Identifi cation of sesquiterpene synthase genes in the genome of Aquilaria sinensis and characterization of anα-humulene synthase

Sesquiterpenes are the major pharmacodynamic components of agarwood,a precious traditional Chinese medicine obtained from the resinous portions of Aquilaria sinensis trees that form in response to environmental stressors.To characterize the sesquiterpene synthases responsible for sesquiterpene production in A.sinensis,a bioinformatics analysis of the genome of A.sinensis identifi ed six new terpene synthase genes,and 16 sesquiterpene synthase genes were identifi ed as type TPS-a in a phylogenetic analysis.The expression patterns for eight of the sesquiterpene synthase genes after treatment with various hormones or hydrogen peroxide were analyzed by real-time quantitative PCR.The results suggest that 100μM methyl jasmonate,ethephon,(±)-abscisic acid or hydrogen peroxide could be eff ective short-term eff ectors to increase the expression of sesquiterpene synthase genes,while 1 mM methyl salicylate may have long-term eff ects on increasing the expression of specifi c sesquiterpene synthase genes(e.g.,As-SesTPS,AsVS,AsTPS12 and AsTPS29).The expression changes in these genes under various conditions refl ected their specifi c roles during abiotic or biotic stresses.Heterologous expression of a novel A.sinensis sesquiterpene synthase gene,AsTPS2,in Escherichia coli produced a major humulene product,so AsTPS2 is renamed AsHS1.AsHS1 is diff erent from ASS1,AsSesTPS,and AsVS,for mainly producingα-humulene.Based on the predicted space conformation of the AsHS1 model,the small ligand molecule may bind to the free amino acid by hydrogen bonding for the catalytic function of the enzyme,while the substrate farnesyl diphosphate(FPP)probably binds to the free amino acid on one side of the RxR motif.Arg450,Asp453,Asp454,Thr457,and Glu461 from the NSE/DTE motif and D307 and D311 from the DDxxD motif were found to form a polar interaction with two Mg^(2+)clusters by docking.The Mg^(2+)-bound DDxxD and NSE/DTE motifs and the free RXR motif are jointly directed into the catalytic pocket of AsHS1.Comparison of the tertiary structural models of AsHS1 with ASS1 showed that they diff ered in structures in several positions,such as surrounding the secondary catalytic pocket,which may lead to diff erences in catalytic products.Based on the results,biosynthetic pathways for specifi c sesquiterpenes such asα-humulene in A.sinensis are proposed.This study provides novel insights into the functions of the sesquiterpene synthases of A.sinensis and enriches knowledge on agarwood formation.

沉香(Aquilaria agallocha Roxb.)茎次生木质部结构的研究

利用光学显微技术和扫描电镜技术,对沉香(Aquilaria agallocha Roxb.)茎次生木质部结构进行解剖研究,探讨其构造特点。结果表明,茎材横切面为散孔材,生长轮不明显,导管分子单穿孔板,木材分子非叠生,绝大多数射线为异形Ⅲ型,管孔排列方式多为茎向复管孔或管孔团,具有内函韧皮部。

沉香(Aquilaria agallocha)次生木质部导管分子研究

对沉香(AquilariaagallochaRoxb.)次生木质部导管分子进行了观察.在沉香次生木质部导管分子中存在着许多不同的样式,分别对其进行了描述,并从导管分子个体发育与系统发育的角度进行了讨论.还发现沉香导管分子中存在着奇特的样式.

History and perspectives of induction technology for agarwood production from cultivated Aquilaria in Asia: a review

Agarwood, the resinous product of Aquilaria spp.(Thymelaeaceae), is highly valued for medicinal and fragrant purposes. Unsustainable forest harvesting contributes to the declining population in the wild, threatening their existence. As a protection effort, cultivation occurred in range countries, mainly in Asia, effectively establishing mass plantations. The success of domesticating Aquilaria relies heavily on technological advancement in agarwood induction, without which the entire thriving industry will collapse. In this paper, we describe efforts since 1929 and current progress and variation in induction as practiced in Asia. The methods include traditional practices and artificial induction via inoculum and chemical inducer, experimented and patented or otherwise. Artificial induction methods are being developed to intensify agarwood production in terms of yield and quality. While traditional methods are commonly applied by rural planters, artificial methods with the use of specific agents/chemicals are seen as more appealing for inducing agarwood, and have a higher demand from entrepreneurs and plantation owners.Several issues related to induction technology faced by stakeholders in agarwood cultivation are presented, such as safety levels, cost, yields, and quality. We conclude by highlighting remaining challenges in induction methods and their associated technologies.

