Phenolic glycosides and indole alkaloids isolated from the roots and rhizomes of Clematis chinensis and their anti-inflammatory activity.

Six undescribed compounds, including three phenolic glycosides (1-3) and three indole alkaloids (4-6), together with ten known alkaloids (7-16) and three known phenolic glycosides (17-19), were isolated from 70% EtOH aqueous extracts of the roots and rhizomes of Clematis chinensis Osbeck. The structures were elucidated by NMR, HRESIMS and X-ray diffraction spectroscopies. The anti-inflammatory activity of these compounds was evaluated, and twelve compounds showed significant inhibitory activity against TNF-α with an inhibition ratio from 47.87% to 94.70% at a dose of 10 µM. Compound 7 exhibited significant inhibitory activity against TNF-α and IL-6 with IC50 values of 3.99 µM and 2.24 µM, respectively. Compound 8 displayed potent anti-inflammatory activity against mouse ear edema induced by croton oil. A mechanistic study suggested that compounds 7 and 8 decreased the activation of the NF-κB signaling pathway to reduce the secretion of inflammatory factors in LPS-induced RAW 264.7 cells.

Separation of a new triterpenoid saponin together with six known ones from Clematis tangutica (Maxim.) Korsh and evaluation of their cytotoxic activities.

A new triterpenoid saponin, 3-O-ß-D-allopyranosyl (1→3)-α-L-rhamnopyranosyl (1→2)-α-L-arabinopyranosyl hederagenin 28-O-α-L-rhamnopyranosyl (1→4)-ß-D-glucopyranosyl (1→6)-ß-D-glucopyranosyl ester (IV), together with six known ones Hederacholichiside F (I), Tanguticoside B (II), Tauroside St-H1 (III), Hederoside H1 (V), Kalopanaxsaponin G (VI), Hederasaponin B (VII) were separated from Clematis tangutica (Maxim.) Korsh. Their cytotoxic activities were evaluated. Saponins IV (new compound) and I showed selective inhibitory activities against HGC-27 with IC50 values of 20.17 and 66.18 µM. Saponin VII exhibited extensive inhibitory action against HGC-27, Hela and SK-OV-3 with IC50 values of 16.47-71.36 µM. Saponin III showed selective inhibitory activity against SK-OV-3 with the IC50 value of 48.70 µM. All isolated saponins were inactive (IC50 >150 µM) to GES-1.

New triterpenoid saponins from the whole plants of Clematis heracleifolia.

Three new triterpenoid saponins, heracleifolianosides A-C (1-3), together with seven known compounds (4-10), were isolated from the whole plants of Clematis heracleifolia. Moreover, three new secondary saponins (1a, 2a and 3a), two known secondary metabolites (5a and 7a) were obtained by alkaline hydrolysis. Their structures were elucidated by extensive spectroscopic analysis and chemical evidences. The cytotoxicity of eight native saponins and five prosapogenins against human breast tumor MDA-MB-231 and gastric carcinoma SGC-7901 cell lines were evaluated by MTT method. Remarkably, the prosapogenin monodesmosidic saponin 7a showed significant cytotoxicity against MDA-MB-231 or SGC-7901 cell lines with IC50 values in the range of 6.05-6.32 µmol/L. It is suggested that it might be a feasible way to change the inactive bisdesmosic triterpenoid saponins to active monodesmosic saponins by a simple procedure of alkaline hydrolysis.

[Effect of silk fibroin microcarrier loaded with clematis total saponins and chondrocytes on promoting rabbit knee articular cartilage defects repair].

