[Identification and analysis of terpene synthase (TPS) gene family in Schizonepeta tenuifolia].

Terpenoids are important secondary metabolites of plants that possess both pharmacological activity and economic value. Terpene synthases(TPSs) are key enzymes in the synthesis process of terpenoids. In order to investigate the TPS gene family members and their potential functions in Schizonepeta tenuifolia, this study conducted a systematic analysis of the TPS gene family of S. tenuifolia based on the whole genome data of S. tenuifolia using bioinformatics methods. The results revealed 57 StTPS members identified from the genome database of S. tenuifolia. The StTPS family members encoded 285-819 amino acids, with protein molecular weights ranging from 32.75 to 94.11 kDa, all of which were hydrophilic proteins. The StTPS family members were mainly distributed in the cytoplasm and chloroplasts, exhibiting a random and uneven physical localization pattern. Phylogenetic analysis showed that the StTPS genes family were divided into six subgroups, mainly belonging to the TPS-a and TPS-b subfamilies. Promoter analysis predicted that the TPS gene family members could respond to various stressors such as light, abscisic acid, and methyl jasmonate(MeJA). Transcriptome data analysis revealed that most of the TPS genes were expressed in the roots of S. tenuifolia, and qRT-PCR analysis was conducted on genes with high expression in leaves and low expression in roots. Through the analysis of the TPS gene family of S. tenuifolia, this study identified StTPS5, StTPS18, StTPS32, and StTPS45 as potential genes involved in sesquiterpene synthesis of S. tenuifolia. StTPS45 was cloned for the construction of an prokaryotic expression vector, providing a reference for further investigation of the function and role of the TPS gene family in sesquiterpene synthesis.

The First Genome-Wide Mildew Locus O Genes Characterization in the Lamiaceae Plant Family.

Powdery mildew (PM) is a widespread plant disease that causes significant economic losses in thousands crops of temperate climates, including Lamiaceae species. Multiple scientific studies describe a peculiar form of PM-resistance associated at the inactivation of specific members of the Mildew Locus O (MLO) gene family, referred to as mlo-resistance. The characterization of Lamiaceae MLO genes, at the genomic level, would be a first step toward their potential use in breeding programs. We carried out a genome-wide characterization of the MLO gene family in 11 Lamiaceae species, providing a manual curated catalog of 324 MLO proteins. Evolutionary history and phylogenetic relationships were studied through maximum likelihood analysis and motif patter reconstruction. Our approach highlighted seven different clades diversified starting from an ancestral MLO domain pattern organized in 18 highly conserved motifs. In addition, 74 Lamiaceae putative PM susceptibility genes, clustering in clade V, were identified. Finally, we performed a codon-based evolutionary analysis, revealing a general high level of purifying selection in the eleven Lamiaceae MLO gene families, and the occurrence of few regions under diversifying selection in candidate susceptibility factors. The results of this work may help to address further biological questions concerning MLOs involved in PM susceptibility. In follow-up studies, it could be investigated whether the silencing or loss-of-function mutations in one or more of these candidate genes may lead to PM resistance.

Evaluation of Candidate Reference Genes for Gene Expression Analysis in Wild Lamiophlomis rotata.

Lamiophlomis rotata (Benth.) Kudo is a perennial and unique medicinal plant of the Qinghai-Tibet Plateau. It has the effects of diminishing inflammation, activating blood circulation, removing blood stasis, reducing swelling, and relieving pain. However, thus far, reliable reference gene identifications have not been reported in wild L. rotata. In this study, we identified suitable reference genes for the analysis of gene expression related to the medicinal compound synthesis in wild L. rotata subjected to five different-altitude habitats. Based on the RNA-Seq data of wild L. rotata from five different regions, the stability of 15 candidate internal reference genes was analyzed using geNorm, NormFinder, BestKeeper, and RefFinder. TFIIS, EF-1α, and CYP22 were the most suitable internal reference genes in the leaves of L. rotata from different regions, while OBP, TFIIS, and CYP22 were the optimal reference genes in the roots of L. rotata. The reference genes identified here would be very useful for gene expression studies with different tissues in L. rotata from different habitats.

A highly contiguous genome assembly of red perilla (Perilla frutescens) domesticated in Japan.

