灵芝多糖精粉制备技术的研究

该项目进行了灵芝液体深层培养和多糖分离提取工艺的研究,并开展了灵芝多糖精粉制备技术的研究。通过动物实验对从灵芝菌丝培养物提取和制备的灵芝多糖精粉的免疫功能和抗肿瘤活性进行了研究,实验结果表明:灵芝多糖精粉作为刺激源,可促进巨噬细胞和B淋巴细胞的免疫功能;与抗癌药物环磷酰胺合用时,还可增强其抑制肿瘤的效能。     

Chemical Composition,Antitumor Properties,and Mechanism of the Essential Oil from Plagiomnium acutum T. Kop.

Plagiomnium acutum T. Kop. (P. acutum) has been used as a traditional Chinese medicine for thousands of years to treat cancer but lacks evidence. The objective of this work was to reveal the chemical composition of P. acutum essential oil (PEO) and explore its potential antitumor activity and molecular mechanism. PEO was prepared by the simultaneous distillation–extraction method and characterized by gas chromatography/mass spectroscopy. CCK8 assay, flow cytometry, western blot, and immunofluorescence techniques were used to analyze the effects and mechanism of PEO against cancer cells. A total of 74 constituents of PEO were identified, with diterpenes (26.5%), sesquiterpenes (23.89%), and alcohols (21.81%) being the major constituents. Two terpenoids, selina-6-en-4-ol and dolabella-3,7-dien-18-ol, were detected in PEO for the first time. PEO showed significant cell growth inhibitory activity on HepG2 and A549 cells by blocking the G1 phase and inducing apoptosis, which may be attributed to its upregulation of p21^Cip1 and p27^Kip1 proteins and interference with mitochondrial membrane potential effect. Dolabella-3,7-dien-18-ol accounts for 25.5% of PEO and is one of the main active components of PEO, with IC₅₀ values in HepG2 and A549 cells of (25.820 ± 0.216) µg/mL and (23.597 ± 1.207) μg/mL, respectively. These results confirmed the antitumor medicinal value of P. acutum and showed great application potential in the pharmaceutical industry.     

Antitumor Effect of Cycloastragenol in Colon Cancer Cells via p53 Activation

Colorectal cancer cell (CRC) is the fourth most common cancer in the world. There are several chemotherapy drugs available for its treatment, though they have side effects. Cycloastragenol (CY) is a compound from Astragalus membranaceus (Fisch.) Bge known to be effective in aging, anti-inflammatory, anticancer, and anti-heart failure treatments. Although many studies have demonstrated the functions of CY in cancer cells, no studies have shown the effects of p53 in colon cancer cells. In this study, we found that CY reduces the viability of colon cancer cells in p53 wild-type cells compared to p53 null cells and HT29. Furthermore, CY induces apoptosis by p53 activation in a dose- and time-dependent manner. And it was confirmed that it affects the L5 gene related to p53. Additionally, CY enhanced p53 expression compared to when either doxorubicin or 5-FU was used alone. Altogether, our findings suggest that CY induces apoptosis via p53 activation and inhibits the proliferation of colon cancer cells. In addition, apoptosis occurs in colon cancer cells due to other factors. Moreover, CY is expected to have a combined effect when used together with existing treatments for colon cancer in the future.     

Structure and Antitumor and Immunomodulatory Activities of a Water-Soluble Polysaccharide from Dimocarpus longan Pulp

A new water-soluble polysaccharide (longan polysaccharide 1 (LP1)) was extracted and successfully purified from Dimocarpus longan pulp via diethylaminoethyl (DEAE)-cellulose anion-exchange and Sephacryl S-300 HR gel chromatography. The chemical structure was determined using Infrared (IR), gas chromatography (GC) and nuclear magnetic resonance (NMR) analysis. The results indicated that the molecular weight of the sample was 1.1 × 10⁵ Da. Monosaccharide composition analysis revealed that LP1 was composed of Glc, GalA, Ara and Gal in a molar ratio of 5.39:1.04:0.74:0.21. Structural analysis indicated that LP1 consisted of a backbone of →4)-α-d-Glcp-(1→4)-α-d-GalpA-(1→4)-α-d-Glcp-(1→4)-β-d-Glcp-(1→ units with poly saccharide side chains composed of →2)-β-d-Fruf-(1→2)-l-sorbose-(1→ attached to the O-6 position of the α-d-Glcp residues. In vitro experiments indicated that LP1 had significantly high antitumor activity against SKOV3 and HO8910 tumor cells, with inhibition percentages of 40% and 50%, respectively. In addition, LP1 significantly stimulated the production of the cytokine interferon-γ (IFN-γ), increased the activity of murine macrophages and enhanced B- and T-lymphocyte proliferation. The results of this study demonstrate that LP1 has potential applications as a natural antitumor agent with immunomodulatory activity.     