Pollination and seed dispersal of Aquilaria sinensis(Lour.)Gilg(Thymelaeaceae):An economic plant species with extremely small populations in China

Pollination and seed dispersal in angiosperms have long been investigated in order to understand the coevolution of plants and animals.However,the signals from flowers and/or seeds to attract pollinators and/or seed dispersers have received comparatively little attention.In this study,the pollination biology and seed dispersal of the vulnerable agarwood plant Aquilaria sinensis(Lour.) Gilg,a traditional medicinal plant in China,was studied in its natural distribution range.The reproductive tactics of A.sinensis were studied in detail by employing various tests dealing with fruit set and also seed dispersal.Dynamic headspace extraction followed by GC-MS analysis was also performed in order to reveal the composition of floral scent.The results showed that noctuids and pyralids are the most effective pollinators of pollinator-dependent A.sinensis.The main compounds of the floral scent were(E,E)-α-Farnesene(61.9 ± 3.2%),trans-Ocimene(16.6 ± 1.2%),and Benzyl salicylate(4.6 ± 1.1%).The results obtained from seed dispersal experiments indicate that hornets are effective seed dispersers and they may play an important role in long-distance seed dispersal of A.sinensis.Based on our findings,we recommend several protection methods for this threatened agarwood plant in China.

BAIMUXIFURANIC ACID,A NEW SESQUITERPENE FROM THE VOLATILE OIL OF AQUILARIA SINENSIS(LOUR.)GILG.

A new sesquiterpenoid,named baimuxifuranic acid,has been isolatedfrom the volatile oil of Aquilaria sinensis（Lour.）Gilg.belonging to the familyThymelaceae by silica gel column chromatography.Based on spectral analysis itsstructure was deduced as（1）.Reduction of baimuxifuranic acid by LiAlH4 gave

Cloning and Characterization of δ-Guaiene Synthase Genes Encoding a Sesquiterpene Cyclase from Aquilaria microcarpa Cell Cultures

Three cDNA clones encoding δ-guaiene synthase, a sesquiterpene cyclase, were isolated from tissue cultures of Aquilaria microcarpa, and data mining analysis of the orthologous genes suggested that 10 and 9 amino acid residues of N- and C-terminal ends of the translated products of these clones remained undefined. The recombinant enzyme proteins, to which the putative missing Nand C-terminal amino acid sequences (MSSAKLGSAS and ALLRHAIEI, respectively) were ligated, exhibited the catalytic activities of sesquiterpene biosynthesis. Among these three δ-guaiene synthases, two isoforms were capable of liberating α-guaiene, δ-guaiene, β-elemene plus α-humulene as a minor product, while remaining one isoenzyme generated α-, δ-guaiene and β-elemene but not α-humulene. Although the enzyme protein solely lacking in the N-terminal 10 amino acid residues was capable of synthesizing the sesquiterpenoids, the protein without 9 amino acids at Cterminal did not exhibit the catalytic activity. These results suggest that two types of δ-guaiene synthase;α-, δ-guaiene, β-elemene-producing type, and α-, δ-guaiene, β-elemene plus α-humulene-producing type;concomitantly occur in A. microcarpa cell cultures, and several amino acid residues at C-terminal of the synthase protein are essential to exhibit the catalytic activities as the sesquiterpene cyclase.

白木香Aquilaria sinensis次生木质部导管分子观察研究

对白木香Aquilariasinensis(Lour.)Gilg次生木质部导管分子进行了观察研究.在白木香次生木质部导管分子中存在着许多不同的样式,分别对其进行了描述,并从导管分子个体发育与系统发育的角度进行了讨论.白木香导管分子中还存在着特殊的样式,对其也进行了讨论.

Sesquiterpenoids and 2-(2-Phenylethyl)chromone Derivatives from the Resinous Heartwood of Aquilaria sinensis

