Isolation and morphological identification of endophytic fungi from leaves and stems of Aquilaria malaccensis Lamk. originating from Java Island, Indonesia

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.

Anxiolytic and antidepressant effects of agarwood inhalation and its mechanism / 中国中药杂志

This study used m-chloropheniperazine(MCPP) and chronic unforeseeable mild stress(CUMS) to induce the rat models of anxiety and depression, respectively. The behaviors of rats were observed by the open field test(OFT), light-dark exploration test(LDE), tail suspension test(TST), and forced swimming test(FST), and the antidepressant and anxiolytic effects of agarwood essential oil(AEO), agarwood fragrant powder(AFP), and agarwood line incense(ALI) were explored. The enzyme-linked immunosorbent assay(ELISA) was used to determine the levels of 5-hydroxytryptamine(5-HT), glutamic acid(Glu), and γ-aminobutyric acid(GABA_A) in the hippocampal area. The Western blot assay was used to determine the protein expression levels of glutamate receptor 1(GluR1) and vesicular glutamate transporter type 1(VGluT1), exploring the anxiolytic and antidepressant mechanism of agarwood inhalation. The results showed that compared with the anxiety model group, the AEO, AFP, and ALI groups decreased the total distance(P<0.05), decreased the velocity of movements(P<0.05), prolonged the immobile time(P<0.05), and reduced the distance and velocity of the rat model of anxiety in the dark box(P<0.05). Compared with the depression model group, the AEO, AFP, and ALI groups increased the total distance and average velocity(P<0.05), reduced the immobile time(P<0.05), and reduced the forced swimming and tail suspension time(P<0.05). In terms of transmitter regulation, the AEO, AFP, and ALI groups decreased the level of Glu in the rat model of anxiety(P<0.05) and increased the levels of GABA_A and 5-HT(P<0.05), while the AEO, AFP, and ALI groups all increased the level of 5-HT in the rat model of depression(P<0.05) and decreased the levels of GABA_A and Glu(P<0.05). At the same time, the AEO, AFP, and ALI groups all increased the protein expression levels of GluR1 and VGluT1 in the hippocampus of the rat models of anxiety and depression(P<0.05). In conclusion, AEO, AFP, and ALI exert anxiolytic and antidepressant effects, and the mechanism might be related to the regulation of the neurotransmitter and the protein expression of GluR1 and VGluT1 in the hippocampus.

Characterization and phylogenetic analysis of complete chloroplast genome of cultivated Qinan agarwood / 中国中药杂志

"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.

Cloning, expression and functional identification of cytochrome P450 reductase gene in Aquilaria sinensis / 药学学报

Cytochrome P450 reductase (CPR) is essential for the electron transport chain of cytochrome P450s, playing an indispensable role in electron transfer in vivo. In this study, one cDNA encoding cytochrome P450 reductase (Ascpr1) was identified from the callus of Aquilaria sinensis. Ascpr1 contains an open reading frame of 2 124 bp. The deduced protein is composed of 707 amino acids, with a predicted molecular weight of 78.82 kD. Phylogenetic analysis revealed that AsCPR1 is a type Ⅱ CPR protein closely related to the CPR from Theobroma cacao. Transmembrane prediction using TMHMM 2.0 indicated that the amino acids 52-71 of AsCPR1 comprise a transmembrane region. After truncating of 67 amino acid residues from N-terminal, the truncated AsCPR1 was successfully expressed in E. coli Transetta (DE3). Further purification of the recombinant AsCPR1 by affinity chromatography and determination of the enzymatic activity allowed the reducing ability of AsCPR1 to cytochrome C in vitro. The results pave the way for further study on the synthesis of defensive chemicals involved in P450s and the functions of CPR in self-defense of A. sinensis.

Anti-asthmatic effect of agarwood alcohol extract in mice and its mechanism / 中国中药杂志

As recorded, agarwood has the function of improving qi reception and relieving asthma, but the underlying mechanism is unclear and rarely reported. Therefore, this study explored the anti-asthmatic effect of the alcohol extract of agarwood produced by the whole-tree agarwood-inducing technique(Agar-Wit) in the asthma mouse model induced by intraperitoneal injection of ovalbumin(OVA) + Al(OH)_3 combined with intranasal administration of OVA and the mechanism, and compared the anti-asthmatic effects of agarwood induced with different methods. Firstly, the anti-inflammatory and anti-asthmatic effects of Agar-Wit agarwood in mice were evaluated based on the asthma frequency, lung tissue injury, and peripheral inflammatory white blood cell(WBC) count and eosinophil count. Then, the levels of interleukin-1β(IL-1β), IL-17, and IL-10 in serum of mice were detected by enzyme-linked immunoassay(ELISA) and the expression of inflammation-and apoptosis-related genes in tissues was measured by reverse transcription polyme-rase chain reaction(RT-PCR) so as to preliminarily explore the anti-asthmatic mechanism. RESULTS:: showed that the alcohol extract of Agar-Wit agarwood significantly reduced asthma frequency, relieved pathological injury, improved peripheral WBC count and eosinophil count, decreased the levels of inflammatory cytokines IL-1β and IL-17, elevated the level of anti-inflammatory cytokine IL-10, and down-regulated the mRNA expression of IL-1 R, tumor necrosis factor receptor R(TNFR), nuclear transcription factor-kappa B(NF-κB), Bax, and caspase 3, but had no significant influence on the expression of high-mobility group box 1(HMGB1) protein, caspase 8, and Bcl-2. The effect of Agar-Wit agarwood alcohol extract was better than that of wild agarwood alcohol extract and alcohol extract of agarwood induced with the burning-chisel-drilling method at the same dose. In conclusion, Agar-Wit agarwood can significantly alleviate inflammation and asthma, which is related to its anti-inflammation and anti-apoptosis activity.

Cloning and expression analysis of chalcone isomerase from Aquilaria sinensis / 药学学报

Chalcone isomerases (CHIs) play an essential role in the biosynthesis of flavonoids important in plant self-defense. Based on the transcriptome data of Aquilaria sinensis Calli, a full-length cDNA sequence of CHI1 (termed as AsCHI1) was cloned by reverse transcription PCR. AsCHI1 contains a complete open frame (ORF) of 654 bp. The deduced protein is composed of 217 amino acids, with a predicted molecular weight of 23.11 kDa. The sequence alignment and phylogenetic analysis revealed that AsCHI1 has conserved most of the active site residues in type I CHIs, indicating a close relationship with the CHI from Gossypium hirsutum. The recombinant AsCHI1 protein was obtained by heterologous expression of AsCHI1 in E. coli BL21(DE3). The purified AsCHI1 protein exhibited CHI activity by catalyzing the production of naringenin from naringenin chalcone. Remarkably, AsCHI1 expression in A. sinensis Calli treated with various abiotic stresses including salt, mannitol, cold, and heavy metals could be markedly increased, and plant hormones such as abscisic acid (ABA), gibberellin (GA3), and salicylic acid (SA) could also increase the expression of AsCHI1, suggesting that AsCHI1 might play an important role in plant self-defense. The results expand our understanding of the biosynthesis of flavonoids in A. sinensis and give further insight into the defensive responses of A. sinensis to abiotic and biotic stresses.

Analysis of bacterial community structure and diversity during mountain-agarwood formation / 中国中药杂志

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.

Microscopic histochemical comparison during mountain-agarwood formation / 中国中药杂志

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.

Analysis of bacteria distribution characteristics in different layers of agarwood based on Hiseq sequencing / 中国中药杂志

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.

Composition analysis,antioxidative and antibacterial activities comparison of agarwood oils extracted by supercritical and steam distillation / 中国中药杂志

Agarwood is a traditional and precious medicinal material and natural spice in China and other southeast Asian countries.As the head of all spices,agarwood has many pharmacological activities such as analgesia,antidiarrheal,anti-inflammatory and antibacterial effects. Due to its high price and scarce resources,there were just a few previous studies on it,mainly focusing on the chemical compositions of the agarwood essential oil and solvent extract mixture. The components of agarwood oils obtained by supercritical extraction and steam distillation were analyzed by using Gas Chromatography-Mass Spectrometer( GC-MS),and then the agarwood oils compositions and contents were compared between the traditional extraction method and the recently emerging supercritical extraction method. Antioxidant experiments of scavenging DPPH,ABTS,hydroxyl radical,total reducing power and MIC experiments of five kinds of tester strains such as staphylococcus aureus were combined to illustrate the differences between these two kinds of agarwood oils in terms of antioxidant and bacteriostatic activities. The results showed that the main components of agarwood oil were sesquiterpenoids( 68. 68%) in steam distillation extraction method,but sesquiterpenoids( 23. 78%) and chromones( 29. 42%) in supercritical extraction method. Fourteen common components included benzyl acetone,α-santalol,γ-eudesmol,agarospirol and guaiol etc. The antioxidant activity and inhibitory MIC of agarwood oils in supercritical extraction method were better than those in steam distillation method,and the inhibitory effect of agarwood oil on the growth of bacillus subtilis was found for the first time.

