Impact of Different Extraction Solvents on Phenolic Content and Antioxidant Potential of Pinus densiflora Bark Extract.

It is well established that various extraction factors, including the method, temperature, time, and solvent system, significantly influence the antioxidant quality of plant-derived products. Previously, we observed that extraction of Pinus densiflora bark (PDB) by the most common traditional Soxhlet method using water at two different temperature conditions 60°C and 100°C for 6-15 h noticeably altered their antioxidant quality. In this study, we examined the impact of different extraction solvents such as ethanol, methanol, isopropanol, acetonitrile, and acetone at a different percentage with water (vol/vol) on antioxidant efficiency as well as the total phenolic content (TPC) of PDB extracts. Among the fourteen different PDB extracts, the extracts obtained from 20% ethanol (E20), 40% ethanol (E40), and 20% acetonitrile (ACN20) showed more significant antioxidant potential, as well as high total phenol content (TPC). Extracts from other aqueous mixtures of organic solvents such as isopropanol, acetone, and methanol, as well as water, showed lesser antioxidant capacity and also had less TPC compared to these three most active extracts, E20, E40, and ACN20. Moreover, using ethanol at 100% for extraction significantly decreased the TPC and antioxidant capacity of PDB extracts. Data are implicating that an increased phenolic content in PDB extracts proportionally increases their antioxidant efficiency.

Pinus densiflora needle supercritical fluid extract suppresses the expression of pro-inflammatory mediators iNOS, IL-6 and IL-1ß, and activation of inflammatory STAT1 and STAT3 signaling proteins in bacterial lipopolysaccharide-challenged murine macrophages.

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-1ß (IL-1ß) 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.

Anti-inflammatory activity of Ternstroemia gymnanthera stem bark extracts in bacterial lipopolysaccharide-stimulated RAW264.7 murine macrophage cells.

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-1ß (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.

Pinus radiata bark extract induces caspase-independent apoptosis-like cell death in MCF-7 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.

Deoxyrhapontigenin, a Natural Stilbene Derivative Isolated From Rheum undulatum L. Induces Endoplasmic Reticulum Stress-Mediated Apoptosis in Human Breast Cancer Cells.

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.

Components of rhizome extract of Cnidium officinale Makino and their in vitro biological effects.

The anti-inflammatory and anticancer activities of a methanol extract of the rhizome of Cnidium officinale were investigated. Four compounds, namely falcarindiol (1), 6-hydroxy-7-methoxy-dihydroligustilide (2), ligustilidiol (3), and senkyunolide H (4) were isolated from the extract of the rhizome of Cnidium officinale and their structures were elucidated by analysis of their spectroscopic data and by comparison with previously reported data. These compounds showed anti-inflammatory activities, measured as inhibition of nitric oxide (NO) release in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells, with IC(50) values of 4.31 ± 5.22, 152.95 ± 4.23, 72.78 ± 5.13, and 173.42 ± 3.22 µM, respectively. They also inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression induced by LPS. Among these compounds, falcarindiol (1) was found to have anti-proliferative effect against MCF-7 human breast cancer cells by induction of a G(0)/G(1) cell cycle block of the cells, with an IC(50) value of 35.67 µM. Typical apoptotic effects were observed by phase contrast microscopy and were also exhibited in fluorescence microscopy with Hoechst 33342 staining. In addition, falcarindiol induced apoptosis through strongly increased mRNA expression of Bax and p53, and slightly reduced Bcl-2 mRNA levels in a dose dependent manner. This study suggested that C. officinale extract and its components would be valuable candidates in therapeutic applications for anti-inflammatory and anti-cancer agents.

Compounds from Viburnum sargentii Koehne and evaluation of their cytotoxic effects on human cancer cell lines.

Compounds were isolated from a methanol extract of the dried stem barks of Viburnum sargentii Koehne. The structures of the compounds, namely 9'-O-methylvibsanol (3), furcatoside A (4) and lareciresinol (5) were elucidated by analysis of spectroscopic data and comparison with values for previously known analogues. In addition, (+)-catechin (1), (+)-epicatechin (2) were also isolated. This work also examined the cytotoxic effects of three compounds 3-5 (25-100 microM) in MCF-7 and A549 cells after 24, 48 and 72 h of exposure. Our results showed that 9'-O-methylvibsanol (3) exhibited strong concentration-dependent anticancer effects according to the MTT assay and produced morphological changes consistent with apoptosis, as confirmed by Ho3342 staining analysis revealed that more apoptotic cells were observed after 9'-Omethylvibsanol (3) treatment.