Abstract:Agarwood is the fragrant resin-infused wood derived from the wounded trees of Aquilaria species. It is a valuable non-timber forest product used in fragrances and as medicine. Reforestation for Aquilaria trees in combination with artificial agarwood-inducing methods serves as a way to supply agarwood and conserve of wild Aquilaria stock. However, the existing agarwood-inducing methods produce poor-quality agarwood at low yield. Our study evaluated a novel technique for producing agarwood in cultivated Aquilaria trees, called the whole-tree agarwood-inducing technique (Agar-Wit). Ten different agarwood inducers were used for comparison of Agar-Wit with three existing agarwood-inducing methods. For Aquilaria trees treated with these ten inducers, agarwood formed and spread throughout the entire tree from the transfusion point in the trunk to the roots and branches of the whole tree. Agarwood yield per tree reached 2,444.83 to 5,860.74 g, which is 4 to 28 times higher than that by the existing agarwood-inducing methods. Furthermore, this agarwood derived from Agar-Wit induction was found to have a higher OPEN ACCESSMolecules 2013, 18 3087 quality compared with the existing methods, and similar to that of wild agarwood. This indicates Agar-Wit may have commercial potential. Induction of cultivated agarwood using this method could satisfy the significant demand for agarwood, while conserving and protecting the remaining wild Aquilaria trees.

Background-Oxidized LDL (ox-LDL) accumulation in the atherosclerotic region may enhance plaque instability. Both accumulation of ox-LDL and expression of its lectin-like receptor, LOX-1, have been shown in atherosclerotic regions. This study was designed to examine the role of LOX-1 in the modulation of metalloproteinases (MMP-1 and MMP-3) in human coronary artery endothelial cells (HCAECs). Methods and Results-HCAECs were incubated with ox-LDL (10 to 80 g/mL) for 1 to 24 hours. Ox-LDL increased the expression of MMP-1 (collagenase) and MMP-3 (stromelysin-1) in a concentration-and time-dependent manner. Ox-LDL also increased collagenase activity. Ox-LDL did not significantly affect the expression of tissue inhibitors of metalloproteinases. Native LDL had no effect on the expression of MMPs. The effects of ox-LDL were mediated by its endothelial receptor, LOX-1, because pretreatment of HCAECs with a blocking antibody to LOX-1 (JTX92, 10 g/mL) prevented the expression of MMPs in response to ox-LDL (PϽ0.01). In parallel experiments, ox-LDL caused the activation of protein kinase C (PKC), which was inhibited by LOX-1 antibody. The PKC-␤ isoform played a critical role in the expression of MMPs, because the PKC-␤ inhibitor hispidin reduced ox-LDL-induced activation of PKC and the expression of MMPs. Other PKC subunits (␣, ␥, and ⑀) did not affect the expression of MMPs. Conclusions-These

LOX-1, a receptor for oxidized low-density lipoprotein (ox-LDL), plays a critical role in endothelial dysfunction and atherosclerosis. LOX-1 activation also plays an important role in monocyte adhesion to endothelial cells. A number of studies show that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) reduce total LDL cholesterol and exert a cardioprotective effect. We examined the modulation of LOX-1 expression and its function by two different statins, simvastatin and atorvastatin, in human coronary artery endothelial cells (HCAECs). We observed that ox-LDL (40 g/ml) treatment upregulated the expression of E-and P-selectins, VCAM-1 and ICAM-1 in HCAECs. Ox-LDL mediated these effects via LOX-1, since antisense to LOX-1 mRNA decreased LOX-1 expression and subsequent adhesion molecule expression. Pretreatment of HCAECs with simvastatin or atorvastatin (1 and 10 M) reduced ox-LDL-induced expression of LOX-1 as well as adhesion molecules (all P Ͻ 0.05). A high concentration of statins (10 M) was more potent than the low concentration (1 M) (P Ͻ 0.05). Both statins reduced ox-LDL-mediated activation of the redox-sensitive nuclear factor-B (NF-B) but not AP-1. These observations indicate that LOX-1 activation plays an important role in ox-LDL-induced expression of adhesion molecules. Inhibition of expression of LOX-1 and adhesion molecules and activation of NF-B may be another mechanism of beneficial effects of statins in vascular diseases.