灵芝的化学成分与药理作用研究进展

灵芝既具有广泛的药理活性又具有食用价值。其化学成分复杂,含有多种对人体有效的物质,药理作用广。通过查阅相关文献,对灵芝的化学成分及其药理作用进行了较全面的综述。为合理开发利用灵芝提供参考。     

Methyl Sartortuoate Inhibits Colon Cancer Cell Growth by Inducing Apoptosis and G2/M-Phase Arrest

The potential anti-neoplastic activity of terpenoids is of continued interest. In this study, we investigate whether methyl sartortuoate, a terpenoid isolated from soft coral, induced cell cycle arrest and apoptosis in a human colon cancer cell line. Culture studies found that methyl sartortuoate inhibited colon cancer cell (LoVo and RKO) growth and caused apoptotic death in a concentration- and time-dependent manner, by activation of caspase-8, caspase-9, caspase-3, p53 and Bax, and inactivation of B-cell lymphoma 2 (Bcl-2) apoptosis regulating proteins. Methyl sartortuoate treatment led to reduced expression of cdc2 and up-regulated p21 and p53, suggesting that Methyl sartortuoate induced G2-M arrest through modulation of p53/p21/cdc2 pathways. Methyl sartortuoate also up-regulated phospho-JNK and phospho-p38 expression levels. This resulted in cell cycle arrest at the G2-M phase and apoptosis in LoVo and RKO cells. Treatment with the JNK inhibitor SP600125 and the p38 MAPK inhibitor SB203580 prevented methyl sartortuoate-induced apoptosis in LoVo cells. Moreover, methyl sartortuoate also prevented neoplasm growth in NOD-SCID nude mice inoculated with LoVo cells. Taken together, these findings suggest that methyl sartortuoate is capable of leading to activation of caspase-8, -9, -3, increasing p53 and Bax/Bcl-2 ratio apoptosis through MAPK-dependent apoptosis and results in G2-M phase arrest in LoVo and RKO cells. Thus, methyl sartortuoate may be a promising anticancer candidate.     

Chemical Composition and Antioxidant Activities of Three Polysaccharide Fractions from Pine Cones

The traditional method of gas chromatography-mass spectrometry for monosaccharide component analysis with pretreatment of acetylation is described with slight modifications and verified in detail in this paper. It was then successfully applied to the quantitative analysis of component monosaccharides in polysaccharides extracted from the pine cones. The results demonstrated that the three pine cone polysaccharides all consisted of ribose, rhamnose, arabinose, xylose, mannose, glucose and galactose in different molar ratios. According to the recovery experiment, the described method was proved accurate and practical for the analysis of pine cone polysaccharides, meeting the need in the field of chemical analysis of Pinus plants. Furthermore; the chemical characteristics, such as neutral sugar, uronic acids, amino acids, molecular weights, and antioxidant activities of the polysaccharides were investigated by chemical and instrumental methods. The results showed that the chemical compositions of the polysaccharides differed from each other, especially in the content of neutral sugar and uronic acid. In the antioxidant assays, the polysaccharide fractions exhibited effective scavenging activities on ABTS radical and hydroxyl radical, with their antioxidant capabilities decreasing in the order of PKP > PAP > PSP. Therefore, although the polysaccharide fractions had little effect on superoxide radical scavenging, they still have potential to be developed as natural antioxidant agents in functional foods or medicine.     