Objective: To prepare the silk fibroin microcarrier loaded with clematis total saponins (CTS) (CTS-silk fibroin microcarrier), and to investigate the effect of microcarrier combined with chondrocytes on promoting rabbit knee articular cartilage defects repair. Methods: CTS-silk fibroin microcarrier was prepared by high voltage electrostatic combined with freeze drying method using the mixture of 5% silk fibroin solution, 10 mg/mL CTS solution, and glycerin. The samples were characterized by scanning electron microscope and the cumulative release amount of CTS was detected. Meanwhile, unloaded silk fibroin microcarrier was also prepared. Chondrocytes were isolated from knee cartilage of 4-week-old New Zealand rabbits and cultured. The 3rd generation of chondrocytes were co-cultured with the two microcarriers respectively for 7 days in microgravity environment. During this period, the adhesion of chondrocytes to microcarriers was observed by inverted phase contrast microscope and scanning electron microscope, and the proliferation activity of cells was detected by cell counting kit 8 (CCK-8), and compared with normal cells. Thirty 3-month-old New Zealand rabbits were selected to make bilateral knee cartilage defects models and randomly divided into 3 groups ( n=20). Knee cartilage defects in group A were not treated, and in groups B and C were filled with the unloaded silk fibroin microcarrier-chondrocyte complexes and CTS-silk fibroin microcarrier-chondrocyte complexes, respectively. At 12 weeks after operation, the levels of matrix metalloproteinase 9 (MMP-9), MMP-13, and tissue inhibitor of MMP 1 (TIMP-1) in articular fluid were detected by ELISA. The cartilage defects were collected for gross observation and histological observation (HE staining and toluidine blue staining). Western blot was used to detect the expressions of collagen type Ⅱ and proteoglycan. The inflammatory of joint synovium was observed by histological staining and inducible nitric oxide synthase (iNOS) immunohistochemical staining. Results: The CTS-silk fibroin microcarrier was spherical, with a diameter between 300 and 500 µm, a porous surface, and a porosity of 35.63%±3.51%. CTS could be released slowly in microcarrier for a long time. Under microgravity, the chondrocytes attached to the surface of the two microcarriers increased gradually with the extension of culture time, and the proliferation activity of chondrocytes at 24 hours after co-culture was significantly higher than that of normal chondrocytes ( P<0.05). There was no significant difference in proliferation activity of chondrocytes between the two microcarriers ( P>0.05). In vivo experiment in animals showed that the levels of MMP-9 and MMP-13 in group C were significantly lower than those in groups A and B ( P<0.05), and the level of TIMP-1 in group C was significantly higher ( P<0.05). Compared with group A, the cartilage defects in groups B and C were filled with repaired tissue, and the repaired surface of group C was more complete and better combined with the surrounding cartilage. Histological observation and Western blot analysis showed that the International Cartilage Repair Scoring (ICRS) and the relative expression levels of collagen type Ⅱ and proteoglycan in groups B and C were significantly better than those in group A, and group C was significantly better than group B ( P<0.05). The histological observation showed that the infiltration of synovial inflammatory cells and hyperplasia of small vessels significantly reduced in group C compared with groups A and B. iNOS immunohistochemical staining showed that the expression of iNOS in group C was significantly lower than that in groups A and B ( P<0.05). Conclusion: CTS-silk fibroin microcarrier has good CTS sustained release effect and biocompatibility, and can promote the repair of rabbit cartilage defect by carrying chondrocyte proliferation in microgravity environment.

Thermal and wine processing enhanced Clematidis Radix et Rhizoma ameliorate collagen â ¡ induced rheumatoid arthritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Clematidis Radix et Rhizoma, a kind of traditional Chinese medicine, is derived from Clematis chinensis Osbeck, Clematis hexapetala Pall. and Clematis manshurica Rupr. This herb shows great effects on expelling wind and dispelling dampness in ancient and it has anti-inflammatory and analgesic activity in modern clinical application. AIM OF THE STUDY: This experiment aimed to research anti-rheumatoid arthritis effect of crude and wine processed RC based on glycolysis metabolism to provide new ideas treating RA. MATERIALS AND METHODS: Network pharmacology was applied to preliminarily forecast the potential pathways of common targets of RC and RA. RAW264.7 macrophages were induced by LPS, NO production, glucose uptake, lactate production, ROS and MMP were detected as instructions in vitro. ELISA was used to measure the content of HK2, PKM2 and LDHA involving in glycolysis process. Gut microbiota was analyzed by 16S rRNA gene amplicon sequencing in CIA rats. RESULTS: Crude and wine processed RC had good anti-inflammatory effect by reducing NO in RAW264.7 macrophages and ameliorating inflammatory infiltration and cartilage surface erosion in CIA rats. Whether in LPS-induced macrophages or CIA rats, crude and wine processed RC could inhibit glycolysis by down-regulating the expression of PKM2, causing less glucose uptake and lactic acid, which lead to less ROS and higher MMP to normal. PI3K-AKT and HIF-1α pathways were deduced to possibly play a crucial part in controlling glycolysis metabolism by network pharmacology analysis. Besides, it was displayed that Firmicutes and Bacteroidetes were prominent gut microbiota in CIA rats feces. CC-H and PZ-H groups could both increase the relative abundance of Firmicutes and decrease Bacteroidetes. These microbiota also played a role in RA pathological process via involving in energy metabolism, carbohydrate metabolism and immune system. CONCLUSION: Crude and wine processed RC have a good influence in ameliorating rheumatoid arthritis by inhibiting glycolysis and modulating gut microbiota together.

