. This work reviews the available literature on the phytochemical profiles, ethnomedicinal uses, and biological activities of the most popular species. These plants are rich sources of many valuable specialized metabolites with beneficial medicinal properties, such as triterpenoid derivatives, iridoids, phenolic acids, and flavonoids. They are also sources of essential oils. The genus Dipsacus has been used for centuries in Chinese and Korean folk medicines to treat bone (osteoporosis) and joint problems (rheumatic arthritis). The Korean Herbal Pharmacopoeia and Chinese Pharmacopoeia include Dipsaci radix, the dried roots of D. asperoides C.Y.Cheng & T.M.Ai. In addition, S. comosa Fisch. ex Roem & Schult. and S. tschiliiensis Grunning are used in traditional Mongolian medicine to treat liver diseases. The current scientific literature data indicate that these plants and their constituents have various biological properties, including inter alia antiarthritic, anti-neurodegenerative, anti-inflammatory, antioxidant, anticancer, and antimicrobial activities; they have also been found to strengthen tendon and bone tissue and protect the liver, heart, and kidney. The essential oils possess antibacterial, antifungal, and insecticidal properties. This paper reviews the key biological values of Dipsacus and Scabiosa species, as identified by in vitro and in vivo studies, and presents their potential pharmacological applications.

Exploring active ingredients of anti-osteoarthritis in raw and wine-processed Dipsaci Radix based on spectrum-effect relationship combined with chemometrics.

ETHNOPHARMACOLOGICAL RELEVANCE: Dipsaci Radix (DR) is the dry root of the Dipsacus asper Wall. ex DC., which has the function of tonifying the liver and kidney, continuing tendons and bones, and regulating blood vessels. However, there are few reports on the main active ingredients. AIM OF THE STUDY: This study aimed to find the main active components of DR in the treatment of osteoarthritis (OA) by spectrum-effect relationship and compare the differences between RDR and WDR. MATERIALS AND METHODS: Firstly, the high-performance liquid chromatography (HPLC) method was used to establish the fingerprint of DR, and 10 peaks of them were determined by UPLC-Q-TOF/MS. Then, the OA rat model was established by injecting sodium iodoacetate to study the effect of DR on OA. The spectrum-effect relationship was analyzed by grey relational analysis (GRA) and Pearson correlation analysis. RESULTS: According to the pharmacological results, compared with the model group, the cartilage score, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6), and Mankin score of rats in low, medium and high dose groups were decreased, and the therapeutic effect of wine-processed DR tended to be better than raw DR at the same dose. Finally, the active components of DR were preliminarily determined as 4 (loganic acid), 6 (chlorogenic acid), 8 (caffeic acid), 14 (dipsanoside B), 16, and 17 (asperosaponin VI) which had a large correlation in GRA and Pearson correlation analysis. CONCLUSION: This study established the spectrum-effect relationship between the raw and wine-processed DR for the first time, which provided a theoretical basis for the study of the pharmacodynamic substance basis of DR before and after processing. This research provided a reference for the subsequent study of DR.

In vitro anticancer, antioxidant and enzyme inhibitory potentials of endemic Cephalaria elazigensis var. purpurea with in silico studies.

In this study, the therapeutic potential and phytochemical composition of ethanolic extract of Cephalaria elazigensis var. purpurea (CE), an endemic species, were investigated. For this purpose, the antiproliferative effect of CE on the MCF-7 human breast cancer cell line was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and its effectiveness on colony formation and cell migration was analyzed with clonogenic assay and wound healing assay, respectively. In addition, the cell death detection ELISA (CDDE) assay was conducted to determine the pro-apoptotic capacity of CE. The IC50 value of the CE was determined as 324.2 ± 14.7 µg/mL. Furthermore, upon 1000 µg/mL CE treatment, there was 4.96-fold increase in the population of cells undergoing apoptosis compared to the untreated control cells. The antioxidant activity tests were performed by DPPH free radical, ABTS cation radical, ferric-ion reducing power (FRAP) and ferrous-ion chelating power (FCAP) assays. Antioxidant activity values for the DPPH, ABTS and FRAP assays were found to be 125.6 ± 6.3, 34.09 ± 0.1 and 123.4 ± 4.2 µmol TE/mg DE, respectively. We further determined the effect of CE ethanolic extract against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes. CE plays an effective inhibitory role in AChE and BuChE (AChE: IC50: 10.54 µg/mL, BuChE: IC50: 6.84 µg/mL) respectively. Further, molecular docking stuy was conducted to understand the nature of the all compound against AChE an BChE. It is revealed that α-Linolenic acid shows lowest binding energy (-7.90 kcal/mol) towards AChE, on the other side, Linoleic acid shows good binding affinity (-7.40 kcal/mol) for BChE.Communicated by Ramaswamy H. Sarma.

