Tandem mass spectrometry (MS/MS) molecular networking guided profiling of small molecules from Aquilaria sinensis (Lour.) Gilg leaves and their bioactivity evaluation.

INTRODUCTION: Agarwood, a fragrant resinous wood mainly formed by Aquilaria spp., is used worldwide as a natural fragrance and traditional medicine. A large amount of Aquilaria sinensis (Lour.) Gilg leaves are underutilised during the process of the agarwood industry, and the development of A. sinensis leaves as tea has recently attracted more and more attention. However, the small molecule profile of A. sinensis leaves and their bioactivities has been rarely reported. OBJECTIVE: To conduct a rapid untargeted liquid chromatography-mass spectrometry (LC-MS) analysis of A. sinensis leaves with a molecular networking (MN) strategy and evaluate its antioxidant and antidiabetic value. METHOD: A MN-assisted tandem mass spectrometry (MS/MS) analysis strategy was used to investigate the small molecule profile of A. sinensis leaves. Additionally, the integration of antioxidant and α-glucosidase inhibitory assays with MN analysis was executed to expeditiously characterise the bioactive compounds for potential prospective application. RESULTS: Five main chemical groups including phenol C-glycosides, organic acids, 2-(2-phenylethyl) chromones, benzophenone O-glycosides and flavonoids were rapidly revealed from the A. sinensis leaves. Eighty-one compounds were provisionally identified by comparing their MS/MS fragments with canonical pathways. The featured xanthone C-glycosides and benzophenone C-glycosides were recognised as the primary components of A. sinensis leaves. Several dimers and a trimer of mangiferin were reported firstly in A. sinensis leaves. Furthermore, 17 and 14 potential bioactive molecules were rapidly annotated from antioxidant and α-glucosidase inhibitory fraction, respectively. CONCLUSION: Our findings will help expand the utilisation of A. sinensis leaves and thus promote the high-quality development of agarwood industry.

Quality Similarity between Induced Agarwood by Fungus and Wild Agarwood.

To prevent the exploitation of wild agarwood, the development of artificial agarwood through fungal inoculation is a promising method, but finding species that produce efficient high-quality agarwood remains difficult. In this study, a fungal inducer was prepared using wild agarwood containing fungi and high-throughput sequencing was performed to determine its species makeup. Subsequently, it was used to inoculate Aquilaria sinensis(Lour.) Spreng. The induced agarwood (IA), wild agarwood (WA), and nonresinous whitewood (WW) were analyzed for the extract content. In addition, liquid and gas chromatography-mass spectrometry was used to determine the chemical composition of the samples. The results were used to evaluate the quality of the IA. Mortierella humilisLinnem. ex W.Gams, Oidiodendron maius(Barron), and Tolypocladium album(W. Gams) Quandt, Kepler, and Spatafora were the fungal inducers that were discovered to produce agarwood. The extracts from the IA and WA contained 64 and 69 2-(2-phenylethyl)chromones, respectively, while there were none in the WW. Furthermore, 20 (relative content 36.19%) and 27 (relative content 54.92%) sesquiterpenes were identified in the essential oils of the IA and WA, respectively, and none were identified in the WW. The fungal inducer that was prepared from the WA effectively improves the quality of the agarwood, which is extremely similar to that of the WA.

Temporal characteristics of agarwood formation in Aquilaria sinensis after applying whole-tree agarwood-inducing technique.

Objective: Agarwood-a resinous wood produced by Aquilaria plants in response to injury or artificial induction-is a valuable medicinal and fragrance resource. Whole-Tree Agarwood-Inducing Technique (Agar-WIT) has been widely used to produce agarwood. However, the time-dependent characteristics of agarwood formation induced by Agar-WIT are yet to be clarified. To promote technologically efficient utilization and upgradation of Agar-WIT, the dynamic process and mechanism of agarwood formation were analyzed for one year. Methods: Agarwood formation percentage, barrier layer microscopic properties, extract levels, compound level, and characteristic chromatograms of agarwood were examined by referring to the Chinese Pharmacopeia (2020 version). Results: Agar-WIT could maintain a high percentage of agarwood formation over one year compared with that of healthy plants. Alcohol-soluble extract and agarotetrol levels showed fluctuating cyclic changes with peaks occurring first during the fifth and sixth months, and subsequently in the 11th month. Aquilaria trees subjected to Agar-WIT treatment for 1-12 months showed significant characteristics of a dynamic agarwood formation process. The barrier layer began to appear in the fourth month after treatment. Alcohol-soluble extractive levels in agarwood formed in the second month, and thereafter, exceeded 10.0%, and agarotetrol in agarwood produced after four months or later, exceeded 0.10%. Conclusion: According to the Chinese Pharmacopoeia, alcohol-soluble extractive levels in agarwood should not be less than 10.0% and agarotetrol level should exceed 0.10%. After four months of Agar-WIT treatment, the formed agarwood theoretically met these standards and was suitable for developed and utilization. However, the optimal harvest time was found to be the 11th month, followed by the sixth month after Agar-WIT treatment. Therefore, Agar-WIT resulted in swift agarwood formation and stable accumulation of alcohol-soluble extracts and agarotetrol. Thus, this method is efficient for large-scale cultivation of Aquilaria sinensis to produce agarwood and provide raw materials for the agarwood medicinal industry.