Two new terpenes from the aerial parts of Clematis chinensis Osbeck.

Two new acyclic sesquiterpenoids (1-2) and fourteen known monocyclic monoterpenoids (3-16) were isolated from the aerial parts of Clematis chinensis Osbeck. All compounds were isolated from C. chinensis for the first time. The structures of all compounds were characterized by spectroscopic methods (1 D, 2 D NMR and HRESIMS). In-vitro cytotoxic activity against two human cancer cell lines (MGC-803 and Ishikawa) of all the compounds were evaluated by CCK-8 assay.

Mitochondrial proteomic analysis reveals the regulation of energy metabolism and reactive oxygen species production in Clematis terniflora DC. leaves under high-level UV-B radiation followed by dark treatment.

Clematis terniflora DC. is an important medicinal plant from the family Ranunculaceae. A previous study has shown that active ingredients in C. terniflora, such as flavonoids and coumarins, are increased under ultraviolet B radiation (UV-B) and dark treatment and that the numbers of genes related to the tricarboxylic acid cycle and mitochondrial electron transport chain (mETC) are changed. To uncover the mechanism of the response to UV-B radiation and dark treatment in C. terniflora, mitochondrial proteomics was performed. The results showed that proteins related to photorespiration, mitochondrial membrane permeability, the tricarboxylic acid cycle, and the mETC mainly showed differential expression profiles. Moreover, the increase in alternative oxidase indicated that another oxygen-consuming respiratory pathway in plant mitochondria was induced to minimize mitochondrial reactive oxygen species production. These results suggested that respiration and mitochondrial membrane permeability were deeply influenced to avoid energy consumption and maintain energy balance under UV-B radiation and dark treatment in C. terniflora leaf mitochondria. Furthermore, oxidative phosphorylation was able to regulate intracellular oxygen balance to resist oxidative stress. This study improves understanding of the function of mitochondria in response to UV-B radiation and dark treatment in C. terniflora. SIGNIFICANCE: C. terniflora was an important traditional Chinese medicine for anti-inflammatory. Previous study showed that the contents of coumarins which were the main active ingredient in C. terniflora were induced by UV-B radiation and dark treatment. In the present study, to uncover the regulatory mechanism of metabolic changes in C. terniflora, mitochondrial proteomics analysis of leaves was performed. The results showed that photorespiration and oxidative phosphorylation pathways were influenced under UV-B radiation and dark treatment. Mitochondria in C. terniflora leaf played a crucial role in energy mechanism and regulation of cellular oxidation-reduction to maintain cell homeostasis under UV-B radiation followed with dark treatment.

Insights into heat response mechanisms in Clematis species: physiological analysis, expression profiles and function verification.