Comparative analysis of anti-osteoporosis efficacy in Radix Dipsaci before and after processing with salt based on spectrum-effect relationship.

Radix Dipsaci (RD) is the dry root of the Dipsacus asper Wall. ex DC., which has the effect of strengthening muscles and bones. The purpose of this study was to find the main active ingredients that could improve the anti-osteoporosis efficacy of RD after processing with salt. The fingerprints of raw and salt-processed RD were established by HPLC-DAD to determine the common components. Then, an experimental study on the anti-osteoporosis efficacy was carried out to compared the difference in the efficacy between raw and salt-processed RD. Pharmacological results showed that, compared with the model group, both the raw and salt-processed RD were able to increase the Ca, bone mineral content, bone mineral density, trabeculae bone area and number of trabeculae bone of rats, and reduce the P, alkaline phosphatase, osteocalcin and trabecular bone separation of rats. Under the same dose, the pharmacological effect of salt-processed RD group was better than that of raw RD group. Finally, spectrum-effect relationship between fingerprints and anti-osteoporosis efficacy of RD was assessed by grey relational analysis and entropy method to screening out the ingredients that affect the anti-osteoporosis efficacy in RD after processing with salt. The results showed that the anti-osteoporosis efficacy of salt-processed RD was stronger than that of raw RD, and the pharmacologically active ingredients that improved its anti-osteoporosis efficacy after processing with salt were peak 4, peak 7 (caffeic acid), peak 8 (loganin), peak 12 (isochlorogenic acid C), peak 13 (dipsanoside A) and peak 14. As far as we known, this was the first time to establish the spectrum-effect relationship between RD and anti-osteoporosis efficacy, which laid the foundation for the follow-up research on the pharmacodynamic components and molecular mechanism of RD.

[Cloning and bioinformatics analysis of ß-amyrin synthase in Dipsacus asper].

Dipsaci Radix is one of the commonly used Chinese medicinal materials in China, with a long history. It has the medicinal activities of nourishing liver and kidney, recovering from broken sinews, and treating bone fracture. Triterpenoid saponins are the main functional ingredients of Dipsacus asper. ß-Amyrin synthases(ß-AS) as a superfamily of oxidosqualene cyclases(OSCs) can catalyze the construction of the skeleton structure of oleanane-type triterpenoid saponins. There are only a few studies about the ß-AS in D. asper, and the catalytic mechanism of this enzyme remains to be explored. To enrich the information of ß-AS, according to the transcriptome sequencing results, we cloned DaWß-AS gene from D. asper into a specific vector for heterologous expression in Escherichia coli. In the meantime, real-time PCR was performed to analyze the relative expression of DaWß-AS in four different tissues of D. asper. The results of RT-qPCR showed DaWß-AS had the highest expression level in leaves. Bioinformatics results indicated that DaWß-AS had a conserved domain of PLN03012 superfamily, belonging to the cl31551 superfamily. There was no transmembrane domain or signal peptide in DaWß-AS. This study provides a scientific basis for revealing the biological pathways of triterpenoid saponins in D. asper, which will facilitate the biosynthesis of the associated saponins and afford reference for the cultivation and development of high-quality resources of D. asper.

Extraction of bioactive compounds from Dipsacus fullonum leaves using deep eutectic solvents.

