Juanjuan Yi

Optimization of Purification, Identification and Evaluation of the in Vitro Antitumor Activity of Polyphenols from Pinus Koraiensis Pinecones.

In this study, an efficient purification method for the polyphenols of Pinus koraiensis pinecone (PPP) has been developed. AB-8 resin was verified to offer good adsorption and desorption ratio for PPP. Response surface methodology (RSM) indicated that the optimized purification parameters for PPP were 1.70 mg GAE/mL phenolic sample concentration, 22.00 mL sample volume, and 63.00% ethanol concentration. Under these conditions, the experimental purity of PPP was 27.93 ± 0.14% (n = 3), which matched well with the predicted purity of 28.17%. Next, the antiproliferative effects of PPP on seven cancer cell lines, including A375 (human skin melanoma cancer cell line), A549 (human lung cancer cell line), SH-SY5Y (human neuroblastoma cell line), LOVO (human colon cancer stem cell line), MCF-7 (human breast cancer cell line), HeLa (human cervical cancer line), and HT29 (human colon cancer line), were examined by MTT assays. The results indicated that PPP had the highest capacity for inhibiting LOVO cells growth with an EC50 value of 0.317 ± 0.0476 mg/mL. Finally, Ultra-high performance liquid chromatography- tandem mass spectrometry (UPLC-MS) was used to tentatively identify twenty-four peaks in the purified PPP, of which five representative peaks were identified as catechin, methyl quercetin, o-vanillin, luteolin and coronaric acid. Our results demonstrate that Pinus koraiensis pinecone is a readily available source of polyphenols, and the purified PPP could be a promising natural antitumor agent for applications in functional foods.

Polyphenols from pinecones of Pinus koraiensis induce apoptosis in colon cancer cells through the activation of caspase in vitro

We had previously extracted and purified a polyphenol from P. koraiensis pinecone (PPP), and evaluated its antiproliferative activities against different cancer cells lines. In the present study, we further improved the purity of PPP through the gradient elution method by different concentrations of ethanol (20%, 40% and 60%), so as to study their antitumor effects. Then, the purity and the influence upon cell viability of the purified components from PPP (PPP-20, PPP-40 and PPP-60) were evaluated. The results showed that PPP-40 had the highest phenolic purity (57.25 ± 1.83%) and exhibited the strongest inhibition (EC50 0.21 ± 0.03 mg mL−1) against LOVO cells in a dose-dependent manner. Catechin and taxifolin, the main components of PPP-40, may contribute to its antitumor activity. Moreover, we further investigated the molecular mechanisms of the PPP-40-induced apoptosis. The DNA damage in LOVO cells was observed by PI staining and comet assay. Besides, the apoptosis rates were further detected by flow cytometry. Consequently, the data showed that PPP-40 could significantly disrupt the mitochondrial membrane potential (MMP), reduce the content of adenosine 5′-triphosphate (ATP) and increase the production of reactive oxygen species (ROS). All the results indicated the mitochondrial dysfunction was involved in the PPP-40-induced apoptosis. Meanwhile, the typical markers of apoptosis involving the intrinsic and extrinsic pathways were analyzed as well. This showed that PPP-40 could not only promote the intrinsic apoptosis by increasing the release of cytochrome c (cyt c) and activating caspase-9 and -3, but also induce extrinsic apoptosis by activating caspase-8.

Preparation optimization and protective effect on 60 Co-γ radiation damage of Pinus koraiensis pinecone polyphenols microspheres

Here, the chitosan and the glutaraldehyde (GA) were used to encapsulate pinecones polyphenols of Pinus koraiensis (P. koraiensis) by emulsification cross-linking technology. First, the prepared parameters (crosslinking agent amount, stirring speed, crosslinking temperature and emulsifying time) of the pinecones polyphenols microspheres (PPMs) were optimized by the response surface methodology (RSM). When chitosan concentration and crosslinking time were 2% and 80min, respectively, the optimal conditions were 7.91mL of crosslinking agent, stirring speed of 660.98r/min, crosslinking temperature of 41.18°C and emulsifying time of 198.65min. The prepared PPMs embedding rate was 73.57%. The optimized PPM possessed a distinct core-shell structure and uniform spherical distribution with a particle size value of 3.4μm. In addition, they had the excellent sustained-release characteristics in vitro. We also evaluated the radioprotective effects of PPMs against 60Co-γ radiation in vivo. PPMs improved significantly the activity of the antioxidant enzyme SOD and reduce MDA level in the plasma of irradiated mice. Accordingly, PPMs could also significantly enhance the immunomodulation activity by promoting the proliferation of splenocytes and monocyte phagocytosis of irradiated mice. These results suggested that PPMs exert effective protection against radiation-induced injury by improving the antioxidant and immunomodulation activities.

Protective Effects on 60Co-γ Radiation Damage of Pine Cone Polyphenols from Pinus koraiensis-Loaded Chitosan Microspheres In Vivo

A novel chitosan microsphere for encapsulating pine cone polyphenols (PP) from P. koraiensis was successfully prepared using an emulsion crosslinking technique. The characteristics of pine polyphenol-loaded microspheres (PPM) were determined using scanning electron microscopy (SEM) and a laser particle size detector. It was found that PPMs were spherical in shape with uniform particle size distribution patterns. The drug content and encapsulation rate of the microspheres were 7.47% and 73.6%, respectively, at a Ch/GA mass ratio of 0.7. The animal experiments showed that PPM had a stronger radiation protective effect than PP. PPM significantly increased the immune organ indices, the quantity of marrow DNA, the superoxide dismutase (SOD) activity, the splenocyte proliferation index, and the phagocytosis activity of monocytes. PPM also decreased the numbers of micronuclei in bone marrow cells and malondialdehyde (MDA) levels in plasma in mice exposed to 60Co γ-irradiation. In addition, gender differences in biological responses to exposure to radiation were observed.

Protection of Spleen Tissue of γ-ray Irradiated Mice against Immunosuppressive and Oxidative Effects of Radiation by Adenosine 5′-Monophosphate

The immune system is very sensitive to radiation. This study revealed that adenosine 5′-monophosphate (5′-AMP) increased the DNA contents of the spleen and the spleen index of irradiated mice. Moreover, the exogenous 5′-AMP could significantly repair the ultra-structure of the damaged spleen through transmission electron microscopy. When indicators of the mouse immune system were assessed, the flow cytometry and enzyme-linked immunosorbent assay (ELISA) revealed that the administration of exogenous 5′-AMP could reduce the apoptosis in the splenic cells. It could also regulate the transition of cells towards S phase, increase the proportion of CD4+ and CD8+ cellular subsets, and enhance the secretion of interleukin-2 (IL-2), IL-4, IL-10, and interferon-γ (IFN-γ). These effects were associated with a decrease in oxidative stress, as evidenced by changes in superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), reduced glutathione (GSH), and malondialdehyde (MDA) levels of spleen tissues. These results suggested that exogenous 5′-AMP could repair the damaged spleen, increase the spleen index, and regulate the cell cycles and apoptosis. There was an increase in the production of various cytokines and play a protective role on the immune system of irradiated mice by dynamically adjusting the REDOX balance.