Anti-Proliferative Activities and Apoptosis Induction by Triterpenes Derived from Eriobotrya japonica in Human Leukemia Cell Lines

Eriobotrya japonica leaf is a traditional herbal medicine that contains numerous triterpenes, which have various pharmacological properties. In this study, we investigated the anti-proliferative activity of four triterpenes derived from E. japonica, including corosolic acid (CA), ursolic acid (UA), maslinic acid (MA) and oleanolic acid (OA), in human leukemia cell lines. CA showed the strongest anti-proliferative activity in all of the leukemia cell lines tested, but not in normal human skin fibroblast cell lines. To determine the mechanism underlying the anti-proliferative effect of CA, we examined the effect of CA on molecular events known as apoptosis induction. CA induced chromatin condensation, DNA fragmentation, sub-G₁ phase DNA, activation of caspase-3, -8 and -9 and the cleavage of PARP in HL-60. CA also activated Bid and Bax, leading to the loss of mitochondrial membrane potential (Δψ_m) and cytochrome c release into the cytosol, whereas Bcl-2 and Bcl-xL were unaffected by CA. These results suggest that CA has an anti-proliferative effect on leukemia cells via the induction of apoptosis mediated by mitochondrial dysfunction and caspase activation. CA may be a potential chemotherapeutic agent for the treatment of human leukemia.     

灵芝精粉与灵芝孢子粉对Lewis肺癌模型小鼠免疫功能的影响

目的研究灵芝精粉与灵芝孢子粉对Lewis肺癌模型小鼠免疫功能的影响,并比较二者在肿瘤治疗中的免疫调节作用。方法经C57BL/6小鼠右腋皮下接种1×106个Lewis肺癌细胞,建立小鼠Lewis肺癌模型。于接种肿瘤细胞次日至第20天,分别隔日灌胃灵芝孢子粉(D1)、灵芝精粉001(D2)及灵芝精粉002(D3),剂量均为0.5g/kg,同时设立对照组和肿瘤组(灌胃等体积生理盐水)。监测各组小鼠体重及瘤体积,第21日处死小鼠,称量瘤重,并无菌制备脾细胞悬液,检测小鼠NK细胞杀伤活性,流式细胞术检测小鼠脾细胞中CD4+、CD8+T细胞和B细胞数量,淋巴细胞转化试验检测脾细胞中T、B细胞增殖功能。结果与对照组比较,肿瘤组小鼠脾细胞中T、B细胞增殖功能、细胞数量及NK细胞杀伤活性均有不同程度的下降。与肿瘤组相比,3种灵芝制剂均可提高Lewis肺癌模型小鼠NK细胞的杀伤活性和T、B细胞增殖功能,其中,以D2作用最为明显(P<0.05);3种灵芝制剂均可不同程度地提高Lewis肺癌模型小鼠CD4+、CD8+T细胞数量,其中,以D3作用最为显著(P<0.05)。3种灵芝制剂对Lewis肺癌模型小鼠的体重、瘤重、瘤体积及B细胞数量均无明显影响。结论灵芝制剂对Lewis肺癌小鼠的免疫功能具有正向调节作用,其中灵芝精粉的作用优于灵芝孢子粉。     

Structural Characterization and Antioxidant Activities of Polysaccharides Extracted from the Pulp of Elaeagnus angustifolia L.

In this study, two polysaccharides (Elaeagnus angustifolia L. polysaccharide-1 (PEA-1) and PEA-2) were prepared from Elaeagnus angustifolia L. Then, the preliminary structure and antioxidant activities of all the samples were investigated. The results showed that the average molecular weights for PEA-1 and PEA-2 were 9113 and 5020 Da, respectively. And, PEA-1 was mainly composed of rhamnose, xylose, mannose, glucose, and galactose, respectively. The components of PEA-2 were rhamnose, mannose, glucose, and galactose, respectively. Moreover, the Antioxidant assays demonstrated that PEA-1 possessed of strong free radicals scavenging activity and hydroxyl radicals scavenging activities, suggesting that PEA-1 could potentially be used as natural antioxidant.     

In Vitro and In Silico Characterization of an Antimalarial Compound with Antitumor Activity Targeting Human DNA Topoisomerase IB

Human DNA topoisomerase IB controls the topological state of supercoiled DNA through a complex catalytic cycle that consists of cleavage and religation reactions, allowing the progression of fundamental DNA metabolism. The catalytic steps of human DNA topoisomerase IB were analyzed in the presence of a drug, obtained by the open-access drug bank Medicines for Malaria Venture. The experiments indicate that the compound strongly and irreversibly inhibits the cleavage step of the enzyme reaction and reduces the cell viability of three different cancer cell lines. Molecular docking and molecular dynamics simulations suggest that the drug binds to the human DNA topoisomerase IB-DNA complex sitting inside the catalytic site of the enzyme, providing a molecular explanation for the cleavage-inhibition effect. For all these reasons, the aforementioned drug could be a possible lead compound for the development of an efficient anti-tumor molecule targeting human DNA topoisomerase IB.     

5-氟尿嘧啶甘氨酸类化合物的合成、表征及抗癌活性

常温下,以5-氟尿嘧啶为起始原料,以1-乙基-3-(3-二甲氨基丙基)碳二亚胺(EDCI)和1-羟基苯并三氮唑(HOBt)为缩合剂,通过液相偶联法,合成了2-(5-氟尿嘧啶-1-基乙酰基)氨基乙酸甲酯,收率72%;控制pH=10～11,温度20～30℃水解得到相应的2-(5-氟尿嘧啶-1-基乙酰基)氨基乙酸,收率75%。两个化合物的结构通过1HNMR、13CNMR、MS、IR确证;通过紫外吸收光谱初步揭示了化合物的抗肿瘤机制;委托国家新药筛选中心进行了体外抗白血病细胞株HL-60和肝癌细胞株BEL-7402活性测试,结果表明,该化合物具有一定的体外抑制肿瘤作用。     

Allyl Isothiocyanate (AITC) Induces Apoptotic Cell Death In Vitro and Exhibits Anti-Tumor Activity in a Human Glioblastoma GBM8401/luc2 Model

Some clinically used anti-cancer drugs are obtained from natural products. Allyl isothiocyanate (AITC), a plant-derived compound abundant in cruciferous vegetables, has been shown to possess an anti-cancer ability in human cancer cell lines in vitro, including human brain glioma cells. However, the anti-cancer effects of AITC in human glioblastoma (GBM) cells in vivo have not yet been examined. In the present study, we used GBM8401/luc2 human glioblastoma cells and a GBM8401/luc2-cell-bearing animal model to identify the treatment efficacy of AITC. Here, we confirm that AITC reduced total cell viability and induced cell apoptosis in GBM8401/luc2 cells in vitro. Furthermore, Western blotting also showed that AITC induced apoptotic cell death through decreased the anti-apoptotic protein BCL-2, MCL-1 expression, increased the pro-apoptotic protein BAX expression, and promoted the activities of caspase-3, -8, and -9. Therefore, we further investigated the anti-tumor effects of AITC on human GBM8401/luc2 cell xenograft mice. The human glioblastoma GBM8401/luc2 cancer cells were subcutaneously injected into the right flank of BALB/c nude mice to generate glioblastoma xenograft mice. The animals were randomly divided into three groups: group I was treated without AITC (control); group II with 0.1 mg/day of AITC; and group III with 0.2 mg/day of AITC every 3 days for 27 days. Bodyweight, and tumor volume (size) were recorded every 3 days. Tumors exhibiting Luc2 intensity were measured, and we quantified intensity using Living Image software on days 0, 12, and 24. After treatment, tumor weight from each mouse was recorded. Tumor tissues were examined for histopathological changes using H&E staining, and we analyzed the protein levels via immunohistochemical analysis. Our results indicate that AITC significantly inhibited tumor growth at both doses of AITC due to the reduction in tumor size and weight. H&E histopathology analysis of heart, liver, spleen, and kidney samples revealed that AITC did not significantly induce toxicity. Body weight did not show significant changes in any experiment group. AITC significantly downregulated the protein expression levels of MCL-1, XIAP, MMP-9, and VEGF; however, it increased apoptosis-associated proteins, such as cleaved caspase-3, -8, and -9, in the tumor tissues compared with the control group. Based on these observations, AITC exhibits potent anti-cancer activity in the human glioblastoma cell xenograft model via inhibiting tumor cell proliferation and the induction of cell apoptosis. AITC may be a potential anti-GBM cancer drug that could be used in the future.     

Natural Products of Marine Origin for the Treatment of Colorectal and Pancreatic Cancers: Mechanisms and Potential

Gastrointestinal cancer refers to malignancy of the accessory organs of digestion, and it includes colorectal cancer (CRC) and pancreatic cancer (PC). Worldwide, CRC is the second most common cancer among women and the third most common among men. PC has a poor prognosis and high mortality, with 5-year relative survival of approximately 11.5%. Conventional chemotherapy treatments for these cancers are limited due to severe side effects and the development of drug resistance. Therefore, there is an urgent need to develop new and safe drugs for effective treatment of PC and CRC. Historically, natural sources—plants in particular—have played a dominant role in traditional medicine used to treat a wide spectrum of diseases. In recent decades, marine natural products (MNPs) have shown great potential as drugs, but drug leads for treating various types of cancer, including CRC and PC, are scarce. To date, marine-based drugs have been used against leukemia, metastatic breast cancer, soft tissue sarcoma, and ovarian cancer. In this review, we summarized existing studies describing MNPs that were found to have an effect on CRC and PC, and we discussed the potential mechanisms of action of MNPs as well as future prospects for their use in treating these cancers.     

Two-stage extraction of antitumor,antioxidant and antiacetylcholinesterase compounds from Ganoderma lucidum fruiting body

Ganoderma lucidum has been used in oriental medicine for its contribution to vitality and longevity. None of them report about targeted antitumor activity against adenocarcinoma cells, or antioxidant and antiacetylcholinesterase activity for potential application in treatment of Alzheimer's and other neurodegenerative diseases. To date, there are a few studies available concerning supercritical carbon dioxide extraction of biologically active compounds from G. lucidum fruiting body. In our study, two stage extractions of biologically active compounds from G. lucidum were performed. First, supercritical carbon dioxide was used as extraction solvent. Next, the same material was used for hot water isolation of biologically active polysaccharides. Cytotoxicity, antioxidant and antiacetylcholinesterase activity were tested for all obtained extracts. Additionally, the effect of extraction process conditions on the biological activity of extracts was assessed.     

Extraction,Preliminary Characterization and Evaluation of in Vitro Antitumor and Antioxidant Activities of Polysaccharides from Mentha piperita

This study describes the extraction, preliminary characterization and evaluation of the in vitro antitumor and antioxidant activities of polysaccharides extracted from Mentha piperita (MPP). The optimal parameters for the extraction of MPP were obtained by Box-Behnken experimental design and response surface methodology (RSM) at the ratio of water to raw material of 20, extraction time of 1.5 h and extraction temperature at 80 °C. Chemical composition analysis showed that MPP was mainly composed of glucuronic acid, galacturonic acid, glucose, galactose and arabinose, and the molecular weight of its two major fractions were estimated to be about 2.843 and 1.139 kDa, respectively. In vitro bioactivity experiments showed that MPP not only inhibited the growth of A549 cells but possessed potent inhibitory action against DNA topoisomerase I (topo I), and an appreciative antioxidant action as well. These results indicate that MPP may be useful for developing safe natural health products.     

Pro-Apoptotic and Immunotherapeutic Effects of Carbon Nanotubes Functionalized with Recombinant Human Surfactant Protein D on Leukemic Cells

Nanoparticles are efficient drug delivery vehicles for targeting specific organs as well as systemic therapy for a range of diseases, including cancer. However, their interaction with the immune system offers an intriguing challenge. Due to the unique physico-chemical properties, carbon nanotubes (CNTs) are considered as nanocarriers of considerable interest in cancer diagnosis and therapy. CNTs, as a promising nanomaterial, are capable of both detecting as well as delivering drugs or small therapeutic molecules to tumour cells. In this study, we coupled a recombinant fragment of human surfactant protein D (rfhSP-D) with carboxymethyl-cellulose (CMC) CNTs (CMC-CNT, 10–20 nm diameter) for augmenting their apoptotic and immunotherapeutic properties using two leukemic cell lines. The cell viability of AML14.3D10 or K562 cancer cell lines was reduced when cultured with CMC-mwCNT-coupled-rfhSP-D (CNT + rfhSP-D) at 24 h. Increased levels of caspase 3, 7 and cleaved caspase 9 in CNT + rfhSP-D treated AML14.3D10 and K562 cells suggested an involvement of an intrinsic pathway of apoptosis. CNT + rfhSP-D treated leukemic cells also showed higher mRNA expression of p53 and cell cycle inhibitors (p21 and p27). This suggested a likely reduction in cdc2-cyclin B1, causing G2/M cell cycle arrest and p53-dependent apoptosis in AML14.3D10 cells, while p53-independent mechanisms appeared to be in operation in K562 cells. We suggest that CNT + rfhSP-D has therapeutic potential in targeting leukemic cells, irrespective of their p53 status, and thus, it is worth setting up pre-clinical trials in animal models.     

Anti-Apoptotic and Antioxidant Activities of the Mitochondrial Estrogen Receptor Beta in N2A Neuroblastoma Cells

Estrogens are steroid hormones that play a crucial role in the regulation of the reproductive and non-reproductive system physiology. Among non-reproductive systems, the nervous system is mainly affected by estrogens due to their antioxidant, anti-apoptotic, and anti-inflammatory activities, which are mediated by membranous and nuclear estrogen receptors, and also by non-estrogen receptor-associated estrogen actions. Neuronal viability and functionality are also associated with the maintenance of mitochondrial functions. Recently, the localization of estrogen receptors, especially estrogen receptor beta, in the mitochondria of many types of neuronal cells is documented, indicating the direct involvement of the mitochondrial estrogen receptor beta (mtERβ) in the maintenance of neuronal physiology. In this study, cell lines of N2A cells stably overexpressing a mitochondrial-targeted estrogen receptor beta were generated and further analyzed to study the direct involvement of mtERβ in estrogen neuroprotective antioxidant and anti-apoptotic actions. Results from this study revealed that the presence of estrogen receptor beta in mitochondria render N2A cells more resistant to staurosporine- and H₂O₂-induced apoptotic stimuli, as indicated by the reduced activation of caspase-9 and -3, the increased cell viability, the increased ATP production, and the increased resistance to mitochondrial impairment in the presence or absence of 17-β estradiol (E2). Thus, the direct involvement of mtERβ in antioxidant and anti-apoptotic activities is documented, rendering mtERβ a promising therapeutic target for mitochondrial dysfunction-associated degenerative diseases.     

竹材剩余物纳米纤维素晶体的制备及性能表征

以竹材加工剩余物为原材料,通过硫酸酸解法成功制备了竹纳米纤维素晶体（竹CNC）,考察酸解时间对竹CNC产率、形态及稳定性的影响。透射电镜（TEM）观察表明,竹CNC为刚性棒状结构,长度为160nm左右,直径为5-15nm;Zeta电位测试结果表明,竹CNC悬浮液具有较好的储存稳定性。随着酸解时间的延长,竹CNC产率出现先上升后下降的变化,酸解时间为60min时竹CNC产率最大,为34．4％。采用Simha理论,采用乌氏黏度计测定竹CNC悬浮液的特性黏数,估算了竹CNC的长径比为20左右,所得结果与TEM观察结果基本一致。     

Isolation and Characterization of Human Colon Adenocarcinoma Stem-Like Cells Based on the Endogenous Expression of the Stem Markers

Background: Cancer stem cells’ (CSCs) self-maintenance is regulated via the pluripotency pathways promoting the most aggressive tumor phenotype. This study aimed to use the activity of these pathways for the CSCs’ subpopulation enrichment and separating cells characterized by the OCT4 and SOX2 expression. Methods: To select and analyze CSCs, we used the SORE6x lentiviral reporter plasmid for viral transduction of colon adenocarcinoma cells. Additionally, we assessed cell chemoresistance, clonogenic, invasive and migratory activity and the data of mRNA-seq and intrinsic disorder predisposition protein analysis (IDPPA). Results: We obtained the line of CSC-like cells selected on the basis of the expression of the OCT4 and SOX2 stem cell factors. The enriched CSC-like subpopulation had increased chemoresistance as well as clonogenic and migration activities. The bioinformatic analysis of mRNA seq data identified the up-regulation of pluripotency, development, drug resistance and phototransduction pathways, and the downregulation of pathways related to proliferation, cell cycle, aging, and differentiation. IDPPA indicated that CSC-like cells are predisposed to increased intrinsic protein disorder. Conclusion: The use of the SORE6x reporter construct for CSCs enrichment allows us to obtain CSC-like population that can be used as a model to search for the new prognostic factors and potential therapeutic targets for colon cancer treatment.