One novel spirolactone,aquilarisinolide(1),three new sesquiterpenoids,(2R,4S,5R,7R)-2-hydroxyeremophila-9,11-dien-8-one(2),(1R,4S,5S,7R,11R)-13-hydroxyepidaphnauran-9-en-8-one(3),and(4R,5S,7R,8S,10S,13R)-8,13-dihydroxyrotunda-1,11-dien-3-one(4),together with 13 known compounds(5-17)were isolated from the resinous heartwood of Aquilaria sinensis(Thymelaeaceae).The structures of the new compounds were elucidated based on the analysis of NMR and MS data and theoretical calculations their ECD spectra.The isolated compounds were evaluated for their protective activities against PC12 cell injury induced by corticosterone(CORT)and 1-methyl-4-phenylpyridine ion(MPP+),as well as inhibitory activities against BACE1.Compound 4,5,6-dihydroxy-2-(2-phenylethyl)chromone(5),daphnauranol B(7),6-methoxy-2-[2-(3-methyoxyphenyl)ethyl]chromone(10),isoagarotetrol(14),and 1-hydroxy-1,5-diphenylpentan-3-one(16)showed significant protective effects on CORT-induced injury in PC12 cells at a concentration of 20μM(P<0.001).Isoagarotetrol(14)showed a significant protective effect on MPP+-induced injury in PC12 cells at a concentration of 20μM(P<0.001),while compound 4 showed a moderate activity(P<0.01).The BACE1-inhibitory activities of all tested compounds were very weak with less than 30%inhibition at a concentration of 20μM.

Medical Culture about Lignum Aquilariae Resinatum and its Maritime Silk Road

Initially,Chen Xiang(沉香Lignum Aquilariae Resinatum)was exotic,but it has been playing an important role in the development of traditional Chinese culture and medicine.This article explores the reason for differences in the quality of ancient and modern Chen Xiang(沉香Lignum Aquilariae Resinatum)by analyzing the essential factors including the prosperity and decline of Maritime Silk Road,the relocation of the producing area,and the process of aroma forming.Based on the development and application of Chen Xiang(沉香Lignum Aquilariae Resinatum)in Chinese literature and art,traditional Chinese medicine,and folk life over the past 2000-odd years,this article puts contends that Chen Xiang(沉香Lignum Aquilariae Resinatum),which is endowed with the connotation of Chinese culture and health,should assume its new role as the traditional Chinese medical and cultural carrier on the New Silk Road.

Correction to:Dynamic analysis of gene expression and determination of chemicals in agar wood in Aquilaria sinensis

The article "Dynamic analysis of gene expression and determination of chemicals in agarwood in Aquilaria sinensis",written by Zeqing Wu,Wanzhen Liu,Jing Li,Liangwen Yu and Li Lin was originally published elec-tronically on the publisher’s internet portal(currently SpringerLink)on 10th May 2019 without open access.

Identification of chemical compounds in agarwood-producing species Aquilaria malaccensis and Gyrinops versteegii

Agarwood is a non-timber forest product found in tropical rain forests.It is a black and fragrant resin valued for the perfume industry and demand continues to increase.However,the Indonesian agarwood-producing species,Aquilaria malaccensis and Gyrinops versteegii do not automatically produce such quality resin.Bio-induction technology or inoculation using Fusarium solani is usually applied to these species to trigger resin production.This research aims to identify agarwood compounds formed in seedlings and trees of A.malaccensis and G.versteegii after these species were inoculated with the fungus F.solani.The chemical compounds were identified by comparing the patterns of mass spectra fragmentation in the sample and in previous studies.Five groups of agarwood compounds were identified:(1)sesquiterpen group-cisjasmone and aromadendrenepoxide;(2)chromones group-8-methoxy-2-(2-phenylethyl)chromen-4-one and newly-discovered chromone derivative,7-(benzyloxy)-5-hydroxy-2-methylchromone found only in G.versteegii;(3)aromatic group-benzylacetone,guaiacol,p-ethylguaiacol,phenol,syringaldehyde,vanilin,furfuryl alcohol,and furfural;(4)fatty acid group-palmitic acid,oleic acid,and lauric acid;and,(5)triterpen group-squalene.

Protective effects of Aristolochia longa and Aquilaria malaccensis against lead induced acute liver injury in rats

Objective: To investigate the protective effects of Aristolochia longa (A. longa) and Aquilaria malaccensis (A. malaccensis) on acute hepatotoxicity induced by lead in female albino rats. Methods: Twenty five (25) apparently healthy female Wistar rats were randomly divided into five groups of five rats in each: control, Pb, Pb + A. longa (Ar), Pb+ A. malaccensis (Aq), and Pb+ A. longa (Ar) + A. malaccensis (Aq) lead (100 mg/kg b.w.) as Pb (C2H3O2)2 added in their drinking water for 75 days. A. longa (rhizome powder at a dose of 10 g/kg of diet ) and A. malaccensis (heartwood powder at a dose 10 g/kg of diet) were added to the feed during the last 15 days of lead exposed in the animals. Results: Obtained results revealed that lead treatment caused a significant increase in serum GOT, GPT and ALP activities and in liver of MDA level and CAT activity. In contrast, it led to an decrease in the liver GOT, GPT and GST activities and in GSH level in rats. Also, the results clearly showed that lead causes alterations of hepatic tissue in comparison with controls. Our results showed that treatment with A. malaccensis and A. longa a partial correction of the previous parameters. The histological observations confirmed the hepatoprotection results by the biochemical parameters. Conclusions: Results demonstrated beneficial effects of A. longa and A. malaccensis treatment in Pb-induced oxidative stress and tissue damage in liver.

