A Distinctive Pattern of Beauveria bassiana‐biotransformed Ginsenoside Products Triggers Mitochondria/FasL‐mediated Apoptosis in Colon Cancer Cells

Ginseng is one of the most commonly used adaptogens. Transformation into the minor ginsenosides produces compounds with more effective action. Beauveria bassiana, a teleomorph of Cordyceps bassiana, is a highly efficient producer of mammalian steroids and produces large amounts of sugar‐utilizing enzymes. However, the fermentation of steroid glycosides in ginseng with B. bassiana has never been studied. Thus, we evaluated the bioconversion of the major ginsenosides in white ginseng by B. bassiana. Interestingly, B. bassiana increased the total amount of protopanaxadiols and hydrolyzed Rb1 into minor ginsenosides, exhibiting high levels of Rd and Rg3, as well as moderate levels of Rb2 and Rc analyzed by high‐performance liquid chromatography coupled with evaporative light‐scattering detection. The β‐glucosidase activity was highly increased, which led to the selective elimination of sugar moiety at the 20‐C position of Rb1 to Rd, followed by Rg3. Rb2 and Rc accumulated because of the minimal activities of α‐L‐arabinopyranosidase and α‐L‐arabinofuranosidase, respectively. The fermentation product exerted dose‐dependent cytotoxicity in HCT‐15 cells, which are resistant to ginseng. The product, but not white ginseng, exhibited apoptotic effects via the Fas ligand and caspase 8/9. This study demonstrates for the first time that the B. bassiana‐fermented metabolites have potent apoptotic activity in colon cancer cells, linking to a therapeutic use. Copyright © 2015 John Wiley & Sons, Ltd.     

人参总皂苷对皮肤细胞的修复作用机制研究

目的研究人参总皂苷对皮肤细胞的抗凋亡及对大鼠皮肤光老化的修复作用。方法通过生化实验证明人参总皂苷对皮肤细胞的抗凋亡作用,并通过建立大鼠光老化模型,观察人参总皂苷对大鼠光老化皮肤的修复作用。结果与空白对照组相比,模型组具有明显的细胞凋亡现象(18.36%);而加入人参总皂苷修复后CCC-HSF-1细胞的凋亡率(15.53%,9.09%)与模型组(0.5 μmol/L DOX)相比有明显的下降(P<0.05),与模型组相比,各给药组BaX、Cleaved-caspase3蛋白水平明显降低,Bcl-2蛋白水平上升,证明人参总皂苷能抑制DOX损伤所带来的凋亡,对CCC-HSF-1细胞有一定的修复作用。结论人参总皂苷能有效抑制皮肤细胞凋亡,对紫外线照射引起的皮肤老化具有一定修复作用,为人参的进一步开发利用提供更充分的理论依据。     

A p‐Menth‐1‐ene‐4,7‐diol (EC‐1) from Eucalyptus camaldulensis Dhnh. Triggers Apoptosis and Cell Cycle Changes in Ehrlich Ascites Carcinoma Cells

Anticancer activities of p‐menth‐1‐ene‐4,7‐diol (EC‐1) isolated from Eucalyptus camaldulensis Dhnh. were studied on Ehrlich ascites carcinoma (EAC) cells by MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyl tetrazolium bromide) assay. Anticancer activities also analyzed in EAC‐bearing mice by assessment of cancer growth inhibition, changes in cancer volume, changes in life span, and hematological parameters. Apoptosis was analyzed by fluorescence microscope, DNA fragmentation assay, and flow cytometry. The expression of apoptosis‐related genes, Bcl‐2, Bcl‐X, PARP‐1, p53, and Bax, were analyzed using polymerase chain reaction (PCR). EC‐1 significantly inhibited proliferation of EAC cells in vivo and restored the altered hematological parameters of EAC‐bearing mice. Cytological observation by fluorescence microscope showed apoptosis of EAC cells upon treatment with EC‐1. Also, DNA fragmentation assay revealed EAC cells' apoptosis following EC‐1 treatment. Increased mRNA expressions of p53 and Bax genes and negative expressions of Bcl‐2 and Bcl‐X were observed in cells treated with EC‐1. These findings confirmed the induction of apoptosis by EC‐1. In addition, MTT assay showed dose‐dependent anticancer activity of EC‐1 against EAC cell. Cell cycle analysis revealed that EC‐1 treatment caused suppression of EAC cells at S phase. To conclude, EC‐1 is a novel anticancer compound and showed antiproliferative and apoptotic activities in cellular and mice models. Copyright © 2015 John Wiley & Sons, Ltd.     

Modulating Bcl-2 family proteins and caspase-3 in induction of apoptosis by paeoniflorin in human cervical cancer cells

Paeoniflorin (PF), the principal bioactive component in the paeony root, has been used alone or combined with other herbs for many years in traditional Chinese medicine. New studies have shown that PF possesses an antitumor effect. However, the effect of PF on human cervical cancer cells has not been reported previously. This study determined the effect of PF on human cervical cancer cell line (HeLa) cells by the methyl thiazolyl tetrazolium (MTT) assay, flow cytometry with annexin V‐fluorescein isothiocyanate (FITC)/propidium iodide (PI) technology, the transmission electron microscope (TEM) and immunocytochemical technique. After treatment with PF, the proliferation of HeLa cells was inhibited in a dose and time‐dependent manner (p < 0.05). The apoptosis rate of HeLa cells increased with ascending concentrations of PF (p < 0.05) and the proportion of HeLa cells in S phase showed an increasing trend also. Typical apoptotic changes of HeLa cells exposed to PF were seen under the TEM. Meanwhile, there was a decrease in the expression of Bcl‐2 and an enhancement in the expression of Bax and caspase‐3 genes compared with the control group (p < 0.05). In conclusion, PF can induce significantly the apoptosis of HeLa cells, which may be demonstrated by the down‐regulation of anti‐apoptosis gene Bcl‐2 and the up‐regulation of pro‐apoptosis genes Bax and caspase‐3. Copyright © 2011 John Wiley & Sons, Ltd.     

Effects of ginseng rhizome and ginsenoside Ro on testosterone 5α-reductase and hair re-growth in testosterone-treated mice

This research program on the novel functions of Panax ginseng C. A. Meyer focused on the effects of ginseng rhizome on hair re‐growth in androgenetic alopecia. Extracts of red ginseng rhizome showed greater dose‐dependent inhibitory effects against testosterone 5α‐reductase (5αR) when compared with extracts of the main root. Ginsenoside Ro, the predominant ginsenoside in the rhizome, and ginsenoside Rg₃, a unique ginsenoside in red ginseng, showed inhibitory activity against 5αR with IC₅₀ values of 259.4 and 86.1 µm , respectively. The rhizome of P. japonicus, which contains larger amounts of ginsenoside Ro, also inhibited 5αR. Topical administration of extracts of red ginseng rhizomes (2 mg/mouse) and ginsenoside Ro (0.2 mg/mouse) to shaved skin inhibited hair re‐growth suppression after shaving in the testosterone‐treated C57BL/6 mice. These results suggest that red ginseng rhizomes containing both oleanane‐ and dammarane‐type ginsenosides are a promising raw material for cosmetic use. This is the first report that ginsenoside Ro enhances in vivo hair re‐growth based on their inhibitory activity against 5αR in the androgenetic alopecia model. Copyright © 2011 John Wiley & Sons, Ltd.     

Genistein inhibits Aβ25–35‐induced SH‐SY5Y cell damage by modulating the expression of apoptosis‐related proteins and Ca2+ influx through ionotropic glutamate receptors

In this study, we investigated the protective effects of genistein against SH‐SY5Y cell damage induced by β‐amyloid 25–35 peptide (Aβ_25–35) and the underlying mechanisms. Aβ‐induced neuronal death, apoptosis, glutamate receptor subunit expression, Ca²⁺ ion concentration, amino acid transmitter concentration, and apoptosis‐related factor expression were evaluated to determine the effects of genistein on Aβ‐induced neuronal death and apoptosis. The results showed that genistein increased the survival of SH‐SY5Y cells and decreased the level of apoptosis induced by Aβ_25–35. In addition, genistein reversed the Aβ_25–35‐induced changes in amino acid transmitters, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptors, and N‐methyl‐d ‐aspartate (NMDA) receptor subunits in SH‐SY5Y cells. Aβ_25–35‐induced changes in Ca²⁺ and B‐cell lymphoma‐2 (Bcl‐2) and Bcl‐2‐associated X (Bax) protein and gene levels in cells were also reversed by genistein. Our data suggest that genistein protects against Aβ_25–35‐induced damage in SH‐SY5Y cells, possibly by regulating the expression of apoptosis‐related proteins and Ca²⁺ influx through ionotropic glutamate receptors.     

Emodin Regulates Apoptotic Pathway in Human Liver Cancer Cells

Emodin, a natural anthraquinone, has been reported to possess antiproliferative effects in many cancer cell lines. However, anticancer mechanism against human liver cancer remains unclear. In this study, we observed that emodin induced apoptosis in HepG2 cells and caused a significant accumulation of cells in the G1 phase. Western blot data showed that emodin treatment caused the increasing of release of cytochrome c into cytosol from mitochondria and the activation of caspase‐8 and caspase‐9, which suggest that the intrinsic and extrinsic pathways could be involved. Emodin treatment also resulted in a dose‐dependent accumulation of intracellular reactive oxygen species. Furthermore, emodin increased the protein level of p53 and decreased the protein level of NF‐κB/p65 in HepG2 cells, which indicated these two regulators might play a role in emodin‐induced apoptosis. Computational modeling showed that emodin could directly bind to the BH3 domain of Bcl‐2 through forming one hydrogen bond with Ala146 residue in Bcl‐2. From these examinations, emodin not only significantly downregulated expression of Bcl‐2 but also inhibited the heterodimerization of Bcl‐2 with Bax because of strong interaction between emodin and Bcl‐2. These suggest that emodin induces apoptosis in liver cancer cell line through a multifaceted complex cascade of events. Copyright © 2012 John Wiley & Sons, Ltd.     

人参抗肿瘤作用的有效成分及其机制研究进展

人参是中国延用了两千多年珍贵的传统中药材之一,由于其具有诸多药理作用而临床广泛应用于治疗肿瘤等多种疾病。目前肿瘤已经成为威胁人类健康的重要因素,因而人参抗肿瘤作用也越加受到关注。针对人参抗肿瘤作用的有效成分及其分子作用机制、构效关系进行综述。研究表明人参抗肿瘤作用的主要有效成分为人参皂苷及其肠道菌群代谢产物、人参多糖和人参炔醇,这些活性成分发挥药理作用的机制目前已较为明确,其作用机制主要包括诱导肿瘤细胞周期阻滞、凋亡及分化、增强对肿瘤细胞免疫、抑制肿瘤细胞增殖及侵袭与转移等,而其分子机制涉及许多相关基因、蛋白、蛋白酶、免疫细胞、细胞因子及相关信号通路等的调控与表达。此外,人参有效成分的抗肿瘤作用表现出一定的剂量依赖性,且其化学结构的不同导致抗肿瘤活性有所差异。人参中含有丰富的抗肿瘤活性成分,有望为临床治疗各种肿瘤提供安全有效的天然药物及制剂。     

Protective Effects of Gastrodin Against Autophagy‐Mediated Astrocyte Death

Gastrodin is an active ingredient derived from the rhizome of Gastrodia elata. This compound is usually used to treat convulsive illness, dizziness, vertigo, and headache. This study aimed to investigate the effect of gastrodin on the autophagy of glial cells exposed to lipopolysaccharides (LPS, 1 µg/mL). Autophagy is a form of programmed cell death, although it also promotes cell survival. In cultured astrocytes, LPS exposure induced excessive autophagy and apoptosis, which were significantly prevented by the pretreatment cells with gastrodin (10 μM). The protective effects of gastrodin via autophagy inhibition were verified by the decreased levels of LC3‐II, P62, and Beclin‐1, which are classical markers for autophagy. Furthermore, gastrodin protected astrocytes from apoptosis through Bcl‐2 and Bax signaling pathway. The treatment of astrocytes with rapamycin (500 nM), wortmannin (100 nM), and LY294002 (10 μM), which are inhibitors of mTOR and PI3K, respectively, eliminated the known effects of gastrodin on the inhibited Beclin‐1 expression. Furthermore, gastrodin blocked the down‐regulation of glutamine synthetase induced by LPS exposure in astrocytes. Our results suggest that gastrodin can be used as a preventive agent for the excessive autophagy induced by LPS. Copyright © 2015 John Wiley & Sons, Ltd.     

Ferulate Protects the Epithelial Barrier by Maintaining Tight Junction Protein Expression and Preventing Apoptosis in Tert‐Butyl Hydroperoxide‐Induced Caco‐2 Cells

Epithelial barrier function is determined by both transcellular and paracellular permeability, the latter of which is mainly influenced by tight junctions (TJs) and apoptotic leaks within the epithelium. We investigated the protective effects of ferulate on epithelial barrier integrity by examining permeability, TJ protein expression, and apoptosis in Caco‐2 cells treated with tert‐butyl hydroperoxide (t‐BHP), a strong reactive species inducer. Caco‐2 cells pretreated with ferulate (5 or 15 μM) were exposed to t‐BHP (100 μM), and ferulate suppressed the t‐BHP‐mediated increases in reactive species and epithelial permeability in Caco‐2 cells. Moreover, ferulate inhibited epithelial cell leakage induced by t‐BHP, which was accompanied by decreased expression of the TJ proteins zonula occludens‐1 and occludin. In addition, pretreatment with ferulate markedly protected cells against t‐BHP‐induced apoptosis, as evidenced by decreased nuclear condensation, cytochrome c release, and caspase‐3 cleavage and an increased Bax/Bcl‐2 ratio. These results suggest that ferulate protects the epithelial barrier of Caco‐2 cells against oxidative stress, which results in increased epithelial permeability, decreased TJ protein expression, and increased apoptosis. The most significant finding of our study is the demonstration of protective, ferulate‐mediated antioxidant effects on barrier integrity, with a particular focus on intracellular molecular mechanisms. Copyright © 2012 John Wiley & Sons, Ltd.     

