水线草提取物的体外抗结肠癌作用及其机理初探

分别用水、石油醚、正丁醇和乙酸乙酯萃取水线草乙醇提取物得五个组分。MTT检测法、Wright染色法及TUNEL染色法初步研究各萃取部位对人结肠癌细胞株HCT-8、RKO的体外抑制作用。其中石油醚萃取部位及沉淀部位在浓度低至31．3μg／mL时对两种人结肠癌细胞株均有一定程度的杀伤作用。水线草中具有抗结肠癌活性的化学物质的分子结构及作用机理值得进一步深入研究。     

Establishment of in vitro plants, cell and tissue cultures from Oldenlandia affinis for the production of cyclic peptides

In vitro cultured plants from Oldenlandia affinis were established from seeds and grown on a hormone-free medium. In vitro plants produced the cyclic peptide kalata B1 in concentrations of 0.67 mg g⁻¹ dry weight after growth of 30 days. This was approximately 50% of the concentration analysed in green house plants (shoot tips), where different concentrations have been determined in leaves (1.82 mg g⁻¹), shoot tips (1.36 mg g⁻¹), stems (0.36 mg g⁻¹), and in flowers (0.16 mg g⁻¹). Callus and cell suspension cultures could be initiated from aseptic root, stem and leaf explants of O. affinis seedlings and plants. Different light intensities were shown to affect culture growth as well as chlorophyll synthesis. The friable callus was then used for the establishment of a cell suspension culture. Fresh and dry weight measurements showed that growth was optimal on MS medium supplemented with 0.4 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-d). Leaf suspensions cultured on this medium showed a 4-fold increase of biomass by the first week of incubation. No quantifiable amounts of kalata B1 were produced under these conditions. Morphological differentiation seems to be essential for cyclic peptide production. Therefore, several undifferentiated as well as organised cell lines of O. affinis have been developed. These cell lines will constitute a worthwhile starting point for the optimisation of kalata B1 synthesis in liquid media to the objective of producing cyclic peptides under controlled and defined conditions in bioreactors.     

Bioactive peptides from marine sources: pharmacological properties and isolation procedures

Marine organisms represent a valuable source of new compounds. The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new active substances in the field of the development of bioactive products. In this paper, the molecular diversity of different marine peptides is described as well as information about their biological properties and mechanisms of action is provided. Moreover, a short review about isolation procedures of selected bioactive marine peptides is offered. Novel peptides from sponges, ascidians, mollusks, sea anemones and seaweeds are presented in association with their pharmacological properties and obtainment methods.     

湘西慈竹叶中黄酮的提取与测定

设计正交试验,以溶剂浸提-回流法提取,以分光光度法测定,研究了湘西慈竹叶中总黄酮提取和测定的方法。结果表明:溶剂、溶剂浓度、水浴温度、回流时间4种因素对提取效果影响大小依次为,溶剂>溶剂浓度>回流时间>水浴温度,其中溶剂及其浓度是主要因素,回流时间和水浴温度是次要因素。不同溶剂对慈竹叶总黄酮提取效果影响大小顺序为丙酮>乙醇>乙醚。浸提-回流法提取湘西慈竹叶中黄酮类化合物的优化提取条件(固定料液比为1∶20)是:以75%的丙酮为浸提剂,于70℃下回流加热4 h。在优化提取条件下,测得湘西慈竹叶总黄酮含量为13.0 mg.g-1(春)~12.4 mg.g-1(秋),春季新叶中黄酮含量略高于秋季老叶。湘西慈竹叶总黄酮含量略低于四川南充慈竹叶。     

四川产中菊头蝠喜马拉雅亚种和马铁菊头蝠日本亚种外部形态及头骨的比较

对四川产中菊头蝠喜马拉雅亚种(Rhinolophus affinis himalayanus)和马铁菊头蝠日本亚种(R.ferrumequinum nippon)的23项外部形态指标和22项头骨形态指标进行了测量,并进行数理统计分析。结果表明,(1)中菊头蝠喜马拉雅亚种和马铁菊头蝠日本亚种的23项外部形态测量指标中有2项差异显著(P<0.05),16项差异极显著(P<0.01),其中:中菊头蝠喜马拉雅亚种(n=14)体长小于59 mm(47.30~58.40),尾长小于26 mm(21.69~25.68),耳长小于22 mm(18.36~21.88),前臂长小于56 mm(50.9~54.20),第Ⅲ掌骨第一指节长小于16 mm(13.2~15.53),第Ⅲ掌骨第二指节小于32 mm(27.38~31.74);而马铁菊头蝠日本亚种(n=6)体长大于62 mm(62.43~66.76),尾长大于30 mm(30.56~36.18),耳长大于24 mm(24.47~27.20),前臂长大于56 mm(58.78~63.46),第Ⅲ掌骨第一指节大于19 mm(19.38~21.39),第Ⅲ掌骨第二指节大于34 mm(34.42~38.11),这些差异可作为区分四川产这两个种的依据。(2)中菊头蝠喜马拉雅亚种和马铁菊头蝠日本亚种的22项头骨形态测量指标中,除颅高、听泡间距差异不显著(P>0.05)外,其他20项指标都存在极显著差异(P<0.01)。     

Administered peptides inhibit the degradation of endogenous peptides. The dilemma of distinguishing direct from indirect effects

Virtually all peptides are biologically active following central administration as a consequence of both direct and indirect cellular actions. Direct effects are mainly interactions with specific membrane receptors but may include unions with other components of the receptor/effector complex. Significant indirect biological effects of exogenous peptides, including apparent secretagogue effects on endogenous peptides largely overlooked in practice, result from extensive competition with endogenous peptides for degradative enzymes (peptidases). A consequence of this competition is enhancement of tonic or intermittent activity of endogenous peptides. The pharmacological profile of any peptide reflects or includes, therefore, the spectrum of endogenous peptides that is protected from peptidase action. It is likely that certain pharmacologically active peptides, including a large number of di-, tri- and oligo-peptides, elicit responses mainly or exclusively by competing for peptidases. Therefore, reliable estimates of the relative contributions of direct and indirect actions of exogenous peptides may be difficult, if not impossible, to obtain.     

Bio-inspired synthesis of AgNPs from lichen as potential antibacterial and mosquito larvicidal activity with negligible toxicity on Gambusia affinis

Mosquitoes serve as reservoirs for viruses and other microorganisms, posing a significant health-related issue for both humans as well as livestock. Control of these deadly disease-producing mosquito vectors is of paramount importance. The chemical analysis of Parmotrema reticulatum was examined by GC–MS. Further, lichen-mediated AgNPs were confirmed through UV–vis spectrophotometry, FTIR, TEM-EDX, and XRD. After 24 h post-treatment, the lichen-synthesized AgNPs showed considerable toxicity against distinct Aedes aegypti instars with LC₅₀ values of 44.61 (I instar), 51.27 (II instars), 61.34 (III instars), 72.95 (IV instar), and 89.84 (pupae) μg/mL, respectively. Further, both P. reticulatum extract and AgNPs greatly reduced the survival and reproductive efficiency of A. aegypti adults. Eventually, in conventional laboratory circumstances, the predatory effectiveness of Gambusia affinis against Ae. aegypti II and III instar larvae were 71.35% and 53.40%, respectively. In antibacterial assays, low concentrations of the P. reticulatum synthesized AgNPs inhibited the development of Pseudomonas aeruginosa and Citrobacter freundii. Surface damage, ROS production, and protein leakage are the antibacterial mechanisms of AgNPs. Overall, the lichen-derived AgNPs can be regarded as newer and safer Ae. aegypti control instruments.     

黄鳍金枪鱼抗菌肽YFGAP在大肠杆菌中融合表达及其活性检测

【目的】抗菌肽YFGAP由32个氨基酸组成,分子量为3.4 kD,对革兰氏阳性菌(G+)和革兰氏阴性菌(G?)表现出强效的抑制作用,不具有溶血活性。在大肠杆菌中表达抗菌肽YFGAP,分离纯化抗菌肽并鉴定其生物学活性。【方法】化学合成EK-YFGAP和L-EK-YFGAP基因序列,构建表达载体pET22b-ELP20-EK-YFGAP、pET22b-ELP40-EK-YFGAP和pET22b-ELP40-L-EK- YFGAP,分别转化至大肠杆菌BL21(DE3)中诱导表达,可逆相变循环纯化融合蛋白。肠激酶酶切,经Vivaspin Turbo纯化柱纯化,测定重组抗菌肽的抑菌活性和溶血活性。【结果】纯化出两种融合蛋白ELP40-EK-YFGAP和ELP40-L-EK-YFGAP,肠激酶酶切纯化后获得重组抗菌肽YFGAP,对4种病原菌均有抑制效果,溶血活性较低。【结论】以ELPs作为非色谱纯化标签,实现了抗菌肽YFGAP的融合表达,具有操作简单、成本低、易于扩大的优势,为重组抗菌肽的量化制备及应用提供了理论基础和技术支持。     

