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Yak butter is one of the most important foods for the Tibetan people. Of note, its production yields waste yak milk as a by-product. In this work, waste yak milk protein hydrolysates made via Pepsin hydrolysis were shown to have antimicrobial activity. Furthermore, an innovative method of magnetic liposome adsorption combined with reversed-phase high performance liquid chromatography (RP-HPLC) was developed to screen for and purify the antimicrobial peptides. Two antimicrobial peptides were obtained and their amino acid sequences were determined by N-sequencing, namely Arg-Val-Met-Phe-Lys-Trp-Ala and Lys-Val-Ile-Ser-Met-Ile. The antimicrobial activity spectra of Arg-Val-Met-Phe-Lys-Trp-Ala included Bacillus subtilis, Staphylcoccus aureus, Listeria innocua, Escherichia coli, Enterobacter cloacae and Salmonella paratyphi, while the Lys-Val-Ile-Ser-Met-Ile peptide shows not only bacterial growth inhibition but also of fungi. Haemolytic testing suggested that these two antimicrobial peptides could be considered to have no haemolytic effect at their minimum inhibitory concentrations (MICs).
A phytochemical investigation of <b>Saxifraga tangutica</b> led to the isolation of 11 compounds, including eight diarylheptanoids (<b>1</b>-<b>6</b>, <b>10</b> and <b>11</b>) and three phenylpropanoids (<b>7</b>-<b>9</b>). The chemical structures were established by extensive analysis of their MS and NMR spectroscopic data or comparison with literature data. In the present research, we report the isolated compounds <b>1</b>-<b>11</b>, for the first time, in the species <b>S. tangutica</b>. Moreover, compounds <b>1</b>, <b>2</b> and <b>4</b>-<b>11</b> have not been reported from any species in Saxifragaceae family. Furthermore, we discuss the chemotaxonomic significance of the isolated compounds.<br>• Eight diarylheptanoids and three phenylpropanoids have been isolated from <b>Saxifraga tangutica.</b> • Compounds <b>1</b>-<b>11</b> are firstly reported in the species <b>Saxifraga tangutica.</b> • Compounds <b>1</b>, <b>2</b> and <b>4</b>-<b>11</b> are firstly isolated from genus <b>Saxifraga</b> or family Saxifragaceae.
ETHNOPHARMACOLOGICAL RELEVANCE: Osteon Myospalacem Baileyi, known as Sai long gu (Tibetan language, means "blind rat bone"), is the whole skeleton of Tibet plateau rodentia animal Myospalacem Baileyi. Osteon Myospalacem Baileyi had been widely used in the Tibet region as an anti-osteoporosis drug and since 1991 Osteon Myospalacem Baileyi has been listed in the Pharmacopoeia of People's Republic of China as the first-class animal new medical material. However, the mechanism of its anti-osteoporosis activities is still unclear. It is very desirable to solve this problem for further study.MATERIALS AND METHODS: in this study, preparative chromatography was employed to produce the active fraction ET4 from Osteon Myospalacem Baileyi crude. Flow cytometry and MTT assay were used to evaluate the toxicities of ET4. BMM cells were separated from mouse bone marrow to test the inhibition effects of ET4 on osteoclastogenesis. Western blot was used to find out the pathways, through which ET4 could act on osteoclastogenesis. Q-PCR was used to test the osteoclastogenesis marker genes. At last, immunofluorescence confocal microscopy was used to test the osteoclastogenesis master protein NFATc1 nuclei translocation. RESULTS: In this study we report that ET4, at the dose of 60μg/mL, significantly inhibited the formation of osteoclasts. Notably, ET4 did not affect the BMM viability at that dose. In addition, Osteon Myospalacem Baileyi could inhibit the expression of osteoclast marker genes, including cathepsin K (CTSK), nuclear factor of activated T cells cytoplasmic 1 (NFATc1), tartrate resistant acid phosphatase (TRAP, Acp5) dendrite cell-specific transmembrane protein (DC-STAMP), calcitonin receptor (CTR), osteoclast associated and immunoglobulin-like receptor (OSCAR). Mechanistically, ET4 dose- and time-dependently blocked the RANKL-induced activation of ERK and c-Fos as well as the induction of NFATc1 which is essential for OC formation. CONCLUSIONS: These data suggest that ET4 might be a useful alternative therapy in preventing or treating osteolytic diseases.
Yak milk cheese is one of the most important foods for the Tibetan people. <b>Lactobacillus plantarum</b> SLG1 isolated from yak cheese was shown to produce a novel bacteriocin, plantaricin SLG1. Production of plantaricin SLG1 in MRS medium was maximized after 24 h incubation at 37 °C (the stationary phase of growth). An innovative method, namely magnetic liposome adsorption combined with reversed-phase high performance liquid chromatography (RP-HPLC), was developed to screen for and efficiently purify bacteriocin compounds from the cell free supernatants from <b>Lb. plantarum</b> SLG1. The molecular mass of plantaricin SLG1 is 1083.25 Da and its amino acid sequence Tyr-Gly-Asn-Gly-Val-Phe-Ser-Val-Ile-Lys was determined by N-sequencing. Analyses by Circular dichroism (CD) spectra and predicted 3D structure suggested that the peptide maintains a well-defined conformation. Plantaricin SLG1 exhibited a wide range of antimicrobial activity against many food-borne spoilage and pathogenic bacteria, as well as some fungi. Results using scanning electron microscopy indicated that the mode of action was bactericidal and plantaricin SLG1 was able to damage the cell membrane integrity ultimately causing pathogen lethality.<br>• Bacteriocin-producer <b>Lb. plantarum</b> SLG1 isolated from yak cheese. • Bacteriocin, plantaricin SLG1, was obtained. • Magnetic liposomes adsorption & RP-HPLC were used to purification. • The active spectrum include Gram-positive and negative bacteria and fungi.
Highland barley is one of the most important industrial crops in Tibetan plateau. Previous research indicated that highland barley has many medical functions. In this work, the antibacterial abilities of highland barley were investigated. The protein solutions hydrolyzed by trypsin for 4 h exhibited the highest antibacterial activity. An antibacterial peptide, barleycin, was screened and purified by magnetic liposome extraction combining with the protein profiles of reversed-phase high-performance liquid chromatography (RP-HPLC). Structure, characterization, and safety evaluation of barleycin were further investigated. Amino acids sequence was determined as Lys-Ile-Ile-Ile-Pro-Pro-Leu-Phe-His by N-sequencing. Circular dichroism spectra indicated the a-helix conformation of barleycin. The activity spectrum included <i>Bacillus subtilis, Staphylcoccus aureus, Listeria innocua and Escherichia coli</i> and the MICs were from 4 to 16 μg/mL. Safety evaluations with cytotoxicity and hemolytic suggested this antibacterial peptide could be considered as safe at MICs. Finally, mode of action of barleycin on sensitive cells was primarily studied. The results suggested the damage of cell membrane.