Perilla frutescens (Lamiaceae) is an important herbal plant with hundreds of bioactive chemicals, among which perillaldehyde and rosmarinic acid are the two major bioactive compounds in the plant. The leaves of red perilla are used as traditional Kampo medicine or food ingredients. However, the medicinal and nutritional uses of this plant could be improved by enhancing the production of valuable metabolites through the manipulation of key enzymes or regulatory genes using genome editing technology. Here, we generated a high-quality genome assembly of red perilla domesticated in Japan. A near-complete chromosome-level assembly of P. frutescens was generated contigs with N50 of 41.5 Mb from PacBio HiFi reads. 99.2% of the assembly was anchored into 20 pseudochromosomes, among which seven pseudochromosomes consisted of one contig, while the rest consisted of less than six contigs. Gene annotation and prediction of the sequences successfully predicted 86,258 gene models, including 76,825 protein-coding genes. Further analysis showed that potential targets of genome editing for the engineering of anthocyanin pathways in P. frutescens are located on the late-stage pathways. Overall, our genome assembly could serve as a valuable reference for selecting target genes for genome editing of P. frutescens.

[Cloning of StHD1 and StHD8 from Schizonepeta tenuifolia and function of regulating glandular trichome development].

Hd-Zip, a unique transcription factor in plant kingdom, influences the growth, development, and secondary metabolism of plants. Hd-zip Ⅳ is thought to play an important role in trichome development of Schizonepeta tenuifolia. This study aims to explore the functions of StHD1 and StHD8 in Hd-zip Ⅳ subfamily in peltate glandular trichome development. To be specific, the expression patterns of the two genes and interaction between the proteins encoded by them were analyzed based on transcriptome sequencing and two-hybrid screening. The subcellular localization was performed and functions of the genes were verified in tobacco and S. tenuifolia. The results showed that StHD1 and StHD8 had high similarity to HD-Zip Ⅳ proteins of other plants and they all had the characteristic conserved domains of HD-Zip Ⅳ subfamily. They were located in the nucleus. The two genes mainly expressed in young tissues and spikes, and StHD1 and StHD8 proteins interacted with each other. The density and length of glandular trichomes increased significantly in tobacco plants with the overexpression of StHD1 and StHD8. Inhibiting the expression of StHD1 and StHD8 by VIGS(virus-induced gene silencing) in S. tenuifolia resulted in the reduction in the density of peltate glandular trichomes, the expression of key genes related to mono-terpene synthesis, and the relative content of limonene and pulegone, the main components of monoterpene. These results suggested that StHD1 and StHD8 of S. tenuifolia formed a complex to regulate glandular trichomes and affect the biosynthesis of monoterpenes.

Sequence Analysis of the Complete Mitochondrial Genome of a Medicinal Plant, Vitex rotundifolia Linnaeus f. (Lamiales: Lamiaceae).

In the present study, we depicted the complete mitochondrial genome of a valuable medicinal plant, Vitex rotundifolia. The mitochondrial genome of V. rotundifolia, mapped as a circular molecule, spanned 380,980 bp in length and had a GC content of 45.54%. The complete genome contained 38 protein-coding genes, 19 transfer RNAs (tRNAs), and 3 ribosomal RNAs (rRNAs). We found that there were only 38.73% (147.54 kb), 36.28% (138.23 kb), and 52.22% (198.96 kb) of the homologous sequences in the mitochondrial genome of V. rotundifolia, as compared with the mitochondrial genomes of Scutellaria tsinyunensis, Boea hygrometrica, and Erythranthe lutea, respectively. A multipartite structure mediated by the homologous recombinations of the three direct repeats was found in the V. rotundifolia mitochondrial genome. The phylogenetic tree was built based on 10 species of Lamiales, using the maximum likelihood method. Moreover, this phylogenetic analysis is the first to present the evolutionary relationship of V. rotundifolia with the other species in Lamiales, based on the complete mitochondrial genome.

Alternate gene expression profiling of monoterpenes in Hymenocrater longiflorus as a novel pharmaceutical plant under water deficit.