KEY MESSAGE: Our results provide insights into heat response mechanisms among Clematis species. Overexpressing CvHSFA2 enhanced the heat resistance of yeast and silencing NbHSFA2 reduced the heat resistance of tobacco. Clematis species are commonly grown in western and Japanese gardens. Heat stress can inhibit many physiological processes mediating plant growth and development. The mechanism regulating responses to heat has been well characterized in Arabidopsis thaliana and some crops, but not in horticultural plants, including Clematis species. In this study, we found that Clematis alpina 'Stolwijk Gold' was heat-sensitive whereas Clematis vitalba and Clematis viticella 'Polish Spirit' were heat-tolerant based on the physiological analyses in heat stress. Transcriptomic profiling identified a set of heat tolerance-related genes (HTGs). Consistent with the observed phenotype in heat stress, 41.43% of the differentially expressed HTGs between heat treatment and control were down-regulated in heat-sensitive cultivar Stolwijk Gold, but only 9.80% and 20.79% of the differentially expressed HTGs in heat resistant C. vitalba and Polish Spirit, respectively. Co-expression network, protein-protein interaction network and phylogenetic analysis revealed that the genes encoding heat shock transcription factors (HSFs) and heat shock proteins (HSPs) may played an essential role in Clematis resistance to heat stress. Two clades of heat-induced CvHSFs were further identified by phylogenetic tree, motif analysis and qRT-PCR. Ultimately, we proposed that overexpressing CvHSFA2-2 could endow yeast with high temperature resistance and silencing its homologous gene NbHSFA2 reduced the heat resistance of tobacco. This study provides first insights into the diversity of the heat response mechanisms among Clematis species.

Identification of anti-TMV active flavonoid glycosides and their mode of action on virus particles from Clematis lasiandra Maxim.

BACKGROUND: Tobacco mosaic virus (TMV) is a disreputable plant pathogen that causes a decline in the quality and yield of various economic crops. Natural products are important potential sources of biopesticides to control TMV. This study focuses on the discovery of anti-TMV active flavonoid glycosides and their mode of action on TMV particles from Clematis lasiandra Maxim. RESULTS: A new benzoyl acylated flavonoid glycoside, kaempferol 3-O-(2''-benzoyl)-ß-d-glucopyranosyl-7-O-α-l-rhamnopyranoside (1), and nine known flavonoids (2-10) were identified first from C. lasiandra. The hydroxyl group at C-7, E-p-coumarate at C-6'' in the Glc of C-6, and the glucuronic acid at C-3 were functional groups for the antiviral flavonoid glycosides. Flavonoids 2, 5, and 6 showed higher inactivation efficacies of 64.62% to 82.54% compared with ningnanmycin at 500 µg ml-1 . The protective and curative efficacies for 2 and 5 were 57.44-59.00% and 41.17-43.92% at 500 µg ml-1 , respectively. Compound 5 showed higher TMV systemic resistance with control efficacies of 41.64%, 36.56% and 27.62% at concentrations of 500, 250 and 125 µg ml-1 compared with ningnanmycin in K326 tobaccos, respectively. Compound 5 can directly fracture TMV particles into small fragments combining with the fusion phenomena, and TMV-CP was an important target for 5 to break TMV particles. CONCLUSION: Flavonoid glycosides from C. lasiandra showed potent antiviral activities against TMV with multiple modes of action including inactivation, protective and curative effects, and inducing systemic resistance. TMV-CP was an important target for active flavonoid glycosides to fracture TMV particles. The results provided evidence that flavonoid glycosides from C. lasiandra have the potential to control TMV.

One Heat Shock Transcription Factor Confers High Thermal Tolerance in Clematis Plants.

Clematis plants play an important role in botanical gardens. Heat stress can destroy the activity, state and conformation of plant proteins, and its regulatory pathway has been well characterized in Arabidopsis and some crop plants. However, the heat resistance response mechanism in horticultural plants including Clematis has rarely been reported. Here, we identified a heat-tolerant clematis species, Clematis vitalba. The relative water loss and electrolytic leakage were significantly lower under heat treatment in Clematis vitalba compared to Stolwijk Gold. Differential expression heat-tolerant genes (HTGs) were identified based on nonparametric transcriptome analysis. For validation, one heat shock transcription factor, CvHSF30-2, extremely induced by heat stimuli in Clematis vitalba, was identified to confer tolerance to heat stress in Escherichia coli and Saccharomyces cerevisiae. Furthermore, silencing of HSF30-2 by virus-induced gene silencing (VIGS) led to heat sensitivity in tobacco and Clematis, suggesting that the candidate heat-resistant genes identified in this RNA-seq analysis are credible and offer significant utility. We also found that CvHSF30-2 improved heat tolerance of Clematis vitalba by elevating heat shock protein (HSP) expression, which was negatively regulated by CvHSFB2a. Taken together, this study provides insights into the mechanism of Clematis heat tolerance and the findings can be potentially applied in horticultural plants to improve economic efficiency through genetic approaches.