Tokaramide A, a new cathepsin B inhibitor from the marine sponge Theonella aff. mirabilis

A new cathepsin B inhibitor, tokaramide A (1) has been isolated from the marine sponge Theonella aff. mirabilis. Its structure was determined by spectroscopic and chemical methods. Tokaramide A inhibits cathepsin B with an IC₅₀ value of 29.0 ng/mL.     

Preparation and evaluation of antioxidant peptides from ethanol-soluble proteins hydrolysate of Sphyrna lewini muscle

To get high yield of ethanol-soluble proteins (EP) and the antioxidant peptides from Sphyrna lewini muscle, orthogonal experiments (L(9)(3)(4)) were applied to optimize the best extraction conditions and enzyme hydrolysis conditions. The yield of EP reached 5.903±0.053% under the optimum conditions of ethanol concentration 90%, solvent to material ratio 20:1, extraction temperature of 40°C and extraction time of 80min. The antioxidant SEPH (EP hydrolysate of S. lewini muscle) was prepared by using papain under the optimum conditions of enzymolysis time 2h, total enzyme dose 1.2%, enzymolysis temperature 50°C and pH 6, and its DPPH radical scavenging activity reached 21.76±0.42% at the concentration of 10mg/ml. Two peptides (F42-3 and F42-5) were isolated from SEPH by using ultrafiltration, anion-exchange chromatography, gel filtration chromatography and RP-HPLC. The structures of F42-3 and F42-5 were identified as Trp-Asp-Arg and Pro-Tyr-Phe-Asn-Lys with molecular weights of 475.50Da and 667.77Da, respectively. F42-3 and F42-5 exhibited good scavenging activity on hydroxyl radical (EC(50) 0.15mg/ml and 0.24mg/ml), ABTS radical (EC(50) 0.34mg/ml and 0.12mg/ml), and superoxide anion radical (EC(50) 0.09mg/ml and 0.11mg/ml), but moderate DPPH radical (EC(50) 3.63mg/ml and 4.11mg/ml). F42-3 and F42-5 were also effectively against lipid peroxidation in the model system and peroxyl free radical scavenging in β-carotene linoleic acid assay. Their high activities were due to the smaller size and the presence of antioxidative amino acids within the peptide sequences.     

Molecular cloning and biological characterization of novel antimicrobial peptides, pilosulin 3 and pilosulin 4, from a species of the Australian ant genus Myrmecia

Venom of an Australian ant species of the Myrmecia pilosula species complex (mss. name Myrmecia banksi Taylor) contains two major allergenic peptides, pilosulin 1 and pilosulin 2. To obtain novel cDNA clones that encode the pilosulin-related bioactive peptides, mRNA of another Myrmecia species was subjected to RT-PCR in which the forward primer corresponds to a nucleotide sequence in the leader sequences of pilosulin 1 and pilosulin 2. As a result, we isolated cDNA clones encoding the novel antimicrobial peptides pilosulin 3 and pilosulin 4. The nucleotide and the amino acid sequences of all four pilosulins have high homology except for the mature peptide coding regions. Synthetic pilosulin 3 and pilosulin 4 peptides displayed antimicrobial activity with histamine-releasing and low hemolytic activities.     

