Haihui Zhang

Whole body radioprotective activity of an acetone–water extract from the seedpod of Nelumbo nucifera Gaertn. seedpod

Procyanidins extracted with acetone-water from lotus (Nelumbo nucifera Gaertn.) seedpod (LSPCs) were evaluated for in vivo radioprotective activity against whole body gamma irradiation in Swiss albino mice. Pretreated with LSPCs 200 mg/kg by intragastric (i.g.) for 15 days was found to be the most effective dose in preventing radiation sickness, reducing radiation-induced mortality, increasing mean survival time and elevating radiation median lethal dose (LD(50)) from 8.9 to 10.5 Gy, indicating a dose modifying factor (DMF) of 1.18. Further, administered LSPCs at a dose of 200 mg/kg could effectively maintain spleen index close to normal, stimulate endogenous spleen colony forming units, promote the levels of red blood cells (RBC), white blood cells (WBC), platelets and hemoglobin in peripheral blood, and prevent spleen and skin damage in irradiated mice, reduce the level of radiation-induced micronucleated polychromatic erythrocytes in bone marrow, maintain the polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) ratio (P/N ratio) and significantly decrease bone marrow chromosomal damage. Alternatively, pretreated with LSPCs (200 mg/kg) significantly decreased the lipid peroxidation (LPO) level, and elevated the activities of endogenous antioxidant enzymes in liver after irradiation. Thus LSPCs possess a strong whole body radioprotective activity, and it may be used as a radioprotector.

Structural elucidation and immunostimulatory activity of polysaccharide isolated by subcritical water extraction from Cordyceps militaris

Water-soluble polysaccharides were obtained from Cordyceps militaris (C. militaris) (CMP) by subcritical water extraction (SWE). Two polysaccharides fractions, CMP-W1 and CMP-S1, were isolated from CMP using DEAE-52 cellulose and Sephadex G-150 column chromatography. The structural characteristics of CMP-W1 and CMP-S1 were investigated. The results showed that the molecular weight of CMP-W1 and CMP-S1 are 3.66×105Da and 4.60×105Da, respectively, and both of them were heteropolysaccharides composed of d-mannose, d-glucose, d-galactose with the molar ratios of 2.84:1:1.29 and 2.05:1:1.09, respectively. FT-IR spectra analysis suggested that CMP-W1 and CMP-S1 belonged to pyranose form sugar and protein free. For immunostimulatory activity assay in vitro, CMP-W1 and CMP-S1 significantly promoted lymphatic spleen cell proliferation of mice. Therefore, the polysaccharides obtained from C. militaris by SWE can be used as potential natural immunostimulant in functional foods or medicine.

Cytotoxic effects of procyanidins from Castanea mollissima Bl. shell on human hepatoma G2 cells in vitro.

Significant cytotoxic effects of procynadins from chestnut (Castanea mollissima Bl.) shell (CSPC) on human hepatoma G2 (HepG2) cells were found in vitro. CSPC could inbibit HepG2 proliferation in a dose-dependent manner (100-400 μg/mL), arrest cell cycle in the G0/G1 phase, induce apoptosis and trigger necrosis of HepG2. Proapoptotic effect of CSPC was evidenced by nuclear condensation, internucleosomal DNA fragmentation. Treatment of HepG2 cells with CSPC caused a loss of mitochondrial membrane potential and stimulated reactive oxidative species (ROS) generation. These results suggested CSPC could trigger apoptosis and necrotic cell death in HepG2 cell, which might be associated with ROS generation through the mitochondria-dependent signaling way.

Autophagic cell death of human hepatoma G2 cells mediated by procyanidins from Castanea mollissima Bl. Shell-induced reactive oxygen species generation

The autophagy of human hepatoma G2 (HepG2) cells induced by procyanidins from chestnut (Castanea mollissima Bl.) shell (CSPCs) was investigated, and the inherent relationship between autophagic levels and reactive oxygen species (ROS) generation was studied. The results showed that CSPCs induced HepG2 cell death in a time- and concentration-dependent manner, increased the accumulation of autophagolysosomes and microtubule-associated proteins light chain 3-II (LC3-II, a marker of autophagy). However, these phenomena were not observed in the group pretreated with the autophagy inhibitor 3-MA, suggesting that CSPCs induced HepG2 cell autophagy. Furthermore, we found that CSPCs triggered ROS generation in cells, while the levels of ROS decreased in the N-acetylcysteine (Nac) co-treatment, revealing that CSPCs-mediated autophagy was partly blocked by Nac. In addition, treatment with CSPCs decreased the mitochondrial membrane potential of HepG2 cells. These results suggested CSPCs could trigger autophagy via ROS generation, which may be associated with the mitochondria-dependent signaling way.

Procyanidins, from Castanea mollissima Bl. shell, induces autophagy following apoptosis associated with PI3K/AKT/mTOR inhibition in HepG2 cells

Procyanidins from Castanea mollissima Bl. shell (CSPCs) induced autophagy and apoptosis in HepG2 cells and its mechanism remains to be examined. In this paper, autophagy was measured by the lipid modification of light chain-3 (LC3) and the formation of autophagosomes. Hoechst 33258 staining and flow cytometer analysis were used to measure apoptosis. The western blot analysis was used to examine the effects of CSPCs on the expression of LC3, PI3K, phosphorylation of AKT, mTOR, Bcl-2, Bad, Bax, BID and cleaved caspase 3 in HepG2 cells. The results showed that 3-methyladenine (3-MA) and apoptosis inhibitor (Z-VAD) could inhibited the death of HepG2 induced by CSPCs for 48h (150μg/mL). CSPCs induced the accumulation of autophagosomes and microtubule-associated proteins light chain 3-II (LC3-II, a marker of autophagy). P-AKT, PI3K and mTOR were significantly decreased on CSPCs exposure. However, these phenomena were not observed in the group pretreated with the autophagy inhibitor 3-MA and Z-VAD. CSPCs also induced the expression of Bad, Bax and Beclin-1 proteins and decreased the expression of Bcl-2, which was inhibited by 3-MA and Z-VAD. Moreover the apoptotic cell death could be inhibited by 3-MA. In addition, inhibition of LC3-II by siRNA-dependent knockdown attenuated the cleavage of caspase 3. These results suggested CSPCs could trigger autophagy via inhibition of the PI3K/AKT/mTOR signaling pathway, enhanced apoptosis in HepG2 cells which may be associated with the mitochondria-dependent signaling way.

Chemoprotective action of lotus seedpod procyanidins on oxidative stress in mice induced by extremely low-frequency electromagnetic field exposure.

With the increasing use of electromagnetic technology, the effects of extremely low-frequency electromagnetic fields (ELF-EMF) on biological systems, central neurotransmitter systems, and human health have attracted extensive attention worldwide. In this study, lotus seedpod procyanidins (LSPCs) were evaluated for their protective effects on ELF-EMF induced oxidative stress injury in mice. Sixty male ICR mice were used for the experiment. The mice were randomly divided into five equal groups. The control group did not receive LSPCs or ELF-EMF but orally received normal saline. The ELF-EMF group received ELF-EMF exposure plus normal saline orally. The other three groups received ELF-EMF exposure plus LSPCs orally (60, 90, or 120mg kg(-1).bw, respectively). Each group exposed to ELF-EMF at 8 mT, 4h day(-1) for 28 consecutive days after administration daily of LSPCs or normal saline to mice for 15 consecutive days with the exception of the control group. Thereafter, blood and cerebral cortex of the mice were analyzed for antioxidant indices, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione-S-transferase (GST) and malondialdehyde (MDA). LSPCs administration at different doses significantly inhibited oxidative stress damage of mice induced by ELF-EMF. LSPCs treatment augmented SOD, CAT, GSH-Px, GR and GST activity. Furthermore, administration significantly lowered MDA level in LSPCs treatment groups LSPCs. All results indicated LSPCs can effectively prevent oxidative stress injury induced by ELF-EMF exposure, which may be related to its ability of scavenging free radicals and stimulating antioxidant enzyme activity.

Protective effect of procyanidins extracted from the lotus seedpod on immune function injury induced by extremely low frequency electromagnetic field

This study aimed to evaluate the protective effect of Lotus seedpod procyanidins (LSPCs) from extremely low frequency electromagnetic field (ELF-EMF) exposure (50Hz, 8mT, 28 days) and their protective mechanism against radiation damage. The results showed that LSPCs increased the organ index of mice and made the damaged blood-producing function and cytokine(INF-γ, TNF-α, IL-2, IL-6 and IL-10 in spleen) levels by ELF-EMF-irradiation recovered to normal appearance. And experimental results proved that dosing LSPCs inhibit more stagnation of splenocytes in G0/G1 phase caused by ELF-EMF, thus the spleen cells from G0/G1 phase to S phase shift, restore normal cell metabolism, promote the splenocytes proliferation, reduced the apoptosis of spleen cells, effective protect the damage induced by the ELF-EMF radiation. In addition, LSPCs prevented the decline of DNA content caused by ELF-EMF. Western blot determinated the levels of apoptosis genes including Bcl-2, Bax, Bcl-cl, Caspase-3 and Caspase-9. The results revealed that a significant suppression in Bcl-2 expression and increase in Bax, Caspase-3 and Caspase-9 expression in splenic cells in ELF-EMF group. However, LSPCs restored these changes. Taking these results together, it may be summarized that LSPCs could protect hematopoietic tissues and the immune system from ELF-EMF. And it may be hypothesized that ELF-EMF-induced apoptosis in splenocytes might occur via triggers the trans-activation of Bax and activates caspases-3 and -9, which then cleaves the death substrates, leading to apoptosis in splenocytes of mice treated with ELF-EMF.

Neuroprotective effects of lotus seedpod procyanidins on extremely low frequency electromagnetic field-induced neurotoxicity in primary cultured hippocampal neurons.

The present study investigated the protective effects of lotus seedpod procyanidins (LSPCs) on extremely low frequency electromagnetic field (ELF-EMF)-induced neurotoxicity in primary cultured rat hippocampal neurons and the underlying molecular mechanism. The results of MTT, morphological observation, superoxide dismutase (SOD) and malondialdehyde (MDA) assays showed that compared with control, incubating neurons under ELF-EMF exposure significantly decreased cell viability and increased the number of apoptotic cells, whereas LSPCs evidently protected the hippocampal neurons against ELF-EMF-induced cell damage. Moreover, a certain concentration of LSPCs inhibited the elevation of intracellular reactive oxygen species (ROS) and Ca(2+) level, as well as prevented the disruption of mitochondrial membrane potential induced by ELF-EMF exposure. In addition, supplementation with LSPCs could alleviate DNA damage, block cell cycle arrest at S phase, and inhibit apoptosis and necrosis of hippocampal neurons under ELF-EMF exposure. Further study demonstrated that LSPCs up-regulated the activations of Bcl-2, Bcl-xl proteins and suppressed the expressions of Bad, Bax proteins caused by ELF-EMF exposure. In conclusion, these findings revealed that LSPCs protected against ELF-EMF-induced neurotoxicity through inhibiting oxidative stress and mitochondrial apoptotic pathway.

Procyanidins from Nelumbo nucifera Gaertn. Seedpod induce autophagy mediated by reactive oxygen species generation in human hepatoma G2 cells

In this study, autophagic effect of procyanidins from lotus (Nelumbo nucifera Gaertn.) seedpod (LSPCs) on human hepatoma G2 (HepG2) cells, and the inherent correlation between autophagic levels and reactive oxygen species (ROS) generation were investigated. The results showed that LSPCs increased monodansylcadaverine (MDC) fluorescence intensity and LC3-I/LC3-II conversion in HepG2 cells. In addition, the typically autophagic characteristics (autophagosomes and autolysosomes) were observed in LSPCs-treated cells, but not found in the cells treated with autophagy inhibitor 3-methyladenine (3-MA). Furthermore, the elevated ROS level was in line with the increasing of autophagy activation caused by LSPCs, however, both 3-MA and the ROS scavenger N-acetylcyteine (NAC) inhibitors effectively suppressed the autophagy and ROS generation triggered by LSPCs. As a result, these results indicated that LSPCs induced HepG2 cell autophagy in a time- and dose-dependent manner, and promoted reactive oxygen species (ROS) generation on HepG2 cells. Moreover, we found that LSPCs caused DNA damage, S phase arrest and the decrement of mitochondria membrane potential (MMP) which were associated with ROS generation. In summary, our findings demonstrated that the LSPCs-induced autophagy and autophagic cell death were triggered by the ROS generation in HepG2 cells, which might be associated with ROS generation through the mitochondria-dependent signaling way.

Subcritical water extraction of polyphenolic compounds from sorghum (Sorghum bicolor L.) bran and their biological activities

Subcritical water extraction (SWE), an environment-friendly technique, was applied to extract polyphenolics from sorghum bran. Extraction temperatures (°C), time (min), and solid-liquid ratio (mL/g) were investigated and optimized by Box-Behnken design. The optimized conditions for SWE was 144.5 °C of temperature, 21 min of time, and 35 mL/g of solid-liquid ratio, with a polyphenolics yield of 47.253 ± 0.375 mg GAE/g dw, which was in good agree with the predicted value. Comparing with hot water extraction (HWE), SWE resulted in a higher yield of polyphenolics, higher radical scavenging activities, and more efficient antiproliferative activity. Furthermore, major polyphenolic compositions of the extracts were identified and quantified by HPLC-ESI-MS/MS. Taxifolin, taxifolin hexoside, oligomeric procyanidins, and epicatechin were the most abundant polyphenolic compounds in the extracts. Taken together, SWE can be used as a effective extraction method for polyphenolics from sorghum bran, which could be used as a potential source of natural antioxidants.