In this study, deep eutectic solvent (DES) based systems were evaluated for selective extraction and optimized for increased recovery of chlorogenic acid derivatives, flavone glycosides and iridoid glycosides from Dipsacus fullonum L. leaves. Bioactives from Dipsacus plants has shown great antioxidant and antimicrobial activities as well as effectiveness against several cancer strains and a source for anti-Borrelia compounds. Twelve different hydrophilic and hydrophobic DESs were tested to find the best solvent composition. Choline chloride and betaine were used as hydrogen bond acceptors (HBA) for the preparation of hydrophilic DESs and menthol for hydrophobic DESs. The tested hydrogen bond donors (HBD) were various organic acids and glycerol. The composition of most effective DES was optimized using the Box-Behnken design for each of the three main group of analytes from D. fullonum L. to evaluate possible selectivity and highest recovery. HPLC-DAD-MS was used to identify and quantify the main bioactive compounds extracted from plant material. The optimal extraction for highest overall recovery was achieved using a molar ratio of choline chloride and lactic acid of 1:2.4 with 35% water and 27 mL of the solvent per one gram of dry material. The optimized DES extract gave concentrations 1.8 to 2.2 times higher than traditional organic solvent extracts depending on the group of analytes.

[To explore mechanism of Scabiosa comosa against liver fibrosis based on pharmacodynamics and network pharmacology].

This study aims to systematically elucidate the pharmacodynamics and network pharmacological mechanism of Mongolian medicinal plants Scabiosa comosa, explore their key targets and related pathways, and further clarify the mechanism of the plants in treating liver fibrosis. Wistar rats were assigned into the blank group, carbon tetrachloride-induced liver fibrosis model group, and low-, medium-, and high-dose S. comosa groups. HE staining and Masson staining were performed for the observation of liver tissue under a microscope. Further, Wistar rats were assigned into a control group and a S. comosa group for administration. Seven days later, blood was collected from the abdominal aorta, and different doses of drug-containing serum samples were used to treat hepatic stellate cell-T6(HSC-T6). Flow cytometry was adopted to detect the apoptosis of HSC-T6 cells. Ultra-high performance liquid chromatography-time of flight-mass spectrometry(UHPLC-TOF-MS) was employed to determine the components in Scabiosa comosa. The target of S. comosa and liver fibrosis were obtained from SwissTargetPrediction and GeneCards, respectively, and the common targets were selected as the anti-liver fibrosis targets. Protein-protein interaction was analyzed via STRING. Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment were carried out via Metascape. Phosphatidylinosital 3-kinase(PI3 K), protein kinase B(AKT), p-AKT, p38, and p-p38 targets which are involved in the top-ranked PI3 K/AKT and mitogen activated kinase-like protein(MAPK) signaling pathways were selected for validation via Western blot. The HE and Masson staining results showed that Scabiosa alleviated the hyperplasia of connective tissue and the fibrosis. The serum containing Scabiosa significantly promoted the apoptosis of HSC-T6 in a concentration-dependent manner. A total of 76 chemical components were identified by UHPLC-TOF-MS, among which flavonoids, alkaloids, terpenoids, phenols, and fatty acids were the main components. According to the prediction, there were 63 anti-liver fibrosis targets in Scabiosa comosa, the annotated GO terms of which involved biological processes, cell components, and molecular functions. The KEGG pathway enrichment showed that the targets were mainly involved in PI3 K/AKT, epidermal growth factor receptor(EGFR), RAS-associated protein 1(Rap1), hypoxia-inducible factor 1(HIF-1), resistance to audiogenic seizures(Ras), and MAPK signaling pathways. Western blot results showed that compared with the model group, S. comosa down-regulated the protein levels of α-smooth muscle actin(α-SMA), collagen Ⅰ, PI3 K, AKT, p-AKT, p38, and p-p38 in liver tissue. Compared with the control group, the low-, medium-, and high-dose S. comosa significantly down-regulated the protein levels of α-SMA, collagen Ⅰ, PI3 K, AKT, p-AKT, p38, and p-p38 in HSC-T6. The evidence of pharmacodynamics, network pharmacology, and molecular biology indicated that the plants of S. comosa had significant activity against liver fibrosis, the mechanism of which may involve the regulation of the key targets PI3 K, AKT, and MAPK14 p38 in the PI3 K/AKT and MAPK signaling pathways.

A hederagenin-type triterpene saponin, sumbulianoside a from Cephalaria sumbuliana and its potent immunomodulatory activity against seasonal flu virus H3N2.