E.s.r. study of the effect of oxygen on the irradiation of peptides and polypeptides

Radiation damage induced in polycrystalline peptides and polypeptides in vacuum and at different oxygen partial pressures has been investigated using e.s.r. spectroscopy. For some peptides no alteration in the presence of oxygen was observed. For others, a reduction in the number of stable free radicals was observed as a function of p_O2, time and chain length. In some cases, the presence of oxygen during irradiation produces peroxide radicals. The above observations suggest that oxygen can interact with free radicals, inducing an increase in electron delocalization and the possibility of internal damage migration. The interaction occurs between the radicals which are adjacent to the carboxylic terminals and the oxygen located in the interstitial spaces. It may occur with or without the production of paramagnetic peroxides, depending on the nature of the carboxylic adjacent radicals.     

Secondary structure of peptides: 15. 13C n.m.r. CP/MAS study of solid elastin and proline-containing copolyesters

In order to study the chemical shifts and the cis—trans isomerism of prolyl units neighbouring glycine or other amino acids, 75.4 MH_z¹³C nuclear magnetic resonance (n.m.r.) cross-polarization/magic angel spinning (CP/MAS) spectra of the following solid oligopeptides and sequence polypeptides were measured: Z-Gly-Pro-OH,Z-Gly-Pro-Gly-Gly-OEt,Z-Gly-Pro-Ala-Ala-OMe,(Gly-Pro-Gly)_n,(Gly-Pro-Ala)_n,(β-Ala-Pro)_n and $\text{(δ-Ava-Pro)}{}_{\mathrm{<mtext>n</mtext>}}\text{(δ-Ava=δ-}\text{aminovaleric acid}$). Whereas all these oligo- and polypeptided contain exclusively trans X-Pro bonds, both cis and trans peptide bonds were found in a polypeptide prepared by copolymerization of glycine- and $\text{proline-}\text{N}\text{-carboxyanhydrides}$ in pyridine. On the basis of these model compounds, the ¹³C n.m.r. CP/MAS spectra of solid elastin allows the following conclusions. Almost all X-Pro bonds assume the trans conformation, most alanine and leucine units form α-helical chain segments, whereas only a small fraction of β-sheet structure is present. A 30.3 MHz ¹⁵N n.m.r. CP/MAS spectrum of solid elastin confirms that ～25% of all amino acids assume the α-helical structure. A model of elastin is discussed consisting of an amorphous phase, α-helical chain segments and helical segments of still unknown pitch.     

Design and synthesis of cationic Aib-containing antimicrobial peptides: conformational and biological studies.

Development of antimicrobial peptides has attracted considerable attention in recent years due to the excessive use of antibiotics, which has led to multiresistant bacteria. Cationic amphiphilic Aib-containing peptide models Ac-(Aib-Arg-Aib-Leu)(n)-NH2, n = 1-4, and sequential cationic polypeptides (Arg-X-Gly)(n), X = Ala, Val, Leu, were prepared and studied for their antimicrobial and hemolytic activity, as well as for their proteolytic stability. Ac-(Aib-Arg-Aib-Leu)(n)-NH2, n = 2, 3 and the polypeptide (Arg-Leu-Gly)(n) exhibited significant antimicrobial activity, and they were nontoxic at their MIC values and resistant, in particular the Aib-peptide models, to enzymatic degradation. The conformational characteristics of the peptide models were studied by circular dichroism (CD). Structure-activity relationship studies revealed the importance of the amphipathic alpha-helical conformation of the reported peptides in inducing antimicrobial effects. It is concluded that peptide models comprising cationic amino acids (Arg), helicogenic and noncoding residues (Aib) and/or hydrophobic and helix-promoting components (Leu) may lead to the development of antimicrobial therapeutics.     

Automated multi-dimensional liquid chromatography: sample preparation and identification of peptides from human blood filtrate

A comprehensive on-line sample clean-up with an integrated two-dimensional HPLC system was developed for the analysis of natural peptides. Samples comprised of endogenous peptides with molecular weights up to 20 kDa were generated from human hemofiltrate (HF) obtained from patients with chronic renal failure. The (poly-)peptides were separated using novel silica-based restricted access materials with strong cation-exchange functionalities (SCX-RAM). The size-selective sample fractionation step is followed by cation-exchange chromatography as the first dimension. The subsequent second dimension of separation is based on hydrophobic interaction using four parallel short reversed-phase (RP) columns implemented via a fully automated column switching technique. More than 1000 peaks were resolved within the total analysis time of 96 min. Substances of selected peaks were sampled to analyse their molecular weights by off-line MALDI-TOF mass spectrometry and to determine their amino acid sequence by Edman degradation. The potential for comprehensive peptide mapping and identification is demonstrated.     

