Data_Sheet_1_Characterization of volatile organic compounds with anti-atherosclerosis effects in Allium macrostemon Bge. and Allium chinense G. Don by head space solid phase microextraction coupled with gas chromatography tandem mass spectrometry.docx

IntroductionAllium macrostemon Bge. (AMB) and Allium chinense G. Don (ACGD) are both edible Allium vegetables and named officinal Xiebai (or Allii Macrostemonis Bulbus) in East Asia. Their medicinal qualities involve in lipid lowering and anti-atherosclerosis effects. And steroidal saponins, nitrogenous compounds and sulfur compounds are like the beneficial components responsible for medicinal functions. Sulfur compounds are the recognized main components both in the volatile oils of AMB and ACGD. Besides, few researches were reported about their holistic chemical profiles of volatile organic compounds (VOCs) and pharmacodynamic effects.MethodsIn this study, we first investigated the lipid-lowering and anti-atherosclerotic effects of volatile oils derived from AMB and ACGD in ApoE–/– mice with high fat and high cholesterol diets.ResultsThe results showed the volatile oils of AMB and ACGD both could markedly reduce serum levels of TG, TC, and LDL-C (p 0.05). Pathological results displayed they both could obviously improve the morphology of cardiomyocytes and the degree of myocardial fibrosis in model mice. Meanwhile, oil red O staining results also proved they could apparently decrease the lesion areas of plaques in the aortic intima (p DiscussionTaken together, this study was the first analysis of holistic chemical profiles and anti-atherosclerosis effects of AMB and ACGD volatile oils, and would benefit the understanding of effective components in AMB and ACGD.</p

Representative fluorescent micrographs of the trabecular bone sections show the apposition of the xylenol (red) and calcein (green) labels in the sham-operated (Sham), ovariectomized only (vehicle-treated) (OVX), and OVX mice treated with different XLGB fractions.

<p>The white arrow pointed to the labeled the distance between two sequential fluorescent dyes in a time interval of 8 days. 20 x, bar = 10um.</p

<i>In Vivo</i> Screening for Anti-Osteoporotic Fraction from Extract of Herbal Formula Xianlinggubao in Ovariectomized Mice

<div><p>Background and Objectives</p><p>Traditional Chinese Medicine (TCM) Fufang or formula Xianlinggubao (XLGB) is a prescribed TCM drug in China registered for prevention and treatment of osteoporosis. Fufang in TCM is comprised of a group of herbal compounds contributing in group to the treatment efficacy. The present study aims to identify the bioactive fraction(s) in XLGB extract that account(s) dominantly for its osteogenic effects.</p><p>Methods</p><p>The extract of XLGB formula was separated into three fractions using chromatography, i.e., XLGB-A, XLGB-B and XLGB-C. They were administrated to 4-month old ovariectomized (OVX) mice for 6 weeks to determine which bioactive fraction(s) were more effective for preventing OVX-induced bone loss evaluated by microCT, biomechanical testing and biochemical markers. The main peaks of the key fraction were identified using reference compounds isolated from the fraction. In addition, the effects of the composite compounds in XLGB-B on osteoblasts’ proliferation and mineralization were evaluated in UMR 106 cells.</p><p>Results</p><p>XLGB-B with a yield of 13.0% from herbal Fufang XLGB was identified as the most potential one among the three fractions for prevention of OVX-induced bone loss confirmed with bone mass, bone microarchitecture, bone strength and bone turnover markers. Nine compounds in HPLC fingerprint were identified in the XLGB-B fraction, including phenylpropanoids from <i>Herba Epimedii</i>, terpenes from <i>Radix Dipsaci</i> and coumarins from <i>Fructus Psoraleae</i>. In addition, the identified compounds effectively promoted proliferation and/or mineralization of osteoblast-like UMR 106 cells <i>in vitro</i>.</p><p>Conclusion</p><p>XLGB-B with defined phytochemical structures was screened as the key fraction that demonstrated preventive effects on OVX-induced bone loss in mice. The present study laid down a foundation towards a new generation of herbal Fufang characterized with “less herbal materials for achieving equal treatment efficacy” in development strategy of TCM for prevention of OVX-induced osteoporosis.</p></div

Treatments for five groups of mice (n = 6–8).

<p>Treatments for five groups of mice (n = 6–8).</p

The level of endogenous miR-3162-3p, β-catenin mRNA and protein expressions in the lung in mice varies at different stages of asthma pathogenesis.

<p><b>(A)</b> Responsiveness to aerosolized methacholine was assessed by PenH value one day after the last process in each group of mice. The PenH values indicate airway hyperresponsiveness when mice inhaled the aerosolized methacholine. Data is shown as the mean ± SEM. *p < 0.05 vs. other groups; **p<0.05 vs. normal or sensitization (n = 5–6 per group). <b>(B)</b> Representative lung sections were H&E-stained to estimate the degree of inflammation (blue arrows) in the terminal bronchioles and alveolar regions of each group of mice (200× magnification). <b>(C)</b> Representative airway tissues were stained with PAS to assess the relative amount of intraepithelial mucus production (black arrows). Abundant mucus hypersecretion was found in asthma mice (200× magnification). <b>(D)</b> Number of BALF total cells and proportion of eosinophils (EOS) in BALF mice. *p, **p<0.05 vs. challenge. <b>(E-F)</b> Expression level of miR-3162-3p in peripheral blood and lung from each group of mice. Results from three independent experiments (n = 3) are shown. *p<0.05 vs. other groups of mice. <b>(G)</b> β-catenin mRNA expression profile in the lung for each group is shown. *p < 0.05 vs other groups. <b>(H)</b> β-catenin protein level in the lung of asthma mice showed a marked decrease in response to allergen challenge which resulted in upregulation of endogenous miR-3162-3p level.</p

β-catenin is a direct target of miR-3162-3p.

<p><b>(A)</b> The three mRNA transcript variants of β-catenin possess different 3’-UTRs, which contain either one or two miR-3162-3p pairing sites. Site 2 is a common pairing site for all three transcript variants. Transcript variants 2 and 3 have only one pairing site (site 2) and transcript variant 1 has two pairing sites, sites 1 and 2. <b>(B)</b> Potential matching target sites of miR-3162-3p in the 3’-UTR of β-catenin mRNA transcript and sequence information of the wild type and mutant luciferase report constructs. Two fragments which comprise miR-3162-3p target sites on the 3′-UTR of β-catenin mRNA transcript were separately inserted into the luciferase gene of the pmirGLO vector at the <i>NheI</i> and <i>SalI</i> restriction sites. <b>(C)</b> The physical interaction between miR-3162-3p and the predicted target sites on β-catenin 3′-UTR in A549 cells was detected by luciferase reporter assay. Each wild type reporter construct was co-transfected with mimic-miR-3162-3p, syn-miR control, anti-miR-3162-3p or anti-miR control. The mutant reporter construct was co-transfected with mimic-miR-3162-3p or syn-miR control. Luciferase activity was measured at 24 h after transfection. Activity of the 3’-UTR reporter constructs was normalized to Renilla. Results are shown as the mean ± SD (n = 3). *p < 0.05 vs. syn-miR control.</p

Effects of miR-3162-3p, its mimics and inhibitor on the endogenous β-catenin expression level in A549, H1299 and Beas-2B cell lines.

<p><b>(A)</b> Synthetic oligonucleotides—mimic-miR-3162-3p, syn-miR control, anti-miR-3162-3p or anti-miR control—were transfected into A549, H1299 and Beas-2B cell lines, respectively. β-catenin mRNA was quantified by real-time PCR (β-actin as an internal control). Results are shown as the mean ± SD (n = 3), *p<0.05 vs. syn-miR control; **p<0.05 vs. anti-miR control. <b>(B)</b> Western blot analysis of cell lysates was performed to determine the relative β-catenin protein expression level.</p

Representative fluorescent micrographs of the trabecular bone sections show the apposition of the xylenol (red) and calcein (green) labels in the sham-operated (Sham), ovariectomized only (vehicle-treated) (OVX), and OVX mice treated with different XLGB fractions.

<p>The white arrow pointed to the labeled the distance between two sequential fluorescent dyes in a time interval of 8 days. 20 x, bar = 10um.</p

Effects of XLGB fractions on biomechanical properties, bone apposition rate and bone turnover marker in OVX mice (mean values ± standard deviations, n = 10).

<p>Sham: sham-operated; OVX: ovariectomized; XLGB-A, OVX mice treated with 140 mg XLGB-A/kg body weight/day; XLGB-B, OVX mice treated with 31 mg XLGB-B/kg body weight/day; XLGB-C, OVX mice treated with 55 mg XLGB-C/kg body weight/day; Combined XLGB: OVX mice treated with 140 mg XLGB-A, 31 mg XLGB-B and 55 mg XLGB-C kg body weight/day.</p><p>Mean value was significantly different from that of the OVX group</p><p>* P<0.05</p><p>** P<0.01</p><p>#: Mice treated for 6 weeks.</p><p>Effects of XLGB fractions on biomechanical properties, bone apposition rate and bone turnover marker in OVX mice (mean values ± standard deviations, n = 10).</p

Effects of three doses of XLGB extracts on bone parameters as measured by microCT at the sixth lumbar vertebrae in OVX mice (mean values ± standard deviations, n = 10).

<p>BMD: bone mineral density; BV/TV: bone volume/tissue volume; Tb.Th: trabecular thickness; Sham: sham-operated; OVX: ovariectomized; XLGB-L: OVX mice treated with 118 mg XLGB extract/kg body weight/day; XLGB-M: OVX mice treated with 236 mg XLGB extract/kg body weight/day; XLGB-H: OVX mice treated with 472 mg XLGB extract/kg body weight/day.</p><p>Mean value was significantly different from that of the OVX group</p><p>* P<0.05</p><p>** P<0.01</p><p>#Mice were subjected to the treatment for 6 weeks.</p><p>Effects of three doses of XLGB extracts on bone parameters as measured by microCT at the sixth lumbar vertebrae in OVX mice (mean values ± standard deviations, n = 10).</p