Anti-inflammatory sesquiterpenes from agarwood produced via whole-tree agarwood-inducing technique of Aquilaria sinensis / 中国中药杂志

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.

One new 2-(2-phenylethyl) chromone derivative from agarwood of Aquilaria crassna in Cambodia / 中草药

Objective: To study the chemical constituents of agarwood originating from Aquilaria crassna in Cambodia. Methods: The compounds were isolated and purified by silica gel column chromatography and semi-preparetive HPLC and so on. Their structures were identified by spectroscopic data. All compounds were tested for cytotoxic activities against two human cancer cell lines by MTT method. Results: Four compounds were isolated and elucidated as 6-methoxy-8-hydroxy-2-(2-phenylethyl) chromone (1), 6-methoxy-7-hydroxy-2-[2-(4-methoxyphenyl) ethyl] chromone (2), 7β,8β-epoxy-6α-hydroxy-5α-methoxy-5,6,7,8-tetrahydro-2-[2-(4- methoxyphenyl) ethyl] chromone (3), and rel-(1aR,2R,3R,7bS)-5,6-epoxy-7,8-dihydroxy-5,6,7,8-tetrahydro-2-[2-(4-methoxyphenyl) ethyl] chromone (4). Conclusion: Compound 1 is a new compound, while compounds 2-4 are isolated from this agarwood for the first time. Among them, compound 4 showed inhibitory activities against SGC-7901 and A549 cells.

Species Identification of Natural Adhesives and Evaluation of the Performance for Incense and Combustibility of Joss Stick / 中国药学杂志

OBJECTIVE: To identify five kinds of natural fragrant adhesives on the market and evaluate their performance for making joss stick. METHODS: The common fragrance-making adhesives on the market were collected and their primitives were identified by DNA barcode technology. Five kinds of agarwood incenses (standard φ: 1.5 mm, L: 18 cm) were made by the traditional method. The performance indexes such as color, viscosity, weight, burning time, the burning rate, odor, ash weigh and so on were determined. RESULTS: Five common natural adhesives were identified to be Ulmus glabra, Litsea glutinosa (Lour.) C.B. Rob, Phoebe puwenensis Cheng, Machilus balansae (Airy Shaw) F.N. Wei and S.C. Tang, and Endlicheria sp.. Nanmu powder was better than the other adhesives in burning time, burning rate, odor, and ash weigh. Combustion experiments showed that the heavier the weight and the bigger the diameter, the longer the burning time. CONCLUSION: The five common plant adhesives are identified. Nanmu powder is the best in all the adhesives. The burning time of joss stick is proportional to its weight, length and diameter.

Quality of Agarwood Produced by Whole Tree Agarwood-inducing Technique (Agar-Wit) from China and Southeast Asian Countries / 中国药学杂志

OBJECTIVE: To investigate the similarities and differences of the quality of agarwood produced by agar-wit from China and Southeast Asian countries. METHODS: The sensory characteristic, identification item, extract, content determination, characteristic chromatogram and total chromone content of agarwood produced by agar-wit from 11 place of origins of China and Southeast Asian countries were mainly evaluated, according to the Chinese Pharmacopoeia (2015 edition). RESULTS: The morphological characteristics, TLC and characteristic chromatograms of 11 batches of agarwood produced by agar-wit were similar. Special structures such as included phloem, vessel and rays were observed in all samples. The extract and total chromone contents of agarwood produced by agar-wit from the six producing areas in China were higher than those from the five producing areas in Southeast Asia. Compared with other agarwood-inducing method, the sensory characteristics of the agarwood produced by agar-wit were relatively stable, and the average contents of the extract and total chromone were significantly higher, but the average content of agarotetrol was slightly lower than that of the agarwood produced by peripheral-inducing and iron-inducing. CONCLUSION: In terms of quality, the agar-wit is superior to the traditional agarwood-inducing method. It is not only applicable to the agarwood production of Aquilaria sinensis in China, but also applicable to the species of Aquilaria Lam. in Southeast Asian countries. The quality of agarwood produced by agar-wit is good in general, but the quality of the agarwood produced by A. sinensis is better than the species of Aquilaria Lam. in the Southeast Asian.