Hymenocrater longiflorus (surahalala) is a wild plant species with potential pharmaceutical and ornamental interest. To date, the genomics of this plant is unknown and the gene expression profiling of the genes related to its metabolite has never been studied before. In order to study the responses of in vitro-grown surahalala plants to abiotic stresses and the differential expression of the genes related to its essential oils under exogenous proline application; three levels of PEG600 (0, 10, and 20%) and five levels of proline (0, 5, 10, 15, and 20 µm) were combined in the culture media. Thus, water deficit increased oxidants levels and decreased fresh weight of surahalala tissues, whereas addition of proline up to 15 µm was able to relatively compensate the negative effect of water deficit. Contrarily, high proline level (20 µm) had a negative effect on surahalala plants probably due to the stress simulation (nutrition) under high proline concentration. In addition, the best combination for achieving highest essential oils content was 10 µm proline plus 10% PEG. The expressional profiling of the genes TPS27, L3H, TPS2, TPS1, OMT and GDH3 were successfully carried out and their involvement in 1,8-cineole, carvone, α-pinene, thymol, estragole and ß-Citronellol biosynthesis, respectively, was verified. In addition, our results indicated that these genes could also be involved in the synthesis of other metabolites under water deficit condition.

The complete chloroplast genome sequence of Clerodendranthus spicatus, a medicinal plant for preventing and treating kidney diseases from Lamiaceae family.

BACKGROUND: Clerodendranthus spicatus (Thunb.) C. Y. Wu ex H. W. Li is one of the most important medicines for the treatment of nephrology in the southeast regions of China. To understand the taxonomic classification of Clerodendranthus species and identify species discrimination markers, we sequenced and characterized its chloroplast genome in the current study. METHODS AND RESULTS: Total genomic DNA were isolated from dried leaves of C. spicatus and sequenced using an Illumina sequencing platform. The data were assembled and annotated by the NOVOPlasty software and CpGAVAS2 web service. The complete chloroplast genome of C. spicatus was 152,155 bp, including a large single-copy region of 83,098 bp, a small single-copy region of 17,665 bp, and a pair of inverted repeat regions of 25,696 bp. The Isoleucine codons are the most abundant, accounting for 4.17% of all codons. The codons of AUG, UUA, and AGA demonstrated a high degree of usage bias. Twenty-eight simple sequence repeats, thirty-six tandem repeats, and forty interspersed repeats were identified. The distribution of the specific rps19, ycf1, rpl2, trnH, psbA genes were analyzed. Analysis of the genetic distance of the intergenic spacer regions shows that ndhG-ndhI, accD-psaI, rps15-ycf1, rpl20-clpP, ccsA-ndhD regions have high K2p values. Phylogenetic analysis showed that C. spicatu is closely related to two Lamiaceae species, Tectona grandis, and Glechoma longituba. CONCLUSIONS: In this study, we sequenced and characterized the chloroplast genome of C. spicatus. Phylogenomic analysis has identified species closely related to C. spicatus, which represent potential candidates for the development of drugs improving renal functions.

Antibacterial Activity and Epigenetic Remodeling of Essential Oils from Calabrian Aromatic Plants.

Natural compounds have historically had a wide application in nutrition. Recently, a fundamental role has been identified for essential oils extracted from aromatic plants for their nutritional, antimicrobial, and antioxidant properties, and as food preservatives. In the present study, essential oils (EOs) from ten aromatic plants grown in Calabria (Italy), used routinely to impart aroma and taste to food, were evaluated for their antibacterial activity. This activity was investigated against Escherichia coli strain JM109, and its derived antibiotic-resistant cells selected by growing the strain at low concentrations of ampicillin, ciprofloxacin, and gentamicin by measuring the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). Although all the essential oils showed bactericidal activity, those from Clinopodium nepeta, Origanum vulgare, and Foeniculum vulgare displayed the greatest inhibitory effects on the bacterial growth of all cell lines. It is plausible that the antibacterial activity is mediated by epigenetic modifications since the tested essential oils induce methylation both at adenine and cytosine residues in the genomes of most cell lines. This study contributes to a further characterization of the properties of essential oils by shedding new light on the molecular mechanisms that mediate these properties.

Do we have infraspecific taxa of Salvia macrosiphon Boiss. (Lamiaceae) in Iran?