Traditional Chinese medicines and their active ingredients sensitize cancer cells to TRAIL-induced apoptosis.

The rapidly developing resistance of cancers to chemotherapy agents and the severe cytotoxicity of such agents to normal cells are major stumbling blocks in current cancer treatments. Most current chemotherapy agents have significant cytotoxicity, which leads to devastating adverse effects and results in a substandard quality of life, including increased daily morbidity and premature mortality. The death receptor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can sidestep p53-dependent pathways to induce tumor cell apoptosis without damaging most normal cells. However, various cancer cells can develop resistance to TRAIL-induced apoptosis via different pathways. Therefore, it is critical to find an efficient TRAIL sensitizer to reverse the resistance of tumor cells to TRAIL, and to reinforce TRAIL's ability to induce tumor cell apoptosis. In recent years, traditional Chinese medicines and their active ingredients have shown great potential to trigger apoptotic cell death in TRAIL-resistant cancer cell lines. This review aims to collate information about Chinese medicines that can effectively reverse the resistance of tumor cells to TRAIL and enhance TRAIL's ability to induce apoptosis. We explore the therapeutic potential of TRAIL and provide new ideas for the development of TRAIL therapy and the generation of new anti-cancer drugs for human cancer treatment. This study involved an extensive review of studies obtained from literature searches of electronic databases such as Google Scholar and PubMed. "TRAIL sensitize" and "Chinese medicine" were the search keywords. We then isolated newly published studies on the mechanisms of TRAIL-induced apoptosis. The name of each plant was validated using certified databases such as The Plant List. This study indicates that TRAIL can be combined with different Chinese medicine components through intrinsic or extrinsic pathways to promote cancer cell apoptosis. It also demonstrates that the active ingredients of traditional Chinese medicines enhance the sensitivity of cancer cells to TRAIL-mediated apoptosis. This provides useful information regarding traditional Chinese medicine treatment, the development of TRAIL-based therapies, and the treatment of cancer.

Uses, chemical compositions, pharmacological activities and toxicology of Clematidis Radix et Rhizome- a Review.

ETHNOPHARMACOLOGICAL RELEVANCE: Clematis chinensis Osbeck (C. chinensis), Clematis hexapetala Pall (C. hexapetala) and Clematis terniflora var. mandshurica Rupr (C. mandshurica) are collectively referred to as Clematidis Radix et Rhizome (CRR) in China. CRR is widely distributed in China, which is used as a traditional Chinese medicine to treat rheumatic arthralgia, limb numbness, tendon constriction and inconvenience in flexion and extension. AIMS OF THIS REVIEW: This review systematically summarized the research progress on uses, chemical components, pharmacological activities and toxicology of CRR, listed the chemical structures of main compounds for clarifying the differences in chemical compositions. Meanwhile, the review will provide a theoretical and practical basis for the further research and development of CRR. MATERIALS AND METHODS: The available information on CRR was collected using published materials and electronic databases, including ancient and modern books, Chinese Pharmacopoeia, Ph.D. and M. Sc. dissertations, CNKI, SciFinder, WanFang data, PubMed, ScienceDirect and Web of Science. The starting and ending years of references is 1965-2020, the search strategy was conducted by key words such as uses, chemical components, pharmacology and toxicology of CRR. RESULTS: Up to now, CRR has been used to treat various diseases/disorders, such as relieving rheumatism pain, treating cervical spondylopathy and scapulohumeral periarthritis, treating hepatic carcinoma and gastrointestinal, etc. In addition, more than 200 compounds have been isolated from the three plant species of Clematidis. Moreover, the crude extracts and isolated compounds of CRR have been reported to have a wide range of pharmacological activities, such as anti-inflammatory, anti-tumor, antimicrobial and antioxidant activities, etc. Toxicity studies have shown that CRR can cause oral burning, swelling, abdominal pain or severe diarrhea, difficulty breathing, dilated pupils, renal tissue structural changes, and severe death. CONCLUSIONS: Researches in recent years mainly focused on C. chinensis and C. mandshurica, while there are a few reports on the pharmacological studies of C. hexapetala. Therefore, it is necessary to conduct further research on C. hexapetala. Meanwhile, it is important to pay attention to pursue research on the similarities and differences between the three plant species of Clematidis to find their respective advantages and make rational use of CRR. In addition, there is no report on the mechanism of toxicity research, which needs more attention.

Sesquiterpenoids, phenolic and lignan glycosides from the roots and rhizomes of Clematis hexapetala Pall. and their bioactivities.

Approximately 17 compounds were isolated from a 60% EtOH aqueous extract of the roots and rhizomes of Clematis hexapetala Pall., including three new guaianolide sesquiterpenoids with 5/7/5-fused rings and 3S-configuration (1-3), five new prenylated tetra-substituted phenolic glycosides (4-8) with 6/6-fused 9H-benzopyran skeleton (5) and 6/7-fused 7,10-dihydro-benzoxepin skeleton (6-8), one new isoferulyl glucoside (9), two new furofuran lignan diglucosides (10-11), and six known compounds. The chemical structures of the new compounds were elucidated via spectroscopic data and electronic circular dichroism (ECD) analyses in combination with a modified Mosher's method. The possible biosynthetic relationships of prenylated tetra-substituted phenols were postulated. In the in vitro assays, compound 16 exhibited moderate TNF-α secretion inhibitory activity with IC50 value of 3.419 µM. Compounds 14-16 displayed potent PTP1B enzymatic inhibitory activities with inhibition ratios of 48.30-86.00%. And compound 16 showed significant PTP1B enzymatic inhibition with IC50 value of 4.623 µM.

Effect of total flavones of Clematis filamentosa Dunn on the oxLDL-induced injury of vascular smooth muscle cells by regulating miR-455-5p.

This experiment was conducted to investigate whether total flavones of Clematis filamentosa Dunn affect the inflammatory response and apoptosis of vascular smooth muscle cells induced by oxidized low-density lipoprotein (oxLDL) by regulating microRNA-455-5p (miR-455-5p). 50 mg/mL oxLDL was performed to stimulate the injury of vascular smooth muscle cells, and the total flavones of Clematis filamentosa Dunn were added at concentrations of 75, 150, and 300 µg/mL. The expressions of inflammatory factors IL-1ß and TNF-α were analyzed by ELISA, the apoptosis was evaluated by flow cytometry, the expression of Bcl-2 and Bax was determined by western blot, and the real-time fluorescence quantitative PCR (qRT-PCR) was applied to detect miR-455-5p expression. MiR-455-5p mimic was transfected into vascular smooth muscle cells and then induced injury with oxLDL; miR-455-5p inhibitor was transfected into vascular smooth muscle cells and treated with oxLDL and 300 µg/mL total flavones of Clematis filamentosa Dunn. The above methods were employed to investigate the inflammatory response and apoptosis of cells. The total flavones of Clematis filamentosa Dunn significantly inhibited the expression of IL-1ß, TNF-α, apoptosis rate, Bax protein expression of oxLDL induced vascular smooth muscle cells, and remarkably promoted the expression of Bcl-2 protein and miR-455-5p, which all showed concentration dependence (p<0.05). Overexpression of miR-455-5p reduced IL-1ß, TNF-α expression, apoptosis rate, Bax protein expression, and greatly increased Bcl-2 protein expression in oxLDL injured vascular smooth muscle cells (p<0.05). After interfering with the expression of miR-455-5p, the inhibitory effect of total flavones of Clematis filamentosa Dunn on the expression of IL-1ß, TNF-α, apoptosis, Bax protein expression of oxLDL-induced vascular smooth muscle cells was reversed, and its promotion effect on Bcl-2 protein expression was also reversed. Total flavones of Clematis filamentosa Dunn can reduce oxLDL-induced vascular smooth muscle cell inflammation and inhibit its apoptosis. The mechanism of action is related to the up-regulation of miR-455-5p expression.

A botanical drug composed of three herbal materials attenuates the sensorimotor gating deficit and cognitive impairment induced by MK-801 in mice.