Analysis and Comparison of HPLC Fingerprints of Natural and Artificial Agarwood / 中国药学杂志

OBJECTIVE: To establish the HPLC fingerprint analysis method for agarwood with compare the natural agarwood and artificial agarwood combined with the contents of ethanol-soluble extraction (E%) and agarotetrol (A%). METHODS: The E% and A% were determined according to the ChP(2015). The chromatographic fingerprints of agarwood were established on a Platisil ODS C18 column (4.6 mm×250 mm, 5 μm) with mobile phase consisting of acetonitrile-0.1% formic acid. Gradient elution was performed at a flow rate of 0.7 mL•min-1, the detection wave length was set at 252 nm, and the column temperature was maintained at 31 ℃. The similarity evaluation system of traditional Chinese medicine chromatographic fingerprint (2004A) was used to establish the common pattern and calculate the similarity of chromatograms. Principal component analysis (PCA) was used with multivariate statistical analysis software for relative peak area of common chromatographic peak. RESULTS: The chromatographic fingerprint similarity of 13 batches of natural agarwood was 0.021-0.856, the E% was 10.1%-31.0%, and the A% was 0.04%-2.83%, respectively. The chromatographic fingerprint similarity of 11 batches of artificial agarwood ranged from 0.079 to 0.453, and the E% and A% were 10.3%-31.3% and 0.14%-1.02%, respectively. Thirteen batches of natural agarwood and 11 batches of artificial agarwwod were divided into two groups respectively, and the reference crude drug was gathered in the natural agarwood group. The similarity between natural and artificial agarwood chromatographic fingerprint was significantly different (P<0.05). Meanwhile, the similarity of artificial agarwood chromatographic fingerprint was positively correlated with the E% (P<0.05). CONCLUSION: The chromatographic fingerprint analysis combined with multivariate statistical analysis can easily and quickly distinguish natural and artificial agarwood, which provides a reference for the comprehensive evaluation of the quality of agarwood.

Teratogenic and Mutagenic Toxicity of Agarwood Extracts Produced by Agar-Wit / 中国药学杂志

OBJECTIVE: To research the teratogenic and mutagenic toxicity of agarwood extracts produced by agar-wit technique for functional food development (slices dose 0.60 g•d-1). METHODS: Pregnant rats of 6 to 15 d were given with agarwood extracts at 45, 105, 150 mg•kg-1 by gastric perfusion directly every day, then killed and anatomized after being pregnant for 20 d. During the whole course of treatment, the activity and body weight of pregnant rats, fetal survival rate, absorption number, malformation type and number of fetus were observed and recorded. RESULTS: The agarwood extracts had no effect on the body weight of pregnant rats at 45, 105, 150 mg•kg-1 dosages, and the numbers of implantation, live fetuses, fetuses and stillbirths of fetus, malformation type and number had no significant difference compared with the control group (P>0.05). CONCLUSION: Agarwood extracts have no teratogenic and mutagenic toxicity for rat fetuses.

Chronic Toxicity of Agarwood Extracts Produced by Agar-Wit on SD Rats / 中国药学杂志

OBJECTIVE: To assess the chronic toxicity of agarwood extracts produced by agar-wit. METHODS: Agarwood usage in Chinese Pharmacopoeia was referred. SD rats were used as the experimental animal and fed with agarwood extracts at pharmaceutical dosages (1 262.4, 631.2 and 157.8 mg•kg-1) and functional food dosages (150, 105 and 45 mg•kg-1) for 90 d. The weight and food utilization were recorded. The rats were killed on the 90th day, blood and urine were collected for routine blood, serum biochemical indicators and urine index tests, and the bodies were dissected for histological examination. RESULTS: The two groups of rats showed same results. The body weight, food utilization, routine blood, serum biochemical indicators and urine index were not significantly changed compared with the control group (P>0.05). Histological examination showed that the male and female rats both had some lesions, but the group of high dose extracts had no significant difference compared with the control group, also indicating that the lesions had no relationship with agarwood extracts. CONCLUSION: The agarwood extracts produced by agar-wit have no chronic toxicity for mammal in regards of body weight, biochemical indicators and pathology at both pharmaceutical dosages and functional food dosages.

