Pogostemon cablin essential oil affects anxiety- and depressive-like behaviors and the gut microbiota in chronic unpredictable mild stress model rats.

The gut microbiota is thought to be an important factor that influences brain processes and behaviors through the gut-brain axis. Pogostemon cablin is used in traditional Chinese medicine (TCM) to treat gastrointestinal symptoms. Patchouli essential oil (PCO), the main active agent in P. cablin, is used in aromatherapy for stress relief. The aim of our study was to investigate the effects of orally administered PCO on anxiety- and depressive-like behaviors and the gut microbiota. We constructed a rat model of chronic unpredictable mild stress (CUMS) and explored the anxiolytic- and antidepressant-like effects of PCO using the open field test (OFT) and forced swim test (FST). Changes in the abundance of the gut microbiota, short-chain fatty acids (SCFAs), and other related molecules were assessed to determine the role of the gut microbiota. Our results showed that CUMS induced an anxiety-like phenotype in the OFT, which was reversed by PCO, and that PCO also significantly mitigated the depression-like behaviors caused by CUMS in the FST. Furthermore, we found that PCO increased the relative abundances of several probiotics, including Bacteroides and Blautia, and decreased the relative abundances of Ruminococcus_1 and Ruminococcus_2, which were increased by CUMS. Regarding SCFAs, the metabolites of the gut microbiota, PCO increased the concentration of propionic acid and decreased that of caproic acid. Finally, PCO restored the serotonin (5-hydroxytryptamine, 5-HT) level in the hippocampus, which had been decreased by CUMS. The results of this study suggested that PCO can improve stress-related anxiety- and depression-like behaviors and might exert its effects on the central nervous system through interactions with the gut microbiota.

Chromosome-level and haplotype-resolved genome provides insight into the tetraploid hybrid origin of patchouli.

Patchouli (Pogostemon cablin (Blanco) Benth.), a member of the Lamiaceae family, is an important aromatic plant that has been widely used in medicine and perfumery. Here, we report a 1.94 Gb chromosome-scale assembly of the patchouli genome (contig N50 = 7.97 Mb). The gene annotation reveals that tandem duplication of sesquiterpene biosynthetic genes may be a major contributor to the biosynthesis of patchouli bioactivity components. We further phase the genome into two distinct subgenomes (A and B), and identify a chromosome substitution event that have occurred between them. Further investigations show that a burst of universal LTR-RTs in the A subgenome lead to the divergence between two subgenomes. However, no significant subgenome dominance is detected. Finally, we track the evolutionary scenario of patchouli including whole genome tetraploidization, subgenome divergency, hybridization, and chromosome substitution, which are the key forces to determine the complexity of patchouli genome. Our work sheds light on the evolutionary history of patchouli and offers unprecedented genomic resources for fundamental patchouli research and elite germplasm development.

Development and Characterization of High-Throughput EST-Based SSR Markers for Pogostemon cablin Using Transcriptome Sequencing.

Simple sequence repeats (SSRs) or microsatellite markers derived from expressed sequence tags (ESTs) are routinely used for molecular assisted-selection breeding, comparative genomic analysis, and genetic diversity studies. In this study, we investigated 54,546 ESTs for the identification and development of SSR markers in Pogostemon cablin (Patchouli). In total, 1219 SSRs were identified from 1144 SSR-containing ESTs. Trinucleotides (80.8%) were the most abundant SSRs, followed by di- (10.8%), mono- (7.1%), and hexa-nucleotides (1.3%). The top six motifs were CCG/CGG (15.3%), AAG/CTT (15.0%), ACC/GGT (13.5%), AGG/CCT (12.4%), ATC/ATG (9.9%), and AG/CT (9.8%). On the basis of these SSR-containing ESTs, a total of 192 primer pairs were randomly designed and used for polymorphism analysis in 38 accessions collected from different geographical regions of Guangdong, China. Of the SSR markers, 45 were polymorphic and had allele variations from two to four. Furthermore, a transferability analysis of these primer pairs revealed a 10⁻40% cross-species transferability in 10 related species. This report is the first comprehensive study on the development and analysis of a large set of SSR markers in P. cablin. These markers have the potential to be used in quantitative trait loci mapping, genetic diversity studies, and the fingerprinting of cultivars of P. cablin.

Aging and/or tissue-specific regulation of patchoulol and pogostone in two Pogostemon cablin (Blanco) Benth. cultivars.

In Pogostemon cablin (Blanco) Benth. essential oil, patchoulol and pogostone are the two major bioactive phytochemicals while their in vivo biosynthesis remains largely unknown. In this study, seven genes of the plastidic methylerythritol 4-phosphate pathway (MEP) and three genes of the cytoplasmic mevalonate pathway (MVA) in two cultivars, HN and YN, were isolated. Gene expression and phytochemical profiles across leaves and stems at different developmental stages of the two cultivars were evaluated using quantitative reverse-transcription polymerase chain reaction and gas chromatography-mass spectrometry, respectively. Hierarchical analysis showed that the expression of MVA- and MEP-related genes was clustered similarly in the two cultivars. Phytochemical assay revealed that the contents of patchoulol in leaves and pogostone in stems were regulated in an aging-dependent manner. Pogostone was only detected in stems but not in leaves of the two cultivars. The Pearson correlation analysis suggested that several genes were presumably involved in the biosynthesis of patchoulol and pogostone. In the YN cultivar, the 1-deoxy-d-xylulose-5-phosphate reductoisomerase and isopentenyl pyrophosphate isomerase 2 genes, and 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase were positively responsible for patchoulol and pogostone biosynthesis, respectively. In the HN cultivar, 3-hydroxy-3-methylglutaryl-coenzyme A reductase and mevalonate diphosphate decarboxylase, and mevalonate kinase expression were positively associated with pogostone and patchoulol biosynthesis, respectively. The genes identified in this study are good candidates for the enhancement of patchoulol content in the leaves or pogostone content in the stems of P. cablin. Taken together, our results lay a solid foundation for better understanding of the mechanism underlying patchoulol and pogostone biosynthesis, which in turn may help to improve their content in P. cablin.

Rapid and Sensitive Determination of the Major Steroidal Saponins of Ypsilandra thibetica Franch by Ultra High-Performance Liquid Chromatography Coupled with Triple Quadrupole Mass Spectrometry.

A rapid and validated method using ultra high-performance liquid chromatography coupled with a triple quadrupole mass spectrometry (UHPLC-QQQ MS) was developed for simultaneous determination of four active steroidal saponins, i.e., dichotomin ( 1: ), pennogenin 3-O-α-l-arabinofuranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-ß-d-glucopyranoside ( 2: ), pennogenin 3-O-α-l-rhamnopyranosyl-(1→2)-[α-l-rhamnopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→4)]-ß-d-glucopyranoside ( 3: ) and diosgenin 3-O-α-l-rhamnopyranosyl-(1→2)-[α-l-rhamnopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→4)]-ß-d-glucopyranosidein ( 4: ), in Ypsilandra thibetica Franch. The optimized sample preparation and UHPLC-QQQ MS conditions were chosen for quantitative analysis. The separation was performed on an Agilent Zorbax Eclipse Plus C18 column (2.1 mm × 50 mm, 1.8 µm) with gradient elution of acetonitrile-0.1% formic acid in water. All calibration curves showed good linear regression (r> 0.9985) within the test range. The limits of detection and quantification were in the range of 0.02-4.40 and 0.04-22.0 ng/mL, respectively. The proposed method was applied to analyze two batches of Y. thibetica samples for target compounds within 10 min. This work promoted the quality control method for raw material or preparations of Y. thibetica.

Genetic differentiation of relictual populations of Alsophila spinulosa in southern China inferred from cpDNA trnL-F noncoding sequences.

The genetic differentiation and phylogeographical pattern of 11 relictual populations of Alsophila spinulosa distributed across Hainan, Guangdong, and Guangxi in southern China were inferred from sequence variations of trnL-F noncoding regions of chloroplast DNA (cpDNA). The length of trnL-F noncoding sequences varied from 863 to 940 bp. The A + T content was 62.23-63.36%. Sequences were neutral in terms of evolution (Tajima's criterion D=-0.62417, P>0.10 and Fu and Li's test D*=-1.45455, P>0.10; F*=-1.32798, P>0.10). Thirty-four haplotypes were identified based on nucleotide variation. Relatively high levels of haplotype diversity (h=0.929) and nucleotide diversity (Dij=0.022263) were detected in A. spinulosa, probably associated with its long evolutionary history which allowed the accumulation of genetic variation within lineages. Both the minimum spanning network and the strict consensus tree of the most parsimonious trees generated for haplotypes demonstrated that the investigated populations of A. spinulosa were subdivided into two geographical groups: Hainan and Guangdong-Guangxi. An analysis of molecular variance (AMOVA) indicated that most of the genetic variation (87.48%, P<0.001) was partitioned among regions. Spatial structure measurements revealed that population genetic structure was not related to geographical distance. This research suggests that blocked gene flow by Qiongzhou strait and an inbreeding system might result in the geographical subdivision between Hainan and Guangdong-Guangxi (F(ST)=0.92, Nm=0.09). Within each region, the "star like" pattern of phylogeography of haplotypes implied a population expansion process during evolutionary history. Gene genealogies together with coalescent theory were useful tools for uncovering the phylogeography of A. spinulosa.