Red mold rice extract represses amyloid beta peptide-induced neurotoxicity via potent synergism of anti-inflammatory and antioxidative effect

Amyloid beta-peptide (Abeta), a risk of Alzheimer's disease (AD), causes cell death by inflammation and oxidative stress. Red mold rice (RMR) fermented by Monascus species is regarded as cholesterol-lowering functional food in virtue of the metabolite monacolin K identified as lovastatin. In addition, RMR is also demonstrated to express antioxidation because of multiple antioxidants. Therefore, this study focuses on the synergism of RMR against Abeta neurotoxicity and compares the effect between lovastatin and RMR including monacolin K and other functional metabolites. In this study, RE 568, an ethanol extract of RMR produced by strain Monascus purpureus NTU 568, is used to protect PC12 cell against Abeta40 neurotoxicity. All tests contain the treatments with lovastatin or RE 568 including equal monacolin K levels in order to compare the effect and investigate whether other metabolites of RE 568 provide potent assistance against Abeta40 neurotoxicity. In the results, monacolin K represses Abeta40 neurotoxicity via repressing small G-protein-mediated inflammation, and other metabolites of RE 568 also exhibit potent antioxidative ability against Abeta-induced oxidative stress. Importantly, stronger effects on repressing the Abeta40-induced cell death, inflammation, and oxidative stress are performed by RE 568 than that by the equal levels of lovastatin, which results from a potent synergism made up of monacolin K, antioxidants, and anti-inflammatory agents. The present study is the first report to demonstrate the potent synergistic protection of RMR against Abeta40 neurotoxicity, which would cause RMR to be developed as potential and novel functional food for the prophylaxis of AD pathogenesis.     

Apolipoprotein E4 potentiates amyloid beta peptide-induced lysosomal leakage and apoptosis in neuronal cells

We assessed the isoform-specific effects of apolipoprotein (apo) E on the response of Neuro-2a cells to the amyloid beta peptide (Abeta1-42). As determined by the intracellular staining pattern and the release of beta-hexosaminidase into the cytosol, apoE4-transfected cells treated with aggregated Abeta1-42 showed a greater tendency toward lysosomal leakage than neo- or apoE3-transfected cells. Abeta1-42 caused significantly greater cell death and more than 2-fold greater DNA fragmentation in apoE4-secreting than in apoE3-secreting or control cells. H2O2 or staurosporine enhanced cell death and apoptosis in apoE4-transfected cells but not in apoE3-transfected cells. A caspase-9 inhibitor abolished the potentiation of Abeta1-42-induced apoptosis by apoE4. Similar results were obtained with conditioned medium from cells secreting apoE3 or apoE4. Cells preincubated for 4 h with a source of apoE3 or apoE4, followed by removal of apoE from the medium and from the cell surface, still exhibited the isoform-specific response to Abeta1-42, indicating that the potentiation of apoptosis required intracellular apoE, presumably in the endosomes or lysosomes. Studies of phospholipid (dimyristoylphosphatidylcholine) bilayer vesicles encapsulating 5-(and-6)-carboxyfluorescein dye showed that apoE4 remodeled and disrupted the phospholipid vesicles to a greater extent than apoE3 or apoE2. In response to Abeta1-42, vesicles containing apoE4 were disrupted to a greater extent than those containing apoE3. These findings are consistent with apoE4 forming a reactive molecular intermediate that avidly binds phospholipid and may insert into the lysosomal membrane, destabilizing it and causing lysosomal leakage and apoptosis in response to Abeta1-42.     

Mitochondrial mechanisms in amyloid beta peptide-induced cerebrovascular degeneration

Prevailing evidence suggests that amyloid beta peptide (Aβ), a key mediator in age-dependent neuronal and cerebrovascular degeneration, activates death signaling processes leading to neuronal as well as non-neuronal cell death in the central nervous system. A major cellular event in Aβ-induced death of non-neuronal cells, including cerebral endothelial cells, astrocytes and oligodendrocytes, is mitochondrial dysfunction. The death signaling cascade upstream of mitochondria entails Aβ activation of neutral sphingomyelinase, resulting in the release of ceramide from membrane sphingomyelin. Ceramide then activates protein phosphatase 2A (PP2A), a member in the ceramide-activated protein phosphatase (CAPP) family. PP2A dephosphorylation of Akt and FKHRL1 plays a pivotal role in Aβ-induced Bad translocation to mitochondria and transactivation of Bim. Bad and Bim are pro-apoptotic proteins that cause mitochondrial dysfunction characterized by excessive ROS formation, mitochnondrial DNA (mtDNA) damage, and release of mitochondrial apoptotic proteins including cytochrome c, apoptosis inducing factor (AIF), endonuclease G and Smac. The cellular events activated by Aβ to induce death of non-neuronal cells are complex. Understanding these death signaling processes will aid in the development of more effective strategies to slow down age-dependent cerebrovascular degeneration caused by progressive cerebrovascular Aβ deposition.     

Overexpression of receptor of advanced glycation end products hypersensitizes cells for amyloid beta peptide-induced cell death

Receptor of advanced glycation end products (RAGE) was identified as one of the receptors for amyloid beta peptide (Abeta). There is evidence for controversial functions of RAGE such as a mediator of cell death or differentiation. In this report, we demonstrate that RAGE mediates Abeta toxicity. Transient transfection of RAGE already induced cell death. For further analysis, stable clones of hemagglutinin (HA)-tagged RAGE were selected. Analysis of cellular localization of HA-tagged RAGE protein revealed, in addition to the expected cell surface expression, a novel intracellular localization. Stable RAGE-expressing cells were hypersensitive to nanomolar amounts of Abeta. Only cells expressing RAGE at the cell surface showed hypersensitivity to Abeta.     

栀子道地性的分子生态学

运用扩增片段长度多态性(AFLP)方法,研究了来自江西5个不同产地栀子的亲缘关系,并运用HPLC方法测定了栀子苷的含量,用ICP法测定了植物药材中的矿质元素含量.结果表明,各地方品系间在DNA 水平上存在明显的多样性变异;运用NTSYS-PC 软件对53个个体聚类分析,地理位置相近的种群聚为一类.各样本间栀子苷含量高低与聚类分支间无明显相关性,说明道地性的产生是基因型和环境饰变共同作用的结果.经微量元素测定,锌与栀子苷含量间呈负相关关系.     

药用植物栀子的组织培养

栀子(Gardenia jasminoides)为药用木本植物。以栀子果皮、种子团和种子为外植体, 研究不同激素配比及不同培养方式对愈伤组织诱导和芽分化的影响。研究结果表明, 培养基成分为MS+0.5 mg·L^–12,4-D+0.25 mg·L^–16-BA较适宜果皮和种子愈伤组织的诱导, 诱导率分别为83.3%和88.5%; 培养基成分为MS+1.0 mg·L^–12,4-D+1.0 mg·L^–16-BA较适宜种子团愈伤组织的诱导, 诱导率为78.1%。3种外植体诱导的愈伤组织中, 只有种子愈伤组织能通过液体培养分化出芽; TDZ对芽分化有明显的促进作用; 最佳的芽分化培养基为MS+0.05 mg·L^–1NAA+0.10 mg·L^–1TDZ, 其愈伤组织分化率为8.75%。该研究以栀子种子为外植体, 并获得了再生植株, 为药用植物栀子转基因体系的建立奠定了基础。     

药用植物栀子的组织培养

栀子(Gardenia jasminoides)为药用木本植物。以栀子果皮、种子团和种子为外植体,研究不同激素配比及不同培养方式对愈伤组织诱导和芽分化的影响。研究结果表明,培养基成分为MS+0.5 mg·L–12,4-D+0.25 mg·L–16-BA较适宜果皮和种子愈伤组织的诱导,诱导率分别为83.3%和88.5%;培养基成分为MS+1.0 mg·L–12,4-D+1.0 mg·L–16-BA较适宜种子团愈伤组织的诱导,诱导率为78.1%。3种外植体诱导的愈伤组织中,只有种子愈伤组织能通过液体培养分化出芽;TDZ对芽分化有明显的促进作用;最佳的芽分化培养基为MS+0.05 mg·L–1NAA+0.10 mg·L–1TDZ,其愈伤组织分化率为8.75%。该研究以栀子种子为外植体,并获得了再生植株,为药用植物栀子转基因体系的建立奠定了基础。     