OBJECTIVES: A botanical drug derived from the ethanolic extract composed of Clematis chinensis Osbeck (Ranunculaceae), Trichosanthes kirilowii Maximowicz (Cucurbitaceae) and Prunella vulgaris Linné (Lamiaceae) has been used to ameliorate rheumatoid arthritis as an ethical drug in Korea. In our study, we investigated the effect of this herbal complex extract (HCE) on schizophrenia-like behaviours induced by MK-801. METHODS: HCE (30, 100 or 300 mg/kg, p.o) was orally administered to male ICR mice to a schizophrenia-like animal model induced by MK-801. We conducted an acoustic startle response task, an open-field task, a novel object recognition task and a social novelty preference task. KEY FINDINGS: We found that a single administration of HCE (100 or 300 mg/kg) ameliorated MK-801-induced abnormal behaviours including sensorimotor gating deficits and social or object recognition memory deficits. In addition, MK-801-induced increases in phosphorylated Akt and GSK-3ß expression levels in the prefrontal cortex were reversed by HCE (30, 100 or 300 mg/kg). CONCLUSIONS: These results imply that HCE ameliorates MK-801-induced dysfunctions in prepulse inhibition, social interactions and cognitive function, partly by regulating the Akt and GSK-3ß signalling pathways.

Antagonizing Effects of Clematis apiifolia DC. Extract against Benzo[a]pyrene-Induced Damage to Human Keratinocytes.

Background. Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon present in the atmosphere, has cytotoxic and carcinogenic effects. There have been no reports to demonstrate involvement of Clematis apiifolia DC. extract (CAE) in B[a]P-induced effects. This study was conducted to investigate the effect of CAE on B[a]P-induced effects and to elucidate its mechanism of action in HaCaT human keratinocytes. CAE inhibited aryl hydrocarbon receptor (AhR) signaling by decreasing both XRE reporter activity and expression of cytochrome P450 1A1 (CYP1A1) induced by B[a]P treatment in HaCaT cells. We also found that B[a]P-induced nuclear translocation of AhR and production of reactive oxygen species (ROS) and proinflammatory cytokines were attenuated by CAE treatment. CAE treatment suppressed B[a]P-induced phosphorylation of Src (Tyr416). In addition, dasatinib, a Src inhibitor, also inhibited B[a]P-induced nuclear translocation of AhR, similar to CAE treatment. In addition, CAE activated antioxidant response element (ARE) signaling by increasing ARE luciferase reporter activity and expression of ARE-dependent genes such as nuclear factor (erythroid-derived 2)-like 2 (Nrf2), NAD(P)H dehydrogenase [quinone] 1 (NQO1), and heme oxygenase-1 (HO-1). Nuclear translocation of Nrf2 by CAE was demonstrated by Western blot analysis and immunocytochemistry. The effects of CAE on ARE signaling were attenuated by knockdown of the Nrf2 gene. Inhibition of AhR signaling and activation of antioxidant activity by CAE operated in a reciprocally independent manner as evidenced by AhR and Nrf2 siRNA experiments. These findings indicate that CAE exerts protective effects against B[a]P by inhibiting AhR signaling and activating Nrf2-mediated signaling, suggesting its potential in protection from harmful B[a]P-containing pollutants.

Phenolic compounds from the aerial parts of Clematis viticella L. and their in vitro anti-inflammatory activities.

Phytochemical investigations on the EtOH extract of Clematis viticella led to the isolation of six flavonoid glycosides, isoorientin (1), isoorientin 3'-O-methyl ether (2), quercetin 7-O-α-L-rhamnopyranoside (3), quercetin 3,7-di-O-α-L-rhamnopyranoside (4), manghaslin (5) and chrysoeriol 7-O-ß-D-glucopyranoside (6), one phenylethanol derivative, hydroxytyrosol (7), along with three phenolic acids, caffeic acid (8), (E)-p-coumaric acid (9) and p-hydroxybenzoic acid (10). The structures of the isolates were elucidated on the basis of NMR and HR-MS data. All compounds were isolated from C. viticella for the first time. Compounds 7 and 8 showed significant anti-inflammatory activity at 100 µM by reducing the release of NO in LPS-stimulated macrophages comparable to positive control indomethacin. Compounds 3 and 7 exhibited anti-inflammatory activity through lowering the levels of TNF-α while 1, 3 and 5 decreased the levels of neopterin better than the positive controls.

A metabolomic strategy revealed the role of JA and SA balance in Clematis terniflora DC. Response to UVB radiation and dark.

Clematis terniflora DC. is a valuable resource with potential high pharmaceutical value. Proteomic, transcriptomic and metabolomic analyses of C. terniflora that has been exposed to high levels of UVB irradiation and dark conditions (HUVB + D) have revealed the mechanisms underlying its medicinal potential. However, the signal transduction pathways and the mechanisms of regulation for the accumulation of secondary metabolites remain unclear. In this study, we show that the jasmonic acid (JA) and salicylic acid (SA) signals were activated in C. terniflora in response to HUVB + D. Metabolomic analysis demonstrated that the perturbation in JA and SA balance led to additional reallocation of carbon and nitrogen resources. Evaluating the fold change ratios of differentially changed metabolites proved that JA signal enhanced the transformation of nitrogen to carbon through the 4-aminobutyric acid (GABA) shunt pathway, which increased the carbon reserve to be utilized in the production of secondary metabolites. However, SA signal induced the synthesis of proline, while avoiding the accumulation of secondary metabolites. Over all, the results indicate that the co-increase of JA and SA reconstructed the dynamic stability of transformation from nitrogen to carbon, which effectively enhanced the oxidative defense to HUVB + D in C. terniflora by increasing the secondary metabolites.

In vivo pathogenesis of colon carcinoma and its suppression by hydrophilic fractions of Clematis flammula via activation of TRAIL death machinery (DRs) expression.

This work focused on characterizing hydrophilic fractions of Clematis flammula (CFl). The data here clearly demonstrated that hydrolate fractions act as a free radical scavengers and inhibited proliferation of different cell lines in a time- and concentration-dependent manner, transwell, and with a significant cytotoxic effect. Treating cells with CFl had the effect of suppressing cell growth attenuated by ROS generation in colonic carcinoma. Moreover, CFl in HCT116 cells suppressed survival, proliferation, invasion, angiogenesis and metastasis in vitro by inhibiting gene expression. Following CFl treatment, caspases and PARP cleavage were detected. The up- and down-regulated genes obtained from the WBA of the effect of CFl showed that several biological processes were associated with apoptosis and induction of G1 cell cycle arrest. CFl synergizes the effect of TRAIL by down-regulating the expression of cell survival proteins involved in apoptosis compared to cells treated with CFl or TRAIL alone. Our findings showed that CFl sensitizes apoptosis in TRAIL-resistant cells by activating MAPKs, SP1, and CHOP, that induced DR5 expression. Overall, our data showed that CFl is a promising antitumor agent through kinases and transcription factor induction, both of which are required to activate TRAIL receptors. Colon inflammation induced by LPS was inhibited by CFl hydrolate.

Comparative Proteomic Analysis Reveals Elevated Capacity for Photosynthesis in Polyphenol Oxidase Expression-Silenced Clematis terniflora DC. Leaves.

Polyphenol oxidase (PPO) catalyzes the o-hydroxylation of monophenols and oxidation of o-diphenols to quinones. Although the effects of PPO on plant physiology were recently proposed, little has been done to explore the inherent molecular mechanisms. To explore the in vivo physiological functions of PPO, a model with decreased PPO expression and enzymatic activity was constructed on Clematis terniflora DC. using virus-induced gene silencing (VIGS) technology. Proteomics was performed to identify the differentially expressed proteins (DEPs) in the model (VC) and empty vector-carrying plants (VV) untreated or exposed to high levels of UV-B and dark (HUV-B+D). Following integration, it was concluded that the DEPs mainly functioned in photosynthesis, glycolysis, and redox in the PPO silence plants. Mapman analysis showed that the DEPs were mainly involved in light reaction and Calvin cycle in photosynthesis. Further analysis illustrated that the expression level of adenosine triphosphate (ATP) synthase, the content of chlorophyll, and the photosynthesis rate were increased in VC plants compared to VV plants pre- and post HUV-B+D. These results indicate that the silence of PPO elevated the plant photosynthesis by activating the glycolysis process, regulating Calvin cycle and providing ATP for energy metabolism. This study provides a prospective approach for increasing crop yield in agricultural production.