A new hederagenin-type triterpene saponin; hederagenin 28-O-ß-D-galactopyranosyl-(1→6)-ß-D-glucopyranosyl ester named sumbulianoside A (1), together with twelve known saponins were isolated from the n-butanol extract of Cephalaria sumbuliana (Caprifoliaceae) from which, one known saponin, dipsacus saponin A (2) was isolated, for the first time from Cephalaria species. The structures of the isolated compounds were elucidated by 1 D and 2 D NMR and HRESIMS analyses. Cytotoxic activities were investigated on A549, Hela, PANC1, SHSY5Y cells and non-cancerous cell HEK293 by MTT method and immunomodulatory activities were evaluated against activated H3N2 seasonal virus in whole blood by measuring IL-4, IFN-γ, IL-1ß cytokine level with ELISA. According to the cytotoxicity results, compounds 1 and 2 did not possess significant cytotoxicity, while only compound 2 induced significant IL-4 production (** p<.001) against H3N2 showing a possible Th2 response and antibody production. All compounds had no effect on cytokine release (p > 0.5).

Occurrence of an endangered grassland butterfly is mainly driven by habitat heterogeneity, food availability, and microclimate.

The Marsh Fritillary (Euphydryas aurinia) was once widespread in large parts of Central Europe. However, in the course of the last century, populations of the butterfly largely collapsed. Here, we surveyed patch and microhabitat occupancy and its drivers in one of the last vital populations in calcareous grasslands. Our study revealed that environmental conditions at the landscape and habitat level determined the occurrence of E. aurinia in a montane agricultural landscape with low land-use intensity. Patch occupancy increased with the cover of Devil's-bit Scabious (Succisa pratensis) grasslands in the surroundings of the patches, habitat heterogeneity and host-plant cover. Microhabitat occupancy was driven by a warm microclimate and high availability of host plants. In the well-connected landscape of nutrient-poor grasslands, patch occupancy of E. aurinia was driven by parameters defining a high habitat quality. Habitat heterogeneity very likely buffers E. aurinia populations against environmental stochasticity and, hence, enhances long-term viability. For the gregariously feeding caterpillars of E. aurinia, host-plant biomass is essential. Due to their more luxuriant growth, S. pratensis plants were clearly preferred, although the Glossy Scabious (Scabiosa lucida) was also widespread. Additionally, the growth of large Succisa plants was favored by soil humidity and grassland abandonment. To cope with the adverse macro- and mesoclimatic conditions of the study area, females of the butterfly selected host plants growing in extraordinarily warm microhabitats for oviposition. To secure long-term viability of E. aurinia populations, we recommend creating mosaics of traditionally managed grasslands and early stages of abandonment within the patches.

The role of genetic diversity and arbuscular mycorrhizal fungal diversity in population recovery of the semi-natural grassland plant species Succisa pratensis.

BACKGROUND: Ecosystem restoration is as a critical tool to counteract the decline of biodiversity and recover vital ecosystem services. Restoration efforts, however, often fall short of meeting their goals. Although functionally important levels of biodiversity can significantly contribute to the outcome of ecosystem restoration, they are often overlooked. One such important facet of biodiversity is within-species genetic diversity, which is fundamental to population fitness and adaptation to environmental change. Also the diversity of arbuscular mycorrhizal fungi (AMF), obligate root symbionts that regulate nutrient and carbon cycles, potentially plays a vital role in mediating ecosystem restoration outcome. In this study, we investigated the relative contribution of intraspecific population genetic diversity, AMF diversity, and their interaction, to population recovery of Succisa pratensis, a key species of nutrient poor semi natural grasslands. We genotyped 180 individuals from 12 populations of S. pratensis and characterized AMF composition in their roots, using microsatellite markers and next generation amplicon sequencing, respectively. We also investigated whether the genetic makeup of the host plant species can structure the composition of root-inhabiting AMF communities. RESULTS: Our analysis revealed that population allelic richness was strongly positively correlated to relative population growth, whereas AMF richness and its interaction with population genetic diversity did not significantly contribute. The variation partitioning analysis showed that, after accounting for soil and spatial variables, the plant genetic makeup explained a small but significant part of the unique variation in AMF communities. CONCLUSIONS: Our results confirm that population genetic diversity can contribute to population recovery, highlighting the importance of within-species genetic diversity for the success of restoration. We could not find evidence, however, that population recovery benefits from the presence of more diverse AMF communities. Our analysis also showed that the genetic makeup of the host plant structured root-inhabiting AMF communities, suggesting that the plant genetic makeup may be linked to genes that control symbiosis development.