An integrated approach utilizing raman spectroscopy and chemometrics for authentication and detection of adulteration of agarwood essential oils.

Agarwood is a precious aromatic plant which has good pharmacological effects such as antidepressant and sedation. It also has good ornamental and collection value. However, due to it is long and complex production process, the output of agarwood essential oils (AEOs) is scarce, so the price is expensive, the quality is uneven, and the adulteration events is endless. From the commercial and pharmaceutical point of view, the authenticity and quality of the commercial products labeled as AEOs is very important. This paper tested the applicability of Raman spectroscopy combined with chemometrics in classification and authenticity identification of AEOs. In this study, Raman spectroscopy and principal component analysis (PCA) combined with partial least square discriminant analysis (PLS-DA) were used to comprehensively evaluate AEOs from different geographical origins and/or extracted by different methods which showed different characteristic bands. The characteristic component of AEOs, chromone derivatives, and two commonly used adulterants were also detected. These characteristic bands provide spectrum information of AEO samples and reference materials, which can be used as Raman spectral markers for the qualitative identification of AEOs. This study can provide a novel, fast and convenient method for identification of AEOs.

The Combination of Aquilaria sinensis (Lour.) Gilg and Aucklandia costus Falc. Volatile Oils Exerts Antidepressant Effects in a CUMS-Induced Rat Model by Regulating the HPA Axis and Levels of Neurotransmitters.

The Aquilaria sinensis (Lour.) Gilg (CX)-Aucklandia costus Falc. (MX) herbal pair is frequently used in traditional Chinese medicine prescriptions for treating depression. The volatile oil from CX and MX has been shown to have good pharmacological activities on the central nervous system, but its curative effect and mechanism in the treatment of depression are unclear. Therefore, the antidepressant effect of the volatile oil from CX-MX (CMVO) was studied in chronic unpredictable mild stress (CUMS) rats. The suppressive effects of CMVO (25, 50, 100 µL/kg) against CUMS-induced depression-like behavior were evaluated using the forced swimming test (FST), open field test (OFT) and sucrose preference test (SPT). The results showed that CMVO exhibited an antidepressant effect, reversed the decreased sugar preference in the SPT and prolongation of immobility time in the FST induced by CUMS, increased the average speed, time to enter the central area, total moving distance, and enhanced the willingness of rats to explore the environment in the OFT. Inhalational administration of CMVO decreased levels of adrenocorticotropic hormone and corticosterone in serum and the expression of corticotropin-releasing hormone mRNA in the hypothalamus, which indicated regulation of over-activation of the hypothalamic-pituitary-adrenal (HPA) axis. In addition, CMVO restored levels of 5-hydroxytryptamine (5-HT), dopamine, norepinephrine and acetylcholine in the hippocampus. The RT-PCR and immunohistochemistry results showed that CMVO up-regulated the expression of 5-HT1A mRNA. This study demonstrated the antidepressant effect of CMVO in CUMS rats, which was possibly mediated via modulation of monoamine and cholinergic neurotransmitters and regulation of the HPA axis.