Antimicrobial properties of two novel peptides derived from Theobroma cacao osmotin

The osmotin proteins of several plants display antifungal activity, which can play an important role in plant defense against diseases. Thus, this protein can be useful as a source for biotechnological strategies aiming to combat fungal diseases. In this work, we analyzed the antifungal activity of a cacao osmotin-like protein (TcOsm1) and of two osmotin-derived synthetic peptides with antimicrobial features, differing by five amino acids residues at the N-terminus. Antimicrobial tests showed that TcOsm1 expressed in Escherichia coli inhibits the growth of Moniliophthora perniciosa mycelium and Pichia pastoris X-33 in vitro. The TcOsm1-derived peptides, named Osm-pepA (H-RRLDRGGVWNLNVNPGTTGARVWARTK-NH₂), located at R23-K49, and Osm-pepB (H-GGVWNLNVNPGTTGARVWARTK-NH₂), located at G28-K49, inhibited growth of yeasts (Saccharomyces cerevisiae S288C and Pichia pastoris X-33) and spore germination of the phytopathogenic fungi Fusarium f. sp. glycines and Colletotrichum gossypi. Osm-pepA was more efficient than Osm-pepB for S. cerevisiae (MIC = 40 μM and MIC = 127 μM, respectively), as well as for P. pastoris (MIC = 20 μM and MIC = 127 μM, respectively). Furthermore, the peptides presented a biphasic performance, promoting S. cerevisiae growth in doses around 5 μM and inhibiting it at higher doses. The structural model for these peptides showed that the five amino acids residues, RRLDR at Osm-pepA N-terminus, significantly affect the tertiary structure, indicating that this structure is important for the peptide antimicrobial potency. This is the first report of development of antimicrobial peptides from T. cacao. Taken together, the results indicate that the cacao osmotin and its derived peptides, herein studied, are good candidates for developing biotechnological tools aiming to control phytopathogenic fungi.     

Influence of plant and environment parameters on phytochemical composition and biological properties of Pistacia atlantica Desf.

The aim of the current study is to analyze the phytochemical, antioxidant and antimicrobial activities of 34 extracts prepared from Pistacia atlantica Desf. subsp. atlantica, according to gender, organ type (roots, buds and fruits), geographical location and stage of ripening. Bud extracts exhibited the highest phenolic content (565.74 ± 9.84 mg GAE/g DM), followed by fruit and root extracts. TFC and TTC ranged from 0.38 ± 0.03 to 1.92 ± 0.11 mg CE/g DM and from 0.37 ± 0.03 to 16.54 ± 0.94 mg CE/g DM, respectively. For DPPH and TAC assays, the values varied from 0.038 ± 0.000 to 1.331 ± 0.114 mg/mL and 1.58 ± 0.06 to 43.64 ± 2.58 mg AAE/g DM, respectively. Besides, bud extracts showed the highest bioactivity against pathogenic bacteria and a slight antifungal effect. Additionally, HPLC-DAD analysis revealed that the caffeic acid and the dimethyl-allyl caffeic acid characterized the bud extract, while the rutin and the hydroxytyrosol were abundant in the red fruit extract. The present evidence suggests that P. atlantica may be considered as a potential source of new additives for therapeutic, food and cosmetic products.     

Cyclic peptides. XIX. Synthesis, conformation, and biological activity of cyclo(-Pro-Val-Pro-Val-) with L-L-L-L and L-D-L-D sequences

cyclo(-L-Pro-L-Val-L-Pro-L-Val-) (1L) and cyclo(-L-Pro-D-Val-L-Pro-D-Val-) (1D) were synthesized by the conventional method for peptide synthesis. Conformations of 1L and 1D in solution were studied. Compound 1L has a cis-trans-cis-trans backbone conformation with C2 symmetry in CDCl3. This conformation is slightly different from that in crystalline state and in DMSO-d6 solution. Compound 1D has a cis-trans-cis-trans conformation in DMSO-d6 and an all-trans conformation in trifluoroethanol-d3. Compound 1L retarded stem-growth of rice seedlings and, in contrast, compound 1D promoted root-growth of rice seedlings.