Acute Toxicity and Genetic Toxicity Evaluation of Agarwood Extracts Produced by Agar-Wit / 中国药学杂志

OBJECTIVE: To evaluate the acute toxicity and genotoxicity of agarwood extracts produced by agar-wit technology for functional food development (slices dose 0.60 g•d-1). METHODS: SD rats were used as animal model, the total dose of 10 g•kg-1 was administered to the rats twice within 24 h, activity and poisoning of rats were observed and recorded in the next 14 d, and Ames test, mammalian erythrocyte micronucleus test and in vitro mammalian cells chromosome aberration test were used to research the genotoxicity comprehensively. RESULTS: The rats showed normal activity and no poisoning symptoms appeared during acute toxicity test at 10 g•kg-1 dose. In the three genotoxicity test, extracts had no significant difference compared with the negative control (P>0.05), but significant difference compared with positive control (P<0.01). CONCLUSION: Under the condition of this study, the agarwood extracts have neither acute toxicity nor genotoxicity on rats.

Prediction and Analysis of the Components and Therapeutic Targets of Agarwood Essential Oil / 中国药学杂志

OBJECTIVE: To predict and analyze the chemical constituents-therapeutic targets of agarwood essential oil by network pharmacology. METHODS: First, the components of agarwood essential oil were determined and analyze by GC-MS and the compound database was built. Then, ADME and target prediction based on the structural data was performed by virtual computation. Finally, the predicted targets were classified and compared with the known therapeutic targets for sleep disorders, anxiety and depression-related diseases, so as to predict the therapeutic targets for diseases and construct the compound-therapeutic target-disease network. RESULTS: Agarwood essential oil can regulate the neural activity through ligand-receptor interaction pathway, cAMP signaling pathway, 5-hydroxytryptamine neural pathway, cholinergic neural pathway, dopaminergic neural pathway and other multiple pathways. Meanwhile, 11 compounds were screened out to have potential effects on 30 therapeutic targets related to insomnia, anxiety and depression. CONCLUSION: Agarwood essential oil has potential preventive and therapeutic effects on sleep disorders, anxiety, depression and other neurological diseases, which provides theoretical basis and data support for in-depth research and development of agarwood essential oil's sedative-hypnotic effects and its mechanism.

Anti-inflammatory Effect of Chinese Agarwood Essential Oil Via Inhibiting p-STAT3 and IL-1β/IL-6 / 中国药学杂志

OBJECTIVE: Eaglewood is a commonly used precious herbal medicine. Due to the multiple drug effects and shortage of medicinal resources, it is currently a hot research topic. Chinese agarwood essential oil (CEEO) is the main active ingredient of eaglewood. To investigate the anti-inflammatory effect and mechanism of CEEO. METHODS: The anti-inflammatory effect of CEEO was evaluated on xylene-induced ear edema in mice and carrageenan-induced paw swelling in rats. The production and transcription levels of pro-inflammatory cytokines IL-1β and IL-6 were detected by ELISA or quantitative RT-PCR in LPS-activated RAW264.7 macrophages. The protein levels of p-transducer and activator of transcription 3 (STAT3) and p-IκBα/p-NF-κBp65 were detected by Western blot analysis. The chemical profiles of CEEO were identified by GC-MS. RESULTS: Intragastric administration with CEEO (60-960 mg•kg-1 in mice and 680 mg•kg-1 in rats) produced significant anti-inflammatory effect in vivo, the highest inhibition percentages were 73% and 51%, respectively (P<0.001). After treatment with CEEO (10, 5, and 2.5 μg•mL-1), ELISA and RT-PCR analysis showed that the production and mRNA level of IL-1β and IL-6 were attenuated by 68% and 39%, 64% and 52% (P<0.001), respectively, at the highest concentration. Western blot analysis showed that CEEO (680 mg•kg-1, ig) significantly restrained the phosphorylation of STAT3 by 21% (P<0.05). Totally 117 compounds were identified in CEEO by GC-MS analysis and the chemical analysis revealed that sesquiterpenoids were the major compounds (68.83%). CONCLUSION: This study shows for the first time that intragastric administration of CEEO has significant anti-inflammatory effect, which may be due to the suppression of pro-inflammatory cytokines IL-1β and IL-6 by reducing the overexpression of p-STAT3, thereby inhibiting the occurrence and development of inflammation. The anti-inflammatory effect of CEEO may be related to the primary component sesquiterpenoids.