Study on sesquiterpenoids from Aquilaria sinensis / 中草药

Objective: To investigate the sesquiterpenoid constituents from Aquilaria sinensis. Methods: The chemical constituents were isolated and purified by various separation techniques such as silica gel, ODS, Sephadex LH-20, and preparative high- performance liquid chromatography, and their structures were determined according to their physicochemical properties, MS, 1D, and 2D NMR. The antibacterial activity of the obtained compounds against methicillin-resistant Staphylococcus aureus was tested by 96-well plate microdilution method. Results: Seven sesquiterpenoids were obtained from 95% ethanol aqueous extract of Aquilaria sinensis and their structures were identified as (+)-4a,5-dimethyl-3-(prop-1-en-2yl)-octahydronaphthalene-2β,8a-diol (1), baimuxinic acid (2), baimuxinol (3), vetaspira-2(11),6-dien-14-al (4), baimuxinal (5), (-)-10-epi-γ-eudesmol (6), and 9β-hydroxyl-α-agarofuran (7). The minimum inhibitory concentration (MIC) of compound 1 against methicillin-resistant Staphylococcus aureus was 210 μmol/L. Conclusion: Compound 1 is a new compound named as 2β,8aα-dihydroxy-11-en-eremophilane, which has a good inhibitory effect against methicillin-resistant Staphylococcus aureus.

LC-MS-guided isolation of anti-inflammatory 2-(2-phenylethyl)chromone dimers from Chinese agarwood (Aquilaria sinensis).

Fifteen previously undescribed 2-(2-phenylethyl)chromone dimers, along with two known analogues were isolated from Chinese agarwood (Aquilaria sinensis) by a LC-MS-guided fractionation procedure. Their structures were elucidated on the basis of spectroscopic and spectrometric data (1D and 2D NMR, IR, and HRESIMS). The isolated compounds exhibited significant inhibition of nitric oxide production in lipopolysaccharide-stimulated RAW264.7 cells with IC50 values in the range 0.6-37.1 µM.

Analysis of Fungi Diversity in Agarwood Wood from Hainan Province and Guangdong Province / 中国药学杂志

OBJECTIVE: To characterize the fungal communities in agarwood wood from Hainan province and Guangdong province. METHODS: Fungal ITS genes in the agarwood wood were analyzed by high-throughput pyrosequencing using Illumina HiSeq system and bioinformatically. RESULTS: The fungal microbiomes exhibited much similarity among the samples from different regions in each province. And there were notable differences between Guangdong province and Hainan province. Moreover, the fungal communities in the agarwood wood from Hainan Province showed higher diversity and richness than that in the agarwood wood from Guangdong province. At the phylum level, Ascomycota most dominated in the agarwood wood, followed by Basidiomycota. Ascomycota dominated in the agarwood wood from Haikou, Wanning, Ledong, Danzhou and Dongguan regions. Basidiomycota dominated in the agarwood wood from Huazhou region. At the class level, agaricomycetes dominated in the agarwood wood from Huazhou region. Sordariomycetes dominated in the agarwood wood from Dondguan and Danzhou regions, Dothideomycetes dominated in the agarwood wood from Haikou, Ledong and Wanning regions. At the order level, Pleosporales dominated in the agarwood wood from Haikou, Ledong, Wanning and Dondguan regions. Trechisporales and Polyporales dominated in the agarwood wood from Huazhou and Danzhou regions, respectively. At the genus level, Lignosphaeria dominated in the agarwood wood from Haikou and Wanning regions, Perenniporia and Pyrigemmula dominated in the agarwood wood from Ledong and Danzhou regions, respectively. Phaeoacremonium dominated in the agarwood wood from Huazhou and Dondguan regions. Agarwood wood from different regions contained different dominate fungal populations. CONCLUSION: The fungi in the agarwood wood from Hainan province has higher diversity and richness than that in the agawood wood from Guangdong province. Agarwood wood from different regions has distinct fungal communities.

Molecular Cloning and Expression Analysis of the Transcription Factor AsWRKY25 in Aquilaria sinensis (Lour.) Gilg / 中国药学杂志