Germination and Growth Inhibitors in Fruits of Gardenia jasminoides

Plant germination- and growth-inhibiting activities of the iridoidglycosides (geniposide and genipin gentiobioside) containedin fruits of Gardenia jasminoides Ellis were studied. They inhibitedthe germination of G. jasminoides seeds and watercress seedsand the growth of Chinese cabbage roots. In addition, genipin,which is the aglycone of the glycosides, showed a strong inhibitingeffect on the growth of Chinese cabbage roots. (Received September 10, 1982; Accepted November 6, 1982)     

Gardenia jasminoides J.Ellis extract GJ-4 alleviated cognitive deficits of APP/PS1 transgenic mice

BackgroundAccumulating evidence demonstrates that traditional Chinese medicines that act on multiple targets could effectively treat various multi-etiological diseases, including cerebrovascular diseases, Alzheimer's disease (AD), Parkinson's disease (PD) and so on. Previous studies have shown that crocin richments (GJ-4), Gardenia jasminoides J.Ellis extract, provide neuroprotective effects on cognitive impairments in AD mouse models. However, the mechanism how GJ-4 improves cognition remains still unclear.PurposeThe aim of this study was to uncover the protective effects and underlying mechanism of GJ-4 on PrP-hAβPPswe/PS1^ΔE9 (APP/PS1) transgenic mice.MethodsAPP/PS1 mice were given GJ-4 (10, 20, and 50 mg/kg), donepezil (5 mg/kg) and memantine (5 mg/kg) orally at eight months of age for 12 consecutive weeks. Morris water maze and novel object recognition were conducted to assess the cognitive ability of mice. The release of inflammatory cytokines was determined by RT-PCR assay, and the pathological features of neurons and microglia were assayed by immunohistochemistry and immunofluorescence assay. The expression of Aβ-related proteins and signaling pathways were determined by Western blot.ResultsThe behavioral results revealed that GJ-4 ameliorated the cognitive deficits of APP/PS1 mice measured by Morris water maze and novel object recognition tests. Mechanism studies indicated that GJ-4 significantly decreased β-amyloid (Aβ) level through reducing Aβ production and promoting Aβ degradation. It has been reported that Aβ plaques trigger the hyper-phosphorylation of tau protein in APP/PS1 mice. Consistent with previous studies, hyper-phosphorylation of tau was also occurred in APP/PS1 mice in the present study, and GJ-4 inhibited Tau phosphorylation at different sites. Overwhelming evidence indicates that neuroinflammation stimulated by Aβ and hyperphosphorylated tau is involved in the pathological progression of AD. We found that GJ-4 suppressed neuroinflammatory responses in the brain through regulating phosphatidylinositide 3-kinase/AKT (PI3K/AKT) signaling pathway activation, and subsequent expression of inflammatory proteins and release of inflammatory cytokines.ConclusionAltogether, GJ-4 ameliorated cognition of APP/PS1 transgenic mice through multiple targets, including Aβ, tau and neuroinflammation. This study provides a solid research basis for further development of GJ-4 as a potential candidate for the treatment of AD.     

Intermediacy of 8-epiiridodial in the biosynthesis of iridoid glucosides by Gardenia jasminoides cell cultures

By administration of ²H-labelled iridodial, its congeners and ¹³C-labelled 10-hydroxygeraniol to Gardenia jasminoides cell suspension cultures it was demonstrated that tarennoside and gardenoside were biosynthesized, after iridodial cation formation, via 8-epiiridodial, 8-epiiridotrial, 8-epiiridotrial glucoside and 7,8-dehydroiridotrial glucoside. However, the coexistence of a pathway via the iridodial cation, 7,8-dehydroiridodial, 7,8-dehydroiridotrial and 7,8-dehydroiridotrial glucoside could not be excluded.     

栀子对自然降温的适应性研究

以多年生栀子为材料,对其在自然降温过程中的适应性进行了研究。结果表明：随温度的降低,低温半致死温度不断降低;渗透调节物可溶性糖、可溶性蛋白含量增加,淀粉含量下降;内源激素ABA含量上升,与低温半致死温度呈负相关（相关系数为 －0.967 3）,GA含量下降与低温半致死温度呈正相关（相关系数为0.812 5）。综上所述在自然降温过程中,这些物质的适应性变化导致低温半致死温度的下降,从而使栀子的抗寒性增强,能很好的适应栽培地的低温条件而安全越冬。     

Glucosylation of Salicyl Alcohol by Gardenia jasminoides Cell Cultures

Cultured cells of Gardenia jasminoides produced both salicinand isosalicin from exogenously supplied salicyl alcohol. Theglucosylation activity of the cells was highest in the exponentialphase of growth and ca. 70% of the added substrate was convertedto the glucosides within 4 days. The rate of glucosylation wasalso dependent on the medium composition such as auxin and sucroseconcentrations. The ratio of salicin to isosalicin formed fromsalicyl alcohol was influenced by the growth stage of the culturedcells. Salicin was converted to isosalicin when exogenouslyadded to the culture. (Received October 11, 1985; Accepted March 10, 1986)     

Clonal multiplication of Gardenia jasminoides Ellis through axillary bud culture

An efficient clonal multiplication system was developed for in vitro propagation of crocin — producing Gardenia jasminoides Ellis plants. Murashige and Skoog's (MS) medium containing 6-benzylaminopurine (BAP 1 mg l^–1) and indole-3-butyric acid (IBA 1 mg l^–1) resulted in multiple shoot initiation at the rate of 21 shoots per explant in 60 d of culture. Transfer of the microshoots into liquid MS medium supplemented with BAP (5 mg l^–1) with two subcultures of 15 d duration in the same medium resulted in 400 ± 25 shoots per explant. Efficient rooting was achieved in MS medium supplemented with -naphthaleneacetic acid (5 mg l^–1). The in vitro raised plants were hardened in a greenhouse and transplanted to the field successfully. The method described will be useful for rapid multiplication of Gardenia for commercial exploitation.Abbreviations MS Murashige and Skoog (1962) medium - BAP 6-benzylaminopurine - Kn kinetin - 2ip 6-(,-dimethylallylamino)purine - NAA -naphthalene- acetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid     

Monoterpenoids from the fruit of Gardenia jasminoides Ellis (Rubiaceae)

The present phytochemical investigations of Gardenia jasminoides Ellis resulted in the isolation of ten iridoids (1–10) and ten pyronane monoterpenoids (11–20). Among them, compounds 11 and 18 were obtained from this species for the first time. The chemotaxonomic importance of these compounds was also summarized.     

大孔吸附树脂对栀子中环烯醚萜苷类成分的富集行为

采用大孔吸附树脂对栀子中环烯醚萜苷类成分的富集行为进行研究,结果表明,D101大孔吸附树脂柱层析适合分离纯化栀子中环烯醚萜苷类成分,采用80%乙醇洗脱,紫外-可见分光光度法进行定量测定。使用该方法富集后,80%乙醇洗脱液干燥后总固体物中环烯醚萜苷类成分含量可达到70%以上。     

Development of photoautotrophy and photoinhibition of Gardenia jasminoides plantlets during micropropagation

This paper reports on the fast fluorescence responses of Gardenia jasminoides Ellis plantlets, at two successive stages (shoot multiplication and root induction) of culture in vitro. We test whether plantlets in vitro suffer photoinhibition during culture and whether the degree of photoautotrophy of these mixotrophic plantlets has any effect on the extent of photoinhibitory impairment. In this regard the effects of different sucrose levels in the medium and PPFD during growth on the development of photoautotrophy and the extent of photoinhibition were evaluated. Plantlets were grown under low, intermediate, and high (50, 100, and 300 mol m^-2 s^-1) PPFD, and at 3 different sucrose concentrations (0.5, 1.5, and 3.0%, w/v) in the medium, during shoot multiplication. During root induction the same growth conditions were assayed except for the high PPFD. The development of photoautotrophy was assessed via the difference between the stable carbon isotope composition of sucrose used as heterotrophic carbon source and that of leaflets grown in vitro. Plantlets from root induction showed more developed photoautotrophy than those from shoot multiplication. For both stages the low-sucrose medium stimulated the photoautotrophy of plantlets in vitro. In addition, intermediate PPFD induced photoautotrophy during shoot multiplication. For plantlets of both culture stages at the lowest PPFD no photoinhibition occurred irrespective of the sucrose concentration in media. However, during the shoot multiplication stage chlorophyll fluorescence measurements showed a decrease in F _v /F _m and in t _1/2 as growing PPFD increased, indicating photoinhibitory damage. The decline of F _v /F _m was caused mostly by an increase in F _o , indicating the inactivation of PSII reaction centers. However plantlets growing under low sucrose showed reduced susceptibility to photoinhibition. During root induction, only plantlets cultured with high sucrose showed a decrease in F _v /F _m as PPFD increased, although t _1/2 remained unchanged. In this case, the decline of F _v /F _m was mostly due to a decrease in F _m , which indicates increased photoprotection rather than occurrence of photodamage. Therefore, growth in low-sucrose media had a protective effect on the resistance of PSII to light stress. In addition, plantlets were more resistant to photoinhibition during root induction than during shoot multiplication. Results suggest that increased photoautotrophy of plantlets reduces susceptibility to photoinhibition during gardenia culture in vitro.Abbreviations AP apparent photosynthesis - Chl total chlorophyll content - Chl a/b chlorophyll a-to-b ratio - Chl/Car total chlorophyll-to-carotenoids ratio - ¹³C ratio of ¹³C/¹²C relative to PeeDee belemnite standard - F _m maximum chlorophyll fluorescence - F _o fluorescence emission when all reaction centres are open and the photochemical quenching is minimal - F _v variable chlorophyll fluorescence (F _m -F _o ) - F _v /F _m the ratio of variable to maximum chlorophyll fluorescence, indicator photochemical efficiency of PSII - MS medium Murashige and Skoog (1962) medium - PPFD photosynthetic photon flux density - Rd dark respiration, t _1/2 the half-time of the increase from F _o to F _m - IAA indole butyric acid     

Methanol extract of Ficus platyphylla ameliorates seizure severity,cognitive deficit and neuronal cell loss in pentylenetetrazole-kindled mice

Decoctions of Ficus plathyphylla are used in Nigeria's folk medicine to manage epilepsy for many years and their efficacies are widely acclaimed among the rural communities of Northern Nigeria. In this study, we examined the ameliorative effects of the standardized methanol extract of Ficus platyphylla (FP) stem bark on seizure severity, cognitive deficit and neuronal cell loss in pentylenetetrazole-kindled mice. The ³⁵S-GTPγS, glutamate and γ-aminobutyric acid receptors binding properties of the extract were also evaluated. Male CD-1 mice were kindled with an initial subeffective dose of pentylenetetrazole (PTZ, 37.5 mg/kg, i.p.) for a total of 13 convulsant injections and the treatment groups concurrently received FP (100 and 200 mg/kg). Control animals received the same number of saline injections. Twenty-four h after kindling completion the animals’ learning performance was tested in a two-way shuttle-box. The animals were challenged with another subeffective dose of PTZ (32.5 mg/kg, i.p.) on day 7 after kindling completion. Animals were sacrificed a day after the challenged experiment and the brains were processed for histological investigation. FP ameliorates seizure severity, cognitive deficits and neuronal cell loss in PTZ kindled mice. Components of the extract showed affinity for GABAergic and glutamatergic receptors. Glutamate release was diminished and the ³⁵S-GTPγS binding assay revealed no intrinsic activity at glutamatergic receptors. Our results revealed that FP contains psychoactive secondary metabolites with anticonvulsant properties, thus supporting the isolation and development of the biologically active components of this medicinal plant as antiepileptic agents.     

SMART技术构建栀子cDNA文库

目的:构建栀子叶片cDNA文库。方法:提取栀子叶片总RNA。利用SMART技术合成双链cDNA。双链cDNA经限制酶Sfil酶切后与pDNR-LIB质粒连接。利用电刺激转化法将重组质粒导入E.coli DH5α而获得文库。利用PCR法检测文库的重组率。结果:原始文库滴度为2.63×105cfu/ml。随机检测文库中的15个克隆,表明重组率约为86.7%。选择14个插入片段的长度在400bp以上的克隆进行测序和生物信息学分析,结果预测的全长基因占所检测序列的64.3%。结论:成功构建了栀子叶片的cDNA文库,为栀子基因的结构和功能的研究提供了基础。