BackgroundAgarwood is an expensive resinous heartwood derived from Aquilaria plants that is widely used in traditional medicines, incense and perfume. Only wounded trees can produce agarwood, and the huge demand for the agarwood products has led all Aquilaria spp. being endangered and listed in the Appendix II of the CITES (http://www.cites.org). The major components of agarwood are sesquiterpenes and phenylethyl chromones. Owing to a lack of genomic information, the molecular basis of wound-induced sesquiterpenes biosynthesis and agarwood formation remains unknown.ResultsTo identify the primary genes that maybe related to agarwood formation, we sequenced 2 cDNA libraries generated from healthy and wounded A. sinensis (Lour.) Gilg. A total of 89,137 unigenes with an average length of 678.65 bp were obtained, and they were annotated in detail at bioinformatics levels. Of those associated with agarwood formation, 30 putatively encoded enzymes in the sesquiterpene biosynthesis pathway, and a handful of transcription factors and protein kinases were related to wound signal transduction. Three full-length cDNAs of sesquiterpene synthases (ASS1-3) were cloned and expressed in Escherichia coli, and enzyme assays revealed that they are active enzymes, with the major products being δ-guaiene. A methyl jasmonate (MJ) induction experiment revealed that the expression of ASS was significantly induced by MJ, and the production of sesquiterpenes was elevated accordingly. The expression of some transcription factors and protein kinases, especially MYB4, WRKY4, MPKK2 and MAPK2, was also induced by MJ and coordinated with ASS expression, suggesting they maybe positive regulators of ASS.ConclusionsThis study provides extensive transcriptome information for Aquilaria spp. and valuable clues for elucidating the mechanism of wound-induced agarwood sesquiterpenes biosynthesis and their regulation.

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The composition and antimicrobial activity of the essential oils which were obtained from agarwood originated from Aquilaria sinensis (Lour.) Gilg stimulated by the chemical method (S1) were characterized, taking wild agarwood (S2) and healthy trees (S3) respectively as the positive and negative controls. The chemical composition of S1 was investigated by gas chromatography-mass spectrometry (GC-MS). The essential oil of S1 showed a similar composition to that of S2, being rich in sesquiterpenes and aromatic constituents. However, the essential oil of S3 was abundant in fatty acids and alkanes. Essential oils of S1 and S2 had better inhibition activities towards Bacillus subtilis and Staphyloccus aureus, compared with essential oil of S3. Escherichia coli was not sensitive to any of them.

Background-Despite increasing appreciation that atherogenesis involves participation of inflammatory cells, information on mediators of communication between different constituents of atherosclerotic plaque remain incomplete. We examined the role of LOX-1, a receptor for oxidized (ox) LDL, in the expression of CD40/CD40L in cultured human coronary artery endothelial cells (HCAECs). Methods and Results-We observed that ox-LDL increased the expression of CD40 and CD40L in a concentration (10 to 80 g/mL)-and time (1 to 24 hours)-dependent manner. These effects of ox-LDL were mediated by activation of LOX-1, because pretreatment of HCAECs with a blocking antibody to LOX-1 (JTX92) prevented the expression of CD40 and CD40L in response to ox-LDL (PϽ0.01). In parallel experiments, HCAECs were incubated with the protein kinase C (PKC) inhibitor bisindolylmaleimide I, and the cells were then exposed to ox-LDL. Both LOX-1 antibody and the PKC inhibitor inhibited PKC activation in response to ox-LDL (PϽ0.01). The PKC inhibitor also blocked the effects of ox-LDL on the expression of CD40 and CD40L (PϽ0.01). In additional experiments, we found that it is the PKC␣, but not PKC␤ and PKC␥, isoform that mediated ox-LDL-induced CD40 and CD40L upregulation. Further experiments showed that upregulation of CD40 mediated induction of proinflammatory genes, because CD40 antibody markedly reduced ox-LDL-induced TNF-␣ generation and P-selectin expression, whereas nonspecific mouse IgG had no effect. Conclusions-These findings indicate that ox-LDL through its receptor LOX-1 triggers the CD40/CD40L signaling pathway that activates the inflammatory reaction in HCAECs. These observations provide novel insight into ox-LDL-mediated inflammation in atherosclerosis.

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