OBJECTIVE: To clone AsWRKY25, a transcription factor gene of Aquilaria sinensis, for bioinformatic analysis and tissue expression analysis, and express its protein in prokaryotic cells, thus to lay a foundation for further study of the biological function of AsWRKY25. METHODS: With the cDNA isolated from A. sinensis callus as template, the full-length coding sequence (CDS) of AsWRKY25 was amplified using PCR method. The recombinant vector pET-28a-AsWRKY25 was transformed into Escherichia coli BL21 (DE3) for prokaryotic expression. The physiochemical properties and bioinformatic characters of AsWRKY25 were calculated by a series of bioinformatics tools and softwares. The expression patterns in different tissues were detected by RT-PCR. RESULTS: The full-length coding sequence of AsWRKY25 transcription factor was cloned from the callus of A. sinensis. The CDS of AsWRKY25 was 1 728 bp in length and contained a 1 728 bp open reading frame (ORF) encoding 575 amino acids. The CDS sequence was codon-optimized and then ligated into the pET-28a expression vector. The optimal induction condition of recombinant pET-28a-AsWRKY25 was 1 mmol•L-1 IPTG at 37℃ for 6 h in E. coli BL21 (DE3). The result of tissue expression analysis showed that AsWRKY25 had the highest expression level in the agarwood layer and the lowest level in roots, stems and branches. CONCLUSION: The WRKY transcription factor AsWRKY25 is successfully cloned from A. sinensis and expressed in E. coil. It is speculated that AsWRKY25 may be involved in the wound-induced agarwood-formation process in A. sinensis.

Differential expression of transcription factors in three generations of excised roots of Aquilaria sinensis under salt stress / 中草药

Objective: To study the expression patterns and levels of transcription factors (TFs) in three generations of excised roots of Aquilaria sinensis under salt stress, and analyze the variation of TFs family genes in each generation in response to salt stress. Methods The excised roots of A. sinensis were used as experimental material, using highthroughput sequencing technology (Illumina Hiseq4000), all the unigene sequences were compared with the plant transcription factor database (PlantTFDB), and the three generation differential expressed TFs between the treated and the control group were analyzed. Results:A total of 48 286 Unigenes were obtained by de novo splicing from three generation of excised roots of A. sinensis, containing 1 156 potential TFs distributed in 54 TF families. Among them, bHLH, ERF, and NAC were the three most enriched families. Totally, 290, 277, and 349 differentially expressed TFs were respectively screened in three successive generations, which were mainly down-regulated. The expression induced by salt stress were different in each TF family, the numbers of up-regulated DEGs increased in NAC, MYB, and WRKY families, and decreased in GRAS family with the increase of stressed generations. There were 70 common TFs differentially expressed in three generations, and the down-regulated expression multiples of eight genes increased with the increase of salt stress generations. Conclusion:The effect of salt stress on the expression of TFs was mainly down-regulatied. The number of differential expressed TFs in the treated and control group increased with the increase of salt stress generations. The expression of different TF family genes was different under salt stress, and some genes might be involved in the transmission of stressful memory. This study is helpful to understand the expression characteristics of TFs at the whole level and provide a reference for further study on the stressful memories and the molecular mechanism of the salt stress response.

Molecular cloning, prokaryotic expression and characterization of a transcription factor AsWRKY62 in Aquilaria sinensis / 中草药

Objective: The molecular cloning and prokaryotic expression of the transcription factor AsWRKY62 of Aquilaria sinensis were carried out, at the same time, the bioinformatics analysis and expression pattern analysis were also performed. The purpose of this study was to lay a foundation for further study on the role of AsWRKY62 in the growth and development of A. sinensis and the formation of agarwood. Methods: With the cDNA isolated from A. sinensis callus as template, the full-length coding sequence (CDS) of AsWRKY62 was amplified using RT-PCR and PCR method. The recombinant vector pET-21a-AsWRKY62 which was built and verified by gene recombination technique was transformed into E. coli BL21 (DE3) for prokaryotic expression and purification. The characteristics of physiochemical properties, conserved domains and subcellular localization of AsWRKY62 were calculated by a series of bioinformatics tools. The analyses of multiple sequence alignment of amino acid and phylogenetic tree were performed using DNAMAN and MEGA 5.0, respectively. The gene expression pattern in different tissues was detected by RNA-seq data. Results: The full length CDS of AsWRKY62 (GenBank accession MH925301) was 1581 bp, encoding a 526-aa protein which belongs to WRKY group I. The optimized induction conditions of recombinant pET-21a-AsWRKY62 were 0.5 mmol/L IPTG at 37 ℃ for 4 h. According to the tissue-specific expression pattern analysis, the AsWRKY62 gene in A. sinensis is mainly expressed in roots and stems, followed by agarwood and flowers. Conclusion: Cloning, expression and characterization of the AsWRKY62 gene for the first time indicated that it may be related to the formation of agarwood, which provided a theoretical basis for further study of its biological function.