Effect of lyophilized aqueous leaf extract of Aquilaria subintegra on aphrodisiacproperties in mice

Objective: To investigate the effects of Aquilaria subintegra leaf aqueous extracts on the aphrodisiac properties including sexual behaviour, testosterone level, percentage of pregnancy, number of offspring and male to female ratio of offspring in ICR mice. Methods: In this experiment, each male cohabitated with one female in a polysulfone cage. 30 ICR male mice were divided into 6 groups that received normal saline (the control group), 50 mg/kg, 100 mg/kg, 200 mg/kg, 500 mg/kg, and 1000 mg/kg body weight of Aquilaria subintegra leaf aqueous extracts orally for 21 days consecutively. Sexual behavior, percentage of pregnancy, number of offspring and male to female ratio of offspring in ICR mice were measured according to the established methods. Testosterone level was measured by using enzyme-linked immunosorbent assay. Results: Mice that received Aquilaria subintegra leaf aqueous extracts at 50 mg/kg body weight (day 0) had significantly higher mount frequency as compared to the control group;groups treated with 100, 500, 1000 mg/kg body weight extracts produced a greater number of offsprings when compared to the control group. All aphrodisiac parameters were similar between the treatment groups and the control group, indicating that Aquilaria subintegra leaf aqueous extract did not significantly alter the aphrodisiac parameters. Conclusions: Aquilaria subintegra leaf aqueous extracts have no effect on the aphrodisiac properties, but could increase the breeding rate in mice.

Bioassay-guided isolation of cytotoxic constituents from the flowers of Aquilaria sinensis

Bioassay-guided fractionation of the EtOH extract from the flowers of Aquilaria sinensis(Lour.)Spreng.(Thyme-laeaceae)led to the isolation of a new cucurbitane-type triterpenoid,aquilarolide A(1),along with five known compounds(2-6).The structure of 1 was elucidated by extensive 1D and 2D nuclear magnetic resonance(NMR)experiments and mass spectrometry(MS)data and theoretical calculations of its electronic circular dichroism(ECD)spectra.Aquilarolide A,cucurbitacin E(3),cucurbitacin B(4),and 7-hydroxy-6-methoxy-2-[2-(4-methoxyphenyl)ethyl]-4H-1-benzopyran-4-one(6)showed significant cytotoxicity against human lung adenocarcinoma SPC-A-1,human lung squamous cell carcinoma NCI-H520,human lung adenocarcinoma A549,and paclitaxel-resistant A549(A549/Taxol)cell lines.All four active compounds,with IC_(50) values ranging from 0.002 to 0.91μM,had better inhibitory activities against A549/Taxol cells than paclitaxel(IC_(50)=1.80μM).Among them,cucurbitacin E(IC_(50)=0.002μM)is the most active.Further studies are needed to evaluate their in vivo antitumor activities and to clarify their mechanisms.

Five new 2-(2-phenylethyl)chromone derivatives and three new sesquiterpenoids from the heartwood of Aquilaria sinensis, an aromatic medicine in China

Five new 2-(2-phenylethyl)chromone derivatives,(5S,6R,7R,8S,7ʹR)-7ʹ-hydroxyagarotetrol(1),(5S,6R,7R,8S,7ʹS)-7ʹ-hydroxyagarotetrol(2),(6S,7S,8R)-2-[2-(4-methoxyphenyl)ethyl]-6,7,8-trihydroxy-5,6,7,8-tetrahydrochromone(3),(6S,7S,8R)-2-(2-phenylethyl)-6,7,8-trihydroxy-5,6,7,8-tetrahydrochromone(4),(5S,6R,7S,8R)-2-(2-phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydro-8-[2-(2-phenylethyl)-7-methoxychromonyl-6-oxy]chromone(5),three new sesquiterpenoids,(4S,5S,7S,8S,10S,13R)-7,8,13-trihydroxyrotunda-1,11-dien-3-one(6),(4S,5S,7S,8S,10S,13S)-7,8,13-trihydroxyrotunda-1,11-dien-3-one(7),and(4R,5S,7S,8S,10S,13S)-7,8,13-trihydroxyrotunda-1,11-dien-3-one(8),along with 14 known compounds were isolated from the resinous heartwood of Aquilaria sinensis(Thymelaeaceae).The chemical structures of these new compounds were elucidated by 1D and 2D NMR and MS data,single-crystal X-ray diffraction analysis,and electronic circular dichroism(ECD)calculations.The neuroprotective activities of these isolates were evaluated using an in vitro model of rat adrenal pheochromocytoma(PC12)cell injury induced by corticosterone.At concentrations from 5 to 40μM,compounds 4 and 6,agarotetrol(9),and 6-hydroxy-2-(2-phenylethyl)chromone(17)showed significant protective activities against corticosterone-induced PC12 cell injury(P<0.001).

Development of Pharmacological Effects of Aquilariae Lignum Resinatum

Aquilariae Lignum Resinatum is a common medicine for regulating qi.As an ancient medicinal material,Aquilariae Lignum Resinatum has been widely used in traditional Chinese medicine.Therefore,this paper introduced the research and progress of pharmacological action of Aquilariae Lignum Resinatum in detail,which lays a foundation for further research and development of Aquilariae Lignum Resinatum.

Morphology and multigene phylogeny revealed Peroneutypa aquilariae sp.nov.(Diatrypaceae,Xylariales)from Aquilaria sinensis in Yunnan Province,China

Dead twigs of Aquilaria sinensis(Thymelaeaceae)with fungal fruiting bodies were collected from Xishuangbanna,Yunnan Province,China.After initial morphological observations,an interesting fungus whose morphologically resembled Peroneutypa was isolated.Molecular phylogeny of combined ITS and tub2 showed our fungal collection is phylogenetically closely related to P.mackenziei.However,in morphology,our fungal collection is distinct from P.mackenziei in having an ostiolar canal without periphyses and the absence of paraphyses.Based on unique morphological characteristics and multigene phylogenetic analyses results,our fungal isolate is described in this paper as Peroneutypa aquilariae sp.nov.In addition,this is the first report of the genus Peroneutypa from the host A.sinensis.Full description,illustrations,and a phylogenetic tree to show the placement of the new species are provided.A synoptic table of morphological characteristics in Peroneutypa reported worldwide is also provided.

Rapid in vitro propagation of medicinally important Aquilaria agallocha

Aquilaria agallocha can produce fragrant agarwood used for incense, traditional medicine and other products. An efficient plant regeneration system was established via organogenesis from shoots developed from seedlings of Aquilaria agal-locha. Shoots generated many buds on MS medium supplemented with 1.3 μmol/L BA (6-benzylaminopurine) in the first 7 weeks, and the buds elongated on MS medium with 1.3 μmol/L BA+0.5 μmol/L NAA (naphthaleneacetic acid) in another 7 weeks, 2.3 shoots 2 cm in length per explant were obtained within 14 weeks. Plantlets were rooted on 1/2 MS medium after being immersed in 5 μmol/L NAA for 48 h, 96.7% of the roots grew up two weeks later. All plantlets that survived acclimatization grew well in the pots.

Assessing the kinetic model of hydro-distillation and chemical composition ofAquilaria malaccensis leaves essential oil

This study aimed to model the kinetic of hydro-distillation of Aquilaria malaccensis leaves oil in order to understand and optimize the extraction process. In addition, this study, for the first time, aimed to identify the chemical compositions of the A. malaccensis leave-oil. By assessing both first-order kinetic model and the model of simultaneous washing and diffusion, the result indicated that the model of simultaneous washing and diffusion better describes the hydro-distillation mechanism of the essential oil from A. malaccensis leaves.The optimum time, solid to liquid ratio, and the heating power for extracting the highest amount of essential oil were found to be around 3 h, 1:10(g·ml-1), and 300 W respectively. Yellow essential oil with a strong smell and a yield of 0.05 v/w was extracted by hydro-distillation Clevenger apparatus. Chemical compounds of the essential oil were analyzed using gas chromatography–mass spectroscopy(GC/MS), which resulted in identification of 42 compounds that constitute 93% of essential oil. Among the identified components,Pentadecanal(32.082%), 9-Octadecenal,(Z)(15.894%), and Tetradecanal(6.927%) were the major compounds.Considering the fact that all the identified major components possess pesticidal properties, A. malaccensis leaves can be regarded as a promising natural source for producing pesticides.