Origin of Red Color in Edible Bird’s Nests Directed by the Binding of Fe Ions to Acidic Mammalian Chitinase-like Protein

The red color of edible bird’s nests (EBNs) has remained a mystery for hundreds of years. Here, different analytical methods were employed to identify the color origin of EBNs. The treatment of white EBNs with NaNO₂/HCl turned them red. In a simulated-gastric-fluid (SGF)-digested EBN, the HPLC chromatogram, NMR spectrum, circular-dichroism spectrum, and Raman spectrum of a NaNO₂-treated white EBN closely resembled those of an authentic red EBN. From the HPLC chromatogram of the SGF-digested EBN, the peptides associated with red color were identified in a red EBN and NaNO₂-treated white EBN. Several lines of evidence indicated that the color-containing peptide could be derived from the acidic mammalian chitinase-like (AMCase-like) protein of EBNs. Additionally, there was a noticeable increase in Fe–O-bonding intensity after the color change. On the basis of the findings, we proposed that the oxidation of Fe ions in AMCase-like proteins contributed significantly to the color change of EBNs

YPFS induces COX-2 expression in macrophages.

<p>(<b>A</b>): Cultured macrophages were treated with herbal extracts as described in Fig. 4 legend for 24 hours and then COX-2 mRNA expression levels were determined using RT-PCR. GAPDH was used as an internal control for normalization and LPS (1 µg/mL) was used as the positive control. (<b>B</b>): Macrophages were treated with herbal extracts for 24 hours after first incubating the cells with or without the NF-κB-specific inhibitor BAY 11-7082 (5 µM) for 3 hours. Cell lysates were collected, diluted to equal protein concentrations, and analyzed by means of western blotting. Representative western blots are shown. (<b>C</b>): Effects of BAY 11-7082 on the expression of COX-2 protein induced by YPFS in cultured macrophages. (<b>D</b>): Effects of BAY 11-7082 on COX-2 protein expression induced by individual herbs in cultured macrophages. Results in (C) and (D) were calculated using the western blots shown in (B). Values were normalized using the internal control GAPDH and are expressed as fold-increases relative to basal reading (untreated cultures); mean ± SD are shown, <i>n</i> = 5, each with triplicate samples. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p

Asarone from Acori Tatarinowii Rhizome prevents oxidative stress-induced cell injury in cultured astrocytes: A signaling triggered by Akt activation

<div><p>Acori Tatarinowii Rhizome (ATR; the dried rhizome of <i>Acori tatarinowii</i> Schott) is a well-known herb being used for mental disorder in China and Asia. Volatile oil is considered as the active ingredient of ATR, and asarones account for more than 90% of total volatile oil. Here, the protective effects of ATR oil and asarones, both α-asarone and β-asarone, were probed in cultured rat astrocytes. The cyto-protective effect of ATR oil and asarones against tBHP-induced astrocyte injury was revealed, and additionally ATR oil and asarones reduced the tBHP-induced intracellular reactive oxygen species (ROS) accumulation. In parallel, the activity of anti-oxidant response element (ARE) promoter construct (pARE-Luc), being transfected in cultured astrocytes, was markedly induced by application of ATR oil and asarones. The mRNAs encoding anti-oxidant enzymes, e.g. glutathione S-transferase (GST), glutamate-cysteine ligase modulatory subunit (GCLM), glutamate-cysteine ligase catalytic subunit (GCLC) and NAD(P)H quinone oxidoreductase (NQO1) were induced by ATR oil and asarones in a dose-dependent manner. The ATR oil/asarone-induced gene expression could be mediated by Akt phosphorylation; because the applied LY294002, a phosphoinositide 3-kinase inhibitor, fully abolished the induction. These results demonstrated that α-asarone and β-asarone could account, at least partly, the function of ATR being a Chinese medicinal herb.</p></div

YPFS stimulates IALP expression and activity in Caco-2 cells.

<p>(<b>A</b>): Caco-2 cells (100,000/well) cultured in 6-well plates were treated with herbal extracts and cells were harvested on various days. IALP mRNA levels were quantified using RT-PCR, and the values were normalized relative to GAPDH expression. (<b>B</b>): Caco-2 cells (50,000/well) cultured in 12-well plates were treated with herbal extracts. Cells were harvested on various days as in (A) and extracts were prepared using a lysis buffer (pH 10.4). IALP activity in Caco-2 cells was measured by mixing samples with 5 mM p-nitrophenyl phosphate, and absorbance was measured at 405 nm; enzyme activity is expressed as µmol cleaved substrate/mg protein. Here, sodium butyrate (1 mM) served as the positive control. Values are expressed as fold-increases relative to basal reading (taken on the first day of culture); mean ± SD are shown, <i>n = </i>3, each with triplicate samples. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p

α-asarone, β-asarone or ATR oil activates the phosphorylation of Akt in cultured astrocytes.

<p>(A) Serum free astrocytes pre-treated with DMEM or LY294002 (10 μM) for 3 hours prior to treatment of α-asarone, β-asarone or ATR oil (both at 15 μg/mL) for indicated time (5, 10 and 30 min). Phosphorylation of Akt was detected by immune blot analysis using specific antibodies. Band density was estimated, densitometrically, and the phosphorylation rates were expressed as the intensity of phosphorylated Akt relative to total Akt (p-Akt/t-Akt). (B) Cultures, transfected with pARE-Luc, were pre-treated with medium or LY294002 (10 μM) for 3 hours prior to treatment of 15 μg/mL α-asarone, β-asarone or ATR oil for 48 hours. (C) Cultures were pre-treated with medium or LY294002 as in (B) for 48 hours. Then, the cells were challenged with tBHP (100 μM) for 3 hours. The cell viability was determined. Values are in mean ± SEM, where <i>n</i> = 3, each with triplicate samples. *<i>p</i> < 0.05; **<i>p</i> < 0.01 compared with control.</p

α-asarone, β-asarone or ATR oil suppresses tBHP-induced ROS formation and total anti-oxidant capacity.

<p>(A) Cultured astrocytes were exposed to tBHP (0–300 μM) for 1 hour. The level of intracellular ROS was measured. (B) Cultured astrocytes were pre-treated with α-asarone, β-asarone or ATR oil (both at 1–15 μg/mL) and then exposed to tBHP (100 μM) for 1 hour. tBHQ (1.5 μM) was used as positive control of having 40% of ROS inhibition. The results were in % of increase against ROS formation relative to the control (with tBHP alone). (C) Total anti-oxidant capacity of α-asarone, β-asarone and ATR oil at varying concentration were calculated and compared with Trolox equivalent expressed as μM. Data were expressed as mean ± SEM, <i>n</i> = 3–5, each with triplicate samples. *<i>p</i> < 0.05; **<i>p</i> < 0.01 compared with control.</p

α-asarone, β-asarone or ATR oil induces the expression of anti-oxidant enzymes in cultured astrocytes.

<p>Cultured astrocytes were treated with α-asarone, β-asarone or ATR oil (both at 5–15 μg/mL) or tBHQ (1.5 μM) for 48 hours. Total RNAs were isolated from cultured astrocytes and then reversed transcribed into cDNAs for the detection of mRNAs encoding GCLC, GCLM, NQO1 and GST by real-time PCR analysis. The 18S served as internal control. Values are expressed as the fold of increase to basal reading (untreated culture), and in mean ± SEM, where <i>n</i> = 3, each with triplicate samples. *<i>p</i> < 0.05; **<i>p</i> < 0.01 compared with control.</p

YPFS suppresses LPS-induced expression of COX-2 in cultured macrophages.

<p>Cultured macrophages were treated with herbal extracts and stimulated with LPS as described in Fig. 1 legend; 10 µM Dex was used as a positive control. The levels of COX-2 mRNA were determined using RT-PCR and normalized relative to the GAPDH signal. Lysed cells were prepared and equal amounts of proteins were subjected to western blotting performed using an antibody specific for COX-2. GAPDH was used as the internal control. (<b>A</b>): Suppression of COX-2 mRNA expression by YPFS in LPS-stimulated RAW264.7 cells. (<b>B</b>): Inhibition of COX-2 mRNA expression by herbal extracts of AR, AMR, and SR in LPS-stimulated RAW264.7 cells. (<b>C</b>): Representative western blots showing COX-2 staining. (<b>D</b>): Suppression of COX-2 protein expression by YPFS in LPS-stimulated RAW264.7 cells. (<b>E</b>): Inhibition of COX-2 protein expression by herbal extracts of AR, AMR, and SR in LPS-stimulated RAW264.7 cells. Results in (D) and (E) were calculated using the western blots shown in (C). Values are expressed as % LPS-induced activation; mean ± SD are shown, <i>n</i> = 4, each with triplicate samples. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p

α-asarone, β-asarone or ATR oil induces ARE transcriptional activity in cultured astrocytes.

<p>(A) Cultured astrocytes, transfected with pARE-Luc, were treated with tBHQ (0–2.5 μM) for 48 hours. The cell lysates were subjected to luciferase assay to measure the activity driven by ARE. (B) α-Asarone, β-asarone or ATR oil (both at 1–15 μg/mL) was applied onto pARE-Luc-expressed astrocytes for 48 hours. The cell lysates were subjected to luciferase assay. tBHQ (1.5 μM) was used as a positive control. Data are expressed as the fold of increase to basal reading (untreated culture), and they are in mean ± SEM, where <i>n</i> = 4, each with triplicate samples. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001 compared with control.</p

YPFS suppresses LPS-induced expression of iNOS in cultured macrophages.

<p>Cultured macrophages were treated with herbal extracts for 3 hours, after which LPS (1 µg/mL) was applied to the cultures for 24 hours to mimic chronic inflammation. Here, 10 µM dexamethasone (Dex) served as a positive control. The levels of mRNAs encoding iNOS were determined using real-time PCR (RT-PCR), performed with GAPDH serving as an internal control for normalization. The expression of iNOS protein was examined using western-blotting analysis. Whole cell lysates of macrophages were collected and equal amounts of total protein were loaded on gels and stained with an anti-iNOS antibody. (<b>A</b>): Inhibition of iNOS mRNA expression by YPFS in LPS-stimulated RAW264.7 cells. (<b>B</b>): Inhibition of iNOS mRNA expression by single herbs in LPS-stimulated RAW264.7 cells; 1 mg/mL of each of the herbal extracts was added to macrophages and the expression level of iNOS mRNA was determined using RT-PCR. (<b>C</b>): Western blots showing iNOS staining. (<b>D</b>): Inhibition of iNOS protein expression by YPFS in LPS-stimulated RAW264.7 cells. (<b>E</b>): Inhibition of iNOS protein expression by individual herbs in LPS-stimulated RAW264.7 cells. Single herbal extracts were added to LPS-stimulated macrophages for 24 hours. Results in (D) and (E) were calculated using the western blots shown in (C). Values are expressed as % LPS-induced activation; mean ± SD are shown, <i>n = </i>4, each with triplicate samples. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p