Full-length cDNA cloning of AsJAZ1 gene and damage-induced expression analysis in Aquilaria sinensis callus / 中草药

Objective: To clone the full-length cDNA of jasmonate-zim-domain protein (JAZ) gene in Aquilaria sinensis to provide the basic information for further study on gene function in sesquiterpenes biosynthesis pathway. Methods: With the total RNA as template, the full-length cDNA of JAZ in A. sinensis was cloned through rapid amplification of cDNA ends (RACE) technique and reverse transcription PCR (qRT-PCR) method. The bioinformatics of the JAZ gene was analyzed as well. The expression of this gene was detected by qRT-PCR method with MeJA and mechanical wounding treatment in A. sinensis callus. Results: The full-length cDNA (1 507 bp) of JAZ gene was named AsJAZ1; GenBank registration number was KP677281. AsJAZ1 was obtained with an open reading frame (ORF) of 990 bp and encoding 330 amino acids. The relative molecular mass of AsJAZ1 calculated was 34 280, and the isoelecric point was 6.89. Real time PCR results indicated that both MeJA treatment and mechanical wounding could stimulate the increase of mRNA expression of AsJAZ1; There was a sharp rise at 0.5 h with about 27 times higher than the control (without MeJA treatment) with MeJA treatment, then dropped significantly. In mechanical wounding treatment, the highest peak presented in 2 h about 17 times compared to the control, then dropped significantly too. The expression of AsJAZ1 gene returned to be normal in 24 h. Conclusion: We have obtained the full-length cDNA sequence of AsJAZ1 gene firstly, which was extremely sensitive to wounding and responded to the early damage.

GC-MS Study of the Chemical Components of Different Aquilaria sinensis (Lour.) Gilgorgans and Agarwood from Different Asian Countries.

As a traditional medicinal herb and valuable natural spice in China, Aquilaria sinensis (Lour.) Gilg has many significant pharmacological effects. Agarwood is the resinous heartwood acquired from wounded A. sinensis trees, and is widely used in pharmaceuticals owing to its excellent medicinal value. In this study, the chemical composition of volatile components and alcohol extracts from different organs of A. sinensis and agarwoods grown in different regions were investigated using GC-MS. The results showed that Vietnam agarwood had the highest moisture content, which was attributed to the local climate, while the fruit and bark of A. sinensis had higher moisture contents than the other organs. The volatile components of A. sinensis organs included 3-ethyl-5-(2-ethylbutyl)-octadecane, oleic acid 3-(octadecyloxy) propyl ester, and docosanoic acid 1,2,3-propanetriyl ester, while the alcohol extracts of A. sinensis organs contained benzoic acid ethyl ester, hexadecanoic acid ethyl ester, oleic acid, and n-hexadecanoic acid. Furthermore, the main active ingredients in agarwood from different habitats were sesquiterpenoids, aromatic species, and chromone compounds. The role of chromone compound 2-phenylethyl-benzopyran as an elicitor and the mechanism of agarwood formation were also investigated. Antioxidant tests showed that essential oils from agarwood and A. sinensis had antioxidant capacities by comparison with butylated hydroxytoluene and vitamin E. An antibacterial activity test showed that the inhibition effect of the essential oil was better against Gram-positive bacteria than against Gram-negative bacteria.

Prokaryotic expression of Aquilaria sinensis AsMAPK3 gene and its localization in transient expression system in onion epidermis / 中草药

Objective To construct a prokaryotic vector of AsMAPK3 gene from Aquilaria sinensis, the original plant of agarwood, and induce the recombinant proteins expression so as to study the subcellular localization of AsMAPK3. This work will prepare materials for antibody preparation and lay a foundation for screening the interaction proteins and further studying their functions. Methods Partial cDNA sequence was amplified by PCR and recombined to pET-28a vector to construct a prokaryotic expression vector pET-28a-AsMAPK3, and induced the expression of the fusion protein. The full-length cDNA of AsMAPK3 was amplified and subcloned to pAN580 vector to construct a pAN580-AsMAPK3 transient expression vector. The recombinant plasmid of pAN580-AsMAPK3 was introduced into the onion epidermis by gold particle bombardment, and GFP fluorescence was observed by luorescence microscope. Results The Escherichia coli BL21 (DE3) containing the recombinant plasmid was induced with 0.5 mmol/L isopropyl-β-D-galactoside (IPTG) at 37 ℃ for 4 h, and a fusion protein about 39 000 was obtained which was expressed in supernatant and inclusion bodies. The results of GFP fluorescence observation of transient transformed onion epidermis showed that AsMAPK3 was mainly expressed in the nucleus and plasma membrane. Conclusion The expression and purification of AsMAPK3 in vitro were successfully carried out, and the subcellular localization of AsMAPK3 gene was confirmed. This work provides a substantial foundation for follow-up function study of AsMAPK3.

Relationship between Expression of Chalcone Synthase Genes and Chromones in Artificial Agarwood induced by Formic Acid Stimulation Combined with Fusarium sp. A2 Inoculation.

Agarwood (gaharu) is a fragrant resin produced in the heartwood of resinous Gyrinops and Aquilaria species. Artificial agarwood samples were obtained from Aquilaria sinensis (Lour.) Gilg using formic acid (FA) stimulation combined with Fusarium sp. A2 inoculation. The relationship between the expression of chalcone synthase genes (CHS) and dynamic changes in chromone content was explored in resin-deposited parts of the trunks of A. sinensis. CHS gene expression levels were detected by qRT-PCR analysis. The chemical composition of agarwood obtained from the heartwood of A. sinensis before and within 1 year after induction was determined by GC-MS. After induction with FA stimulation combined with F. sp. A2 inoculation, the CHS1 gene showed relatively high expression, whereas the CHS2 gene showed low expression. The relative gene expression level of CHS1 peaked at 12 months, with a 153.1-fold increase, and the dominant period of the CHS2 gene expression was 10 months with a 14.13-fold increase. Moreover, chromones were not detected until after 2 months, and a large proportion of chromone compounds were detected after 4 months. Chromone content increased with time and peaked at 12 months. CHS1 gene expression was significantly correlated with 6-hydroxy-2-(2-phenylethyl)chromone accumulation, and CHS2 gene expression was significantly correlated with 5-hydroxy-6-methoxy-2-(2-phenylethyl)chromone accumulation. CHS gene expression was extremely sensitive to FA stimulation combined with F. sp. A2 inoculation and responded to late-onset injury. CHS genes expression also preceded the chromone accumulation. This work laid the foundation for studies on the mechanism by which genes regulate chromone biosynthesis pathways during the formation of agarwood resin in A. sinensis.

Complete Chloroplast Genome Sequence of Aquilaria sinensis (Lour.) Gilg and Evolution Analysis within the Malvales Order.

Aquilaria sinensis (Lour.) Gilg is an important medicinal woody plant producing agarwood, which is widely used in traditional Chinese medicine. High-throughput sequencing of chloroplast (cp) genomes enhanced the understanding about evolutionary relationships within plant families. In this study, we determined the complete cp genome sequences for A. sinensis. The size of the A. sinensis cp genome was 159,565 bp. This genome included a large single-copy region of 87,482 bp, a small single-copy region of 19,857 bp, and a pair of inverted repeats (IRa and IRb) of 26,113 bp each. The GC content of the genome was 37.11%. The A. sinensis cp genome encoded 113 functional genes, including 82 protein-coding genes, 27 tRNA genes, and 4 rRNA genes. Seven genes were duplicated in the protein-coding genes, whereas 11 genes were duplicated in the RNA genes. A total of 45 polymorphic simple-sequence repeat loci and 60 pairs of large repeats were identified. Most simple-sequence repeats were located in the noncoding sections of the large single-copy/small single-copy region and exhibited high A/T content. Moreover, 33 pairs of large repeat sequences were located in the protein-coding genes, whereas 27 pairs were located in the intergenic regions. Aquilaria sinensis cp genome bias ended with A/T on the basis of codon usage. The distribution of codon usage in A. sinensis cp genome was most similar to that in the Gonystylus bancanus cp genome. Comparative results of 82 protein-coding genes from 29 species of cp genomes demonstrated that A. sinensis was a sister species to G. bancanus within the Malvales order. Aquilaria sinensis cp genome presented the highest sequence similarity of >90% with the G. bancanus cp genome by using CGView Comparison Tool. This finding strongly supports the placement of A. sinensis as a sister to G. bancanus within the Malvales order. The complete A. sinensis cp genome information will be highly beneficial for further studies on this traditional medicinal plant. Moreover, the results will enhance our understanding about the evolution of cp genomes of the Malvales order, particularly with regard to the role of A. sinensis in plant systematics and evolution.

Cloning and expression analysis of two small heat shock proteins genes from Aquilaria sinensis / 中草药

Objective: To aim at cloning the open reading frame (ORF) of sHSP1 and sHSP2 genes from Aquilaria sinensis and analyzing the bioinformatics and expression of the two genes. Methods: Two unique sequences containing sHSPs domain were discovered in transcriptome dataset of A. sisnensis. The full-length cDNAs of sHSP1 and sHSP2 were cloned by RT-PCR strategy with the specific primers. Subcellular localization, transmembrane domain, three-dimensional structure, and phylogenetic analysis were predicted by different softwares to analyze the bioinformatics of sHSPs protein. The expression different levels of sHSP1 and sHSP2 isoforms in different tissues and in responds to salt and ABA, SA, MJ treatment were measured by real-time quantitative PCR. Results: The sHSP1 and sHSP2 cDNA sequence consisted of 474 bp ORF, encoding 157 amino acids. Tissue expression analysis indicated that sHSP1 and sHSP2 were primarily expressed in roots, followed by stems and leaves. Salt treatment experiments indicated that salt treatment caused a rapid increase in sHSP1and sHSP2 expression within 36 and 24 h, respectively. Exogenous ABA and MJ treatment experiments indicated that sHSP1 and sHSP2 genes were induced by exogenous ABA and MJ, and all reached the highest expression level at 12 h. Simultaneously, the SA treatment experiments indicated that exogenous SA treatment caused a rapid increase in sHSP1 and sHSP2 expression within 12 and 24 h, respectively. Conclusion: The full-length cDNA sequence of sHSP1 and sHSP2 genes from A. sinensis is obtained. sHSP1 and sHSP2 have the different expression level in different tissues. When subjected to high salt, ABA, SA, and MJ treatment, sHSP1 and sHSP2 show the different expression levels in different time. Cloning and analyzing sHSP1 and sHSP2 genes from A. sinensis will play an important role for further study on plant defense response.

A new 2-(2-phenylethyl) chromone from Chinese eaglewood / 中草药

Objective: To study the chemical constituents of Chinese eaglewood. Methods: The column chromatography on silica gel, Sephadex LH-20, and semi-preparative HPLC were used to separate and purify the compounds, their structures were elucidated by NMR and MS analyses. Results: Four compounds were isolated from the petroleum ether of Chinese eaglewood and identified as 6-methoxy-7-hydroxy-2-(2-phenylethyl) chromone (1), 6,7-dimethoxy-2-(2-phenylethyl) chromone (2), 6,7-dimethoxy-2-[2-(4'- methoxyphenyl) ethyl] chromone (3), and 6-methoxy-2-[2-(3'-methoxy-4'-hydroxyphenyl) ethyl] chromone (4). Conclusion: Compound 1 is a new compound named as Aquilarone J, and compound 4 is isolated from Chinese eaglewood for the first time.

Structural elucidation of two new megastigmane glycosides from the leaves of Aquilaria sinensis.

The present study was designed to determine the chemical constituents and identify new components of the leaves of Aquilaria sinensis (Lour.) Gilg. The compounds were isolated and purified by repeated silica gel, Sephadex LH-20, and ODS column chromatography and their structures were elucidated by NMR and HR-ESI-MS spectrometry. Eight megastigmane glycosides and two cucurbitacins were isolated and identified as (9S) megastigma-4,7-diene-2,3,9-triol 9-O-ß-D-glucopyranoside (1), (9S) megastigma-4(13),7-diene-3,6,9-triol 9-O-ß-D-glucopyranoside (2), macarangloside D (3), corchoionoside C (4), staphylionoside H (5), (+) 3-oxo-α-ionol-ß-D-glucopyranoside (6), (-) 3-oxo-α-ionol-ß-D-glucopyranoside (7), citroside B (8), 2-O-ß-D-glucopyranosyl cucurbitacin I (9), bryoamaride (10). Compounds 1 and 2 were newly identified megstigmane glucosides and reported from this genus for the first time.

Five new eudesmane-type sesquiterpenoids from Chinese agarwood induced by artificial holing.

Five new eudesmane-type sesquiterpenoids (1-5), along with six known ones (6-11), were isolated from Chinese agarwood induced by artificial holing originating from Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae). The structures of the new sesquiterpenoids were established by spectroscopic methods including UV, IR, MS, 1D, and 2D NMR. Compounds 1, 3, 6 and 7 exhibited antibacterial activities against both Staphylococcus aureus and Ralstonia solanacearum, and compound 5 only showed an inhibitory activity towards S. aureus. Compounds 1, 6, 7 and 10 showed weak acetylcholinesterase inhibitory activity.