Mixtures of Uncaria and Tabebuia extracts are potentially chemopreventive in CBA/Ca mice: a long‐term experiment

A long‐term experimental animal model was developed by our research group for the evaluation of potential chemopreventive effects. The inhibitory effects of agents on carcinogen (7,12‐dimethylbenz[a]anthracene (DMBA) induced molecular epidemiological biomarkers, in this case the expression of key onco/suppressor genes were investigated. The expression pattern of c‐myc, Ha‐ras, Bcl‐2, K‐ras protooncogene and p53 tumour suppressor gene were studied to elucidate early carcinogenic and potential chemopreventive effects. The consumption of so‐called Claw of Dragon tea (CoD? tea) containing the bark of Uncaria guianensis, Cat's Claw (Uncaria sp. U. tomentosa) and Palmer trumpet‐tree (Tabebuia sp. T. avellanedae) was able to decrease the DMBA‐induced onco/suppressor gene overexpression in a short‐term animal experiment. In a following study CBA/Ca mice were treated with 20 mg/kg bw DMBA intraperitoneally (i.p.) and the expression patterns of onco/suppressor genes were examined at several time intervals. According to the examined gene expression patterns in this long‐term experiment the chemopreventive effect of CoD? tea consumption could be confirmed. Copyright © 2010 John Wiley & Sons, Ltd.     

不同基质条件下西洋参皂苷提取物的自毒作用

人参皂苷是西洋参Panax quinquefolium中所含的主要次生代谢产物,西洋参生长过程中,植株内的人参皂苷可以通过根系分泌及腐解进入到土壤环境中.研究利用高效液相色谱法(HPLC)测定了土壤中人参皂苷含量,根据西洋参田间的实测数据设计皂苷提取物的系列浓度(0.2 ～125 mg· L-1溶液或0.2 ～ 125 mg·kg-1土壤),通过在溶液及土壤基质中的添加实验,观察环境中的皂苷提取物对西洋参种胚和成株苗生长的作用.结果表明,在北京市怀柔区三至四年生西洋参根际土中,检测到人参皂苷Rb1,Rb2,Rd,总量为0.80 ～ 3.19 mg·kg-,按照田间持水量为20％计算,总皂苷在土壤溶液中的质量浓度为4～ 16 mg·L-1.在溶液基质中,0.2～125 mg·L-1西洋参皂苷提取物对西洋参种胚胚根的抑制作用在6％ ～23％,在125mg·L-1时达到显著水平(P＜0.05);对西洋参胚芽无显著抑制作用.营养液中添加25 mg·L-1皂苷提取物,培养20 d时,三年生西洋参成株苗株高下降28％,地上部的生物量下降50％(P＜0.05);对新生须根生长的抑制作用不显著.在土壤基质中,添加同样浓度的皂苷提取物在灭菌和未灭菌土中对西洋参胚根、胚芽生长均无显著影响.由此推论,西洋参的皂苷成分对自身胚根和成株苗生长有自毒作用,但在田间土壤条件下,这种作用有所下降.     

Flavonoids of Korean Citrus aurantium L. Induce Apoptosis via Intrinsic Pathway in Human Hepatoblastoma HepG2 Cells

Korean Citrus aurantium L. has long been used as a medicinal herb for its anti‐inflammatory, antioxidant, and anticancer properties. The present study investigates the anticancer role of flavonoids extracted from C. aurantium on human hepatoblastoma cell, HepG2. The Citrus flavonoids inhibit the proliferation of HepG2 cells in a dose‐dependent manner. This result was consistent with the in vivo xenograft results. Apoptosis was detected by cell morphology, cell cycle analysis, and immunoblot. Flavonoids decreased the level of pAkt and other downstream targets of phosphoinositide‐3‐kinase/Akt pathway – P‐4EBP1 and P‐p70S6K. The expressions of cleaved caspase 3, Bax, and Bak were increased, while those of Bcl‐2 and Bcl‐xL were decreased with an increase in the expression of Bax/Bcl‐xL ratio in treated cells. Loss of mitochondrial membrane potential was also observed in flavonoid‐treated HepG2 cells. It was also observed that the P‐p38 protein level was increased both dose and time dependently in flavonoid‐treated cells. Collectively, these results suggest that flavonoid extracted from Citrus inhibits HepG2 cell proliferation by inducing apoptosis via an intrinsic pathway. These findings suggest that flavonoids extracted from C. aurantium L. are potential chemotherapeutic agents against liver cancer. Copyright © 2015 John Wiley & Sons, Ltd.     

Ginsenosides Rb1 and Rd Regulate Proliferation of Mature Keratinocytes Through Induction of p63 Expression in Hair Follicles

Ginsenosides Rb₁ and Rd are the two main types of ginsenosides in Panax ginseng and have been used as an additive to against alopecia. However, the mechanisms involved are largely unknown. To determine how ginsenosides prevent hair loss, we topically applied protopanaxadiol‐type ginsenosides Rb₁ and Rd over the shaved skin of B57CL/6 mice, and monitored and assessed them for 35 days. We then investigated the effects of ginsenosides on cell genesis in different phases of adult hair follicles (HFs), using 5‐bromo‐2′‐deoxyuridine as a marker for dividing cells. Moreover, p63, a specific marker and a major regulator of keratinocyte progenitor cells of the multi‐layered epithelia, was detected in epidermis. Results indicated that treatment with ginsenosides Rb₁ and Rd increased cell proliferation in both anagen and telogen of HFs. However, it had no significant effect on the survival of cells in the bulge and upper follicle region. Investigation of p63 demonstrated that up‐regulation of p63 expression in the matrix and outer root sheath might be one of the mechanisms by which ginsenosides Rb₁ and Rd promote cell proliferation in HFs. Our study reveals a novel mechanism by which ginsenoside promotes hair growth through p63 induction in follicular keratinocytes and indicates that ginsenosides Rb₁ and Rd might be developed as a therapeutic agent for the prevention of hair loss. Copyright © 2012 John Wiley & Sons, Ltd.     

Oral administration of Aloe vera and honey reduces walker tumour growth by decreasing cell proliferation and increasing apoptosis in tumour tissue

Cancer is diagnosed in approximately 11 million people and is responsible for almost 8 million deaths worldwide every year. Research in cancer control has shown the importance of co‐adjuvant therapies. Aloe vera may reduce tumour mass and metastasis rates, while honey may inhibit tumour growth. This study verified the influence of Aloe vera and honey on tumour growth and in the apoptosis process by assessing tumour size, the cell proliferation rate (Ki67‐LI) and Bax/Bcl‐2 expression at 7, 14 and 20 days after Walker 256 carcinoma implant in Wistar rats distributed into two groups: the WA group – tumour‐bearing rats that received a gavage with a 670 µL/kg dose of Aloe vera and honey solution daily, and the CW group – tumour‐bearing rats which received only a 0.9% NaCl solution. The effect of Aloe vera and honey against tumour growth was observed through a decrease in relative weight (%) and Ki67‐LI in tumours from the WA group compared with those from the CW group. The Bax/Bcl‐2 ratio increased in tumours from the WA group at all tested timepoints. These data suggest Aloe vera and honey can modulate tumour growth by reducing cell proliferation and increasing apoptosis susceptibility. Copyright © 2010 John Wiley & Sons, Ltd.     

Antitumor activity of water extract of a mushroom,Inonotus obliquus,against HT‐29 human colon cancer cells

In the current study, it was demonstrated that the hot water extract of I. obliquus (IOWE) exerts inhibitory activity against the proliferation of human colon cancer cells (HT‐29). The inhibitory effect of IOWE on the growth of HT‐29 cancer cells was evaluated by treating cells with IOWE at concentrations of 0.25, 0.5 and 1.0 mg/mL for 24 or 48 h. The IOWE inhibited cell growth in a dose‐dependent manner, and this inhibition was accompanied by apoptotic cell death. The maximum inhibitory effect (56%) was observed when IOWE was treated at a concentration of 1.0 mg/mL for 48 h. The apoptotic effect of IOWE on HT‐29 cells was also confirmed by flow cytometric analysis. In addition, the apoptotic cell percentage was closely associated with down‐regulation of Bcl‐2 and up‐regulation of Bax and caspase‐3. The results suggest that IOWE would be useful as an antitumor agent via the induction of apoptosis and inhibition of the growth of cancer cells through up‐regulation of the expression of proapoptotic proteins and down‐regulation of antiapoptotic proteins. Copyright © 2009 John Wiley & Sons, Ltd.