Aims: Endophytic fungi are the remarkable category of host-associated fungal community that invades the intercellular regions of host tissues, benefiting their host while obtaining an advantage. Fungal endophytes have lately attracted prominence as a source of active secondary metabolites. This investigation aimed to identify fungal endophytes that reside inside the leaves and stems of Aquilaria malaccensis. Methodology and results: Healthy A. malaccensis stems and leaves samples were collected. Clean leaves and stems were cut to a size of 1 cm, followed by sterilization using 75% ethanol for 1 min, 3% sodium hypochlorite solution for 1 min, and finally, rinsing with sterile water 3 times for 1 min and drying with sterile paper. The sterile samples were put onto Potato Dextrose Agar (PDA) media containing chloramphenicol for 7-14 days until the mycelium grew for morphological identification under a light microscope. Five endophytic fungi were recovered from leaves, while nine endophytic fungi were obtained from stems. Using morphological approaches, nine of the endophytes had observed to produce conidia fungi, whereas the others did not. Neopestalotiopsis sp., Aspergillus sp., Arthrinium sp., Curvularia sp., Podospora sp., Mucor sp. and Verticillium sp. were identified as nine of the fourteen endophytes. Conclusion, significance and impact of study The number of endophytic fungi discovered in different organs varies. Not all endophytic fungi that grow can create sexual phages. Six genera of endophytic fungi were identified.
Endophytes ; Thymelaeaceae

From callus cultures of Stellera chamaejasme, 17 compounds were isolated. Based on their physical and chemical data and spectroscopic analysis, they were identified as syringaresinol (1), medioresinol (2), pinoresinol (3), (1R, 2S, 5R, 6S)- 2-(4- hydroxyphenyl)-6-(3-methoxy-4-hydroxyphenyl)-3, 7-dioxabicyclo [3, 3, 0] octane (4), epipinoresinol (5), caruilignan D (6), 3-oxo-guai-4-ene-11, 12-diol (7), (-) -lariciresinol (8), tetrahydro-2-(4-hydroxy-3-methoxyphenyl)-4-[(4-hydroxyphenyl) methyl]-3-furanmethanol (9), 5'-methoxylariciresinol (10), vladinol D (11), cyclo (L-Pro-L-Val) (12), oxomatairesinol (13), (+) -guayarol (14); acutissimalignan B (15), isolariciresinol (16), and beta-sitosterol (17), respectively. Among these compounds, 12 was a cyclodipeptide, 7 was a sesquiterpene, and the others except 17 were lignans. All compounds were first isolated from callus cultures of S. chamaejasme.
Lignans ; analysis ; Thymelaeaceae ; chemistry

Mountain-agarwood plays an important role in ethnic medicine in China for its pharmaceutical value. Modern pharmacological researches demonstrated that mountain-agarwood was effective for its anti-myocardial ischemia, antibacterial, anti-inflammatory, antitumor and analgesic effects. Mountain-agarwood derives from the peeled roots, stems or twigs of Syringa pinnatifolia which belongs to Syringa genus. It often depends on the purple substance and fragrance to estimate the formation of mountain-agarwood. However, the mechanism of mountain-agarwood formation has not been reported. To observe the microcosmic change in the process during the formation of mountain-agarwood, this study described the microscopic and histochemical characteristics of mountain-agarwood formation through histochemical staining. Our results showed that a significant difference of the distribution of tyloses existed during mountain-agarwood formation. It was observed that inchoate mountain-agarwood had more starch granules and viable cells than mountain-agarwood formed with high level or low level. The amount of polysaccharide and degree of lignification were increased during the mountain-agarwood formation. The results indicated that the mountain-agarwood, which meets the quality requirements for pharmaceutical use, contained the following characteristics: a large amount of purple tyloses in heartwood; yellow-brown tyloses distributing in heartwood and sapwood which were less in the latter; lignification with high level; a few viable cells; lots of polysaccharide and few starch granules in xylem rays cell. This study is aimed to reveal the change of histochemical characteristics during mountain-agarwood formation, and lay the foundation for exploring the mechanism of mountain-agarwood formation.
China ; Humans ; Myocardial Ischemia ; Syringa ; Thymelaeaceae

The present study is to investigate the chemical constituents and anti-inflammation of agarwood produced via whole-tree agarwood-inducing technique( Agar-Wit) from Aquilaria sinensis by column chromatographic technique and semi-preparation HPLC.Eleven sesquiterpenes were isolated from the agarwood produced by Agar-Wit,and their structures were identified on the basis of physiochemical characteristics and spectroscopic data analysis as baimuxinol( 1),5α,7α( H)-eudesm-11( 13)-en-4α-ol( 2),( 7 S,9 S,10 S)-( +)-9-hydroxy-selina-4,11-dien-14-al( 3),petafolia A( 4),7( 11)-eremophilen-8-one( 5),neopetasane( 6),petafolia B( 7),11-hydroxy-valenc-1( 10)-en-2-one( 8),( 4αβ,7β,8αβ)-3,4,4α,5,6,7,8,8α-octahydro-7-[1-( hydroxymethyl) ethenyl]-4α-methylnaphthalene-1-carboxaldehyde( 9),12-hydroxy-4( 5),11( 13)-eudesmadien-15-al( 10),and( 4 R,5 R,7 S,9 S,10 S)-(-)-eudesma-11( 13)-en-4,9-diol( 11). Among them,compound 1 was a new natural product,and this is the first time to report its13 CNMR spectroscopic data. Compounds 4,9 and 10 were reported from Aquilaria for the first time,and all the compounds are firstly isolated by Agar-Wit from A. sinensis. The anti-inflammatory activity of RAW264. 7 cells with lipopolysaccharide-induced was evaluated.As a result,1,4 and 9 showed potential anti-inflammatory activities with IC50 values( 2. 5±0. 35),( 3. 2±0. 2),( 4. 3±0. 56) μmol·L^-1,respectively. This work provided scientific foundation for quality evaluation of the agarwood produced by Agar-Wit.
Anti-Inflammatory Agents ; Lipopolysaccharides ; Sesquiterpenes ; Thymelaeaceae ; Trees

To investigate the mechanism of agarwood formation in Aquilaria sinensis induced by Lasiodiplodia theobromae, the fermentation liquor of L. theobromae was analyzed qualitatively and quantitatively by gas chromatography-mass spectrometry (GC-MS). JAs were detected in the fermentation liquor. The effect of the fermentation liquor on the abundance of sesquiterpenes in the callus of A. sinensis was analyzed by solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). And the fermentation liquor stimulated alpha-guaiene, alpha-humulene and delta-guaiene biosynthesis in calli. It was inferred that L. theobromae produced JAs, which resulted in a significant increase of sesquiterpenes in A. sinensis.
Ascomycota ; physiology ; Fermentation ; Sesquiterpenes ; metabolism ; Thymelaeaceae ; metabolism ; microbiology

Aims: Polymyxins are an important last-line treatment for infections caused by multidrug-resistant Gram-negative bacteria. Nonetheless, the emergence of polymyxin-resistance and the limiting of polymyxin monotherapy urgently demands its optimisation. Aquilaria malaccensis (Agarwood) has been widely used as traditional medicine. Many parts of the plant including leaves exhibit a considerable in vitro antibacterial activity against microbial pathogens. Exploiting A. malaccensis in combination with polymyxins provides a novel strategy in fighting antimicrobial resistance. The objective of this study was to evaluate the combination effects of A. malaccensis extract with polymyxins against Acinetobacter baumannii and Klebsiella pneumoniae. Methodology and results: In vitro time-kill studies and GC-MS analysis were performed to evaluate the bacterial killing of polymyxin B and extract combination and analyse chemical compounds of the extract, respectively. The combination of polymyxin B (1 mg/L) and A. malaccensis extract (32 mg/mL and 64 mg/mL) treatments exhibited enhanced bacterial killing compared to polymyxin B alone at 4 h and 24 h. Combination treatments also inhibited the bacterial growth of both A. baumannii and K. pneumoniae observed throughout the 24 h. More than sixty compounds including phytol, 9,12-octadecadienal, fatty acid, alkanes and terpenoids were putatively identified as the compounds that likely contributed to the antibacterial activity. Conclusion, significance and impact of study This study was the first to report the potential application of A. malaccensis extract in combination with polymyxin B in treatment against A. baumannii and K. pneumoniae and can be further investigated and optimized for the treatment of bacterial infectious diseases.
Thymelaeaceae ; Polymyxins ; Acinetobacter baumannii--immunology ; Klebsiella pneumoniae--immunology

As an important substitute for agarwood, mountain-agarwood, belonging to the family Oleaceae, comes from the root, stem and thick branch of Syringa pinnatifolia, which has a wide range of application in Inner Mongolia, China. It has good clinical efficacy in the use of cardiovascular diseases. However, the formation speed of mountain-agarwood is extremely slow, and its cultivated seedlings have low resin content. Therefore, how to speed up the formation of mountain-agarwood and increase the resin content is a hot research topic in this field. In this work, 16 S rDNA amplicon sequencing method was used to systematically analyze the bacterial communities of different samples of mountain-agarwood. Our data revealed that the samples of mountain-agarwood had more obvious species diversity than the ones of non-mountain-agarwood, especially the wild mountain-agarwood samples. By analysis of bacterial community composition and species abundance, Sphingomonas, Modestobacter and unidentified Cyanobacteria genus were three dominant bacterial genera in all samples. In addition, there are two identified genera of dominant bacteria, namely Actinoplanes and Microbacterium in both wild and cultivated mountain-agarwood, by bacterial community composition and species richness analysis. Meanwhile, Roseomonas was the dominant bacterial genus in both wild and cultivated non-mountain-agarwood samples. Our work could provides basic data for exploring the mechanism of the mountain-agarwood formation, and help to exploit resource of endophytic bacteria reasonably.
Bacteria ; genetics ; China ; DNA, Ribosomal ; Resins, Plant ; Thymelaeaceae

To explore the diversity of bacterial community structure between different layers of agarwood, Hiseq(high-throughput sequencing) was used to analyze the bacterial community structure of samples from different layers of agarwood. Our results showed that 1 150 096 optimized sequences and 9 690 OTUs were obtained from 15 samples of 5 layers of agarwood, which belonged to 28 bacterial phyla, 61 classes, 110 orders, 212 families and 384 genera. Further analysis revealed that the normal layer(NL) had the lowest bacterial species richness and the smallest number of OTUs. And the total number of OTUs of the agarwood layer(AL) and NL was zero, which was quite different.At the same time, there were significant differences in bacterial community structure and species diversity between NL and the other four layers. While there were some common dominant bacterial genera in both transition layer(TL) and NL. The similarity of bacterial distribution in 4 non-NL layers was relatively high, which had four common genera, such as Acidibacter, Bradyrhizobium, Acidothemus and Sphingomonas. While Acidibacter, Bradyrhizobium and Acidothemus were the dominant bacterial genus of DA and AL, and all of these layers contained volatile oil. In addition, the Bradyrhizobium was the most abundant in agarwood layer. Our results showed that bacterial community diversity and abundance were decreasing from DL to AL, and different layers showed significant differences in bacterial enrichment. It provided the clues to investigate how bacteria participate in the formation of agarwood.
Bacteria ; High-Throughput Nucleotide Sequencing ; Oils, Volatile ; Thymelaeaceae ; genetics

Qualitative and quantitative analysis of 2-(2-phenylethyl) chromones in sodium chloride(NaCl)-treated suspension cells of Aquilaria sinensis was conducted by UPLC-Q-Exactive-MS and UPLC-QQQ-MS/MS. Both analyses were performed on a Waters T3 column(2.1 mm×50 mm, 1.8 μm) with 0.1% formic acid aqueous solution(A)-acetonitrile(B) as mobile phases at gradient elution. MS data were collected by electrospray ionization in positive ion mode. Forty-seven phenylethylchromones was identified from NaCl-treated suspension cell samples of A. sinensis using UPLC-Q-Exactive-MS, including 22 flindersia-type 2-(2-phenylethyl) chromones and their glycosides, 10 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones and 15 mono-epoxy or diepoxy-5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones. Additionally, 25 phenylethylchromones were quantitated by UPLC-QQQ-MS/MS. Overall, the rapid and efficient qualitative and quantitative analysis of phenylethylchromones in NaCl-treated suspension cells of A. sinensis by two LC-MS techniques, provides an important reference for the yield of phenylethylchromones in Aquilariae Lignum Resinatum using in vitro culture and other biotechnologies.
Chromones ; Sodium Chloride ; Chromatography, Liquid ; Flavonoids ; Tandem Mass Spectrometry ; Thymelaeaceae

"Tangjie" leaves of cultivated Qinan agarwood were used to obtain the complete chloroplast genome using high-throughput sequencing technology. Combined with 12 chloroplast genomes of Aquilaria species downloaded from NCBI, bioinformatics method was employed to determine the chloroplast genome characteristics and phylogenetic relationships. The results showed that the chloroplast genome sequence length of cultivated Qinan agarwood "Tangjie" leaves was 174 909 bp with a GC content of 36.7%. A total of 136 genes were annotated, including 90 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Sequence repeat analysis detected 80 simple sequence repeats(SSRs) and 124 long sequence repeats, with most SSRs composed of A and T bases. Codon preference analysis revealed that AUU was the most frequently used codon, and codons with A and U endings were preferred. Comparative analysis of Aquilaria chloroplast genomes showed relative conservation of the IR region boundaries and identified five highly variable regions: trnD-trnY, trnT-trnL, trnF-ndhJ, petA-cemA, and rpl32, which could serve as potential DNA barcodes specific to the Aquilaria genus. Selection pressure analysis indicated positive selection in the rbcL, rps11, and rpl32 genes. Phylogenetic analysis revealed that cultivated Qinan agarwood "Tangjie" and Aquilaria agallocha clustered together(100% support), supporting the Chinese origin of Qinan agarwood from Aquilaria agallocha. The chloroplast genome data obtained in this study provide a foundation for studying the genetic diversity of cultivated Qinan agarwood and molecular identification of the Aquilaria genus.
Phylogeny ; Genome, Chloroplast ; Codon ; Molecular Sequence Annotation ; Thymelaeaceae/genetics*