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This study was aimed to determine and compare the effect of different percentages of extraction solvent ethanol 0% (E0), 20% (E20), and 40% (E40) with water (vol/vol) as well as extraction time 6, 9 and 15 h on the yield and antioxidant potential of Pinus densiflora needle (PDN) and bark (PDB) extracts. Extraction was performed at 60 ± 5 °C. A maximum yield of 10.99 ± 0.96 and 16.86 ± 1.96% from 20 g of PDN and PDB was obtained with E40 at 15 h of extraction, respectively. Extraction of PDN and PDB using E20 or E40 for 6–15 h significantly (P

Context: Ternstroemia gymnanthera Sprague (Theaceae) possesses various known pharmacological properties. However, its anti-inflammatory activity has not been reported. Objective: The anti-inflammatory activity of Ternstroemia gymnanthera stem bark aqueous extract (TGSBE) was evaluated using LPS-stimulated RAW264.7 macrophages. Materials and methods: Cytotoxicity was assessed by MTT assay after 24 h with TGSBE (25–200 μg/mL). Further testing used TGSBE at 100 and 200 μg/mL. Griess and ELISA methods after 24 h with TGSBE determined NO and cytokine levels, respectively; then, mRNA levels (iNOS & cytokines) were analyzed by Quantitative-PCR after 12 h. NF-κB and MAPK were assessed by immunoblotting after TGSBE treatment for 12 h, followed by LPS for 30 min. Immunofluorescence assay was also performed for NF-κB. ROS and MMP, after 12 h with TGSBE, were determined by flow cytometry. The antioxidant potential of TGSBE was analyzed by ABTS assay. The Folin–Ciocalteu method determined the total phenolic content of TGSBE. LPS concentration was 0.5 μg/mL. Results: TGSBE at 200 μg/mL showed about 96.2% viability while suppressing the production of NO (88.99%), TNFα (24.38%), IL-6 (61.70%) and IL-1β (55.12%) and gene expression by 67.88, 45.24, 65.84, and 70.48%, respectively. TGSBE decreased ROS (79.26%) and improved MMP (48.01%); it inhibited translocation of NF-κB and MAPK activation. Radical scavenging activity was 50% at 402.17 μg/mL (ascorbic acid standard: 88.8 μg/mL). Total phenolic content was 240.9 mg GAE/g. Discussion and conclusion: TGSBE suppresses the inflammatory response by inhibiting the NF-κB and MAPK cascades exhibiting therapeutic potential to treat inflammatory diseases associated with increased activation of macrophages.

Although current chemotherapeutic agents are active at the beginning of therapy, the most common risk is the development of resistance during later stages in almost all cancer types including breast cancer. Hence, investigation of novel drugs is still a priority goal for cancer treatment. The objective of the present study is to investigate the anticancer effect of a derivative of stilbene, deoxyrhapontigenin (DR) isolated from Rheum undulatum L. root extracts against the chemoresistant MCF-7/adr and its parental MCF-7 human breast cancer cells. The morphological images indicate that DR induces an extensive cytoplasmic vacuolation in breast cancer cells. Mechanistic investigations revealed that DR treatment causes endoplasmic reticulum (ER) dilation and upregulated the expression of ER stress markers GRP78, IRE1α, eIF2α, CHOP, JNK, and p38. Subsequently, we also identified that DR increases the levels of apoptotic fragment of PARP (89 kDa) in breast cancer cells. Blocking the expression of one of the components of the ER stress–mediated apoptosis pathway, CHOP using siRNA significantly decreased DR-induced apoptotic cleavage of PARP. In summary, the present study suggests that the induction of ER stress–mediated apoptosis by DR may account for its cytotoxic effects in human breast cancer cells.

In the present study, we investigated the anticancer activity of Pinus radiata bark extract (PRE) against MCF-7 human breast cancer cells. First, we observed that PRE induces potent cytotoxic effects in MCF-7 cells. The cell death had features of cytoplasmic vacuolation, plasma membrane permeabilization, chromatin condensation, phosphatidylserine externalization, absence of executioner caspase activation, insensitivity to z-VAD-fmk (caspase inhibitor), increased accumulation of autophagic markers, and lysosomal membrane permeabilization (LMP). Both the inhibition of early stage autophagy flux and lysosomal cathepsins did not improve cell viability. The antioxidant, n-acetylcysteine, and the iron chelator, deferoxamine, failed to restore the lysosomal integrity indicating that PRE-induced LMP is independent of oxidative stress. This was corroborated with the absence of enhanced ROS production in PRE-treated cells. Chelation of both intracellular calcium and zinc promotes PRE-induced LMP. Geranylgeranylacetone, an inducer of Hsp70 expression, also had no significant protective effect on PRE-induced LMP. Moreover, we found that PRE induces endoplasmic reticulum (ER) stress and mitochondrial membrane depolarization in MCF-7 cells. The ER stress inhibitor, 4-PBA, did not restore the mitochondrial membrane integrity, whereas cathepsin inhibitors demonstrated significant protective effects. Collectively, our results suggest that PRE induces an autophagic block, LMP, ER stress, and mitochondrial dysfunction in MCF-7 cells. However, further studies are clearly warranted to explore the exact mechanism behind the anticancer activity of PRE in MCF-7 human breast cancer cells.

Pinus densiflora was screened in an ongoing project to discover anti-influenza candidates from natural products. An extensive phytochemical investigation provided 26 compounds, including two new megastigmane glycosides (1 and 2), 21 diterpenoids (3&ndash, 23), and three flavonoids (24&ndash, 26). The chemical structures were elucidated by a series of chemical reactions, including modified Mosher&rsquo, s analysis and various spectroscopic measurements such as LC/MS and 1D- and 2D-NMR. The anti-influenza A activities of all isolates were screened by cytopathic effect (CPE) inhibition assays and neuraminidase (NA) inhibition assays. Ten candidates were selected, and detailed mechanistic studies were performed by various assays, such as Western blot, immunofluorescence, real-time PCR and flow cytometry. Compound 5 exerted its antiviral activity not by direct neutralizing virion surface proteins, such as HA, but by inhibiting the expression of viral mRNA. In contrast, compound 24 showed NA inhibitory activity in a noncompetitive manner with little effect on viral mRNA expression. Interestingly, both compounds 5 and 24 were shown to inhibit nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in a dose-dependent manner. Taken together, these results provide not only the chemical profiling of P. densiflora but also anti-influenza A candidates.

Background Regulation of a persistently-activated inflammatory response in macrophages is an important target for treatment of various chronic diseases. Pine needle extracts are well known to have potent immunomodulatory effects. The current study was designed to evaluate the effects of Pinus densiflora needle supercritical fluid extract (PDN-SCFE) on bacterial lipopolysaccharide (LPS)-induced inflammatory response in RAW 264.7 murine macrophages. Methods Cytotoxic effect of PDN-SCFE was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The levels of nitric oxide (NO) and the corresponding enzyme, inducible nitric oxide synthase (iNOS), were quantified by Griess and immunoblotting methods, respectively. The levels of cytokines were quantified using commercial ELISA kits. Quantitative real-time PCR (qRT-PCR) analysis was performed to assess the mRNA expression of iNOS and cytokines. To elucidate the mechanism of action, the involvement of nuclear transcription factor-kappa B (NFκB), mitogen activated protein kinases (MAPKs) and Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathways were examined by an immunoblotting method. In addition, the cellular localization of NFκB was analyzed by immunofluorescence staining. Results MTT assay results indicated that PDN-SCFE is non-toxic to RAW 264.7 cells up to a maximum assayed concentration of 40 μg/mL. The PDN-SCFE exhibited a concentration-dependent inhibitory effect on LPS-induced NO production by down regulating the expression of iNOS. In addition, the extract suppressed the LPS-induced expression of interleukin-6 (IL-6) and interleukin-1β (IL-1β) but not tumour necrosis factor-α (TNFα). Mechanistic studies revealed that PDN-SCFE does not influence the NFκB and MAPK pathways. However, it showed a significant inhibitory effect on LPS-induced activation of STAT1 and STAT3 proteins in macrophages. Conclusion The present findings revealed that the anti-inflammatory activity of PDN-SCFE in LPS-challenged RAW 264.7 macrophages is probably caused by the suppression of the JAK-STAT signaling pathway. Graphical Abstract

Falcarindiol (FAD) is a natural polyacetylene compound found rich in many plants of the Umbelliferae family. Previously, we isolated FAD from the rhizome of Cnidium officinale Makino, which belongs to the Umbelliferae family and found it to have a significant inhibitory effect on lipopolysaccharide (LPS)-induced production of nitric oxide, a pro-inflammatory molecule in murine macrophage RAW 264.7 cells. In this study, we investigated its effect on the expression of other major pro-inflammatory molecules as well as the mechanism underlying these effects. Pre-treatment of RAW 264.7 cells with FAD suppressed LPS-stimulated mRNA expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) and thereby reduced the respective protein levels. Mechanistic studies demonstrated that FAD attenuated the LPS-induced activation of JNK, ERK, STAT1, and STAT3 signaling molecules. Moreover, we found that FAD did not influence LPS-induced activation of p38 and NFκB signaling pathways. Collectively, this study provides evidence that FAD inhibits the production of major pro-inflammatory molecules in LPS-challenged murine macrophages via suppression of JNK, ERK, and STAT signaling pathways.