BACKGROUND: Salvia macrosiphon is an aromatic perennial species of Lamiaceae family that grows naturally in different parts of Iran. This herb is widely used in folk and modern medicine. Although in Flora Iranica and Flora of Iran, no infraspecific taxonomic rank has been detected for S. macrosiphon, some infraspecific taxonomic ranks have been reported. In the current study, we evaluated the genetic diversity and structure of 11 populations of this species to detect inter and intrapopulation genetic diversity and to survey the possibility of infraspecific taxonomic ranks in this species. METHODS AND RESULTS: We utilized the modified C-TAB protocol for DNA extraction and amplified the genomes using several SCoT molecular markers. We calculated of genetic diversity and polymorphism parameters using GenAlex 6.4, Geno-Dive ver.2, PopGene, PopART and Structure 2.3.4. The parameters of genetic polymorphism differed between the populations. Moreover, a low rate of gene flow supported a moderate level of population's genetic diversity and differentiation. According to haplotypes network (TCS) analysis, a high level of genetic mutation has occurred among the individuals of some populations leading to high intrapopulation diversity. On the basis of structure analysis and Nei's genetic distance, the examined populations were classified into four genotypes. CONCLUSIONS: The clustering pattern of the populations in each group was not related to geographical distance or phytogeography. It seems that the wide geographic distribution, a small gene flow rate and the occurrence of a high level of genetic mutation lead to infraspecific genetic differentiation in the species and we suppose some infraspecific ranks exist for it.

Integrating RNA-seq with functional expression to analyze the regulation and characterization of genes involved in monoterpenoid biosynthesis in Nepeta tenuifolia Briq.

Nepeta tenuifolia Briq. (Lamiaceae) is a medicinal plant historically used in the East Asia region to treat cold and fever, and it is currently used as a clinically effective treatment for respiratory diseases. We previously found that monoterpenoids are the dominant volatile secondary metabolites in N. tenuifolia and their biosynthesis occurs in peltate glandular trichomes. To gain an insight into the molecular mechanisms underlying monoterpenoid biosynthesis in N. tenuifolia, we conducted transcriptome sequencing and examined the expression differences in monoterpene molecular pathway-related genes in different tissues and growth stages by qRT-RCR. In total, six p-menthane monoterpene biosynthetic genes in the (+)-menthone pathway were identified and cloned successfully based on transcriptome data. Moreover, the major constituents, including (+)-limonene, (-)-pulegone and (+)-menthone showed greater accumulation in the spikes than in other organs, such as the expression levels of related key enzyme genes. Additionally, the relative expression of pulegone reductase was the highest at 84 days, showing an inverse trend from (-)-pulegone relative content and leading to (+)-menthone accumulation in peltate glandular trichomes. Finished cloning of the gene for limonene 3-hydroxylase in N. tenuifolia (NtL3OH), heterologous expression in yeast, and in vitro assays were performed for functional characterization. Our study provides an important resource for further research of secondary metabolism of monoterpenes in peltate glandular trichomes of N. tenuifolia and other homologous species.

DNA barcoding of some medicinally important plant species of Lamiaceae family in India.

Several species of the Lamiaceae family are the primary source of bioactive aromatic oils and secondary metabolites, having broader applications in the cosmetics, pharmaceuticals, food, confectionery and liquor industries. Due to the scarcity of raw materials and high costs of this family's economically vital species, its products often adulterated to cater to the market's high demand. The present study provides a DNA based approach for identifying different species of this family. Henceforth, the performance of three already proposed barcode loci (matK, trnH-psbA and trnL) was examined for their PCR amplification and species recognition efficacy on various Lamiaceae species and cultivars using three different approaches such as pairwise genetic distance method, BLASTn and phylogenetic tree based on maximum likelihood (ML) analysis. Results illustrate that among all the DNA barcoding loci, matK locus can accurately and efficiently distinguish all the studied species followed by trnH-psbA and trnL. Present investigation may help diminish the illegal trade and events of adulteration of medicinally important plants species in genus Mentha, Ocimum and Plectranthus. This investigation will also help fulfil the scarcity of sequences of barcode loci of these species in the NCBI database. Apart from providing a molecular level reference for identifying processed herbal products, this technique also offers a convenient method for species identification and germplasm conservation of the Lamiaceae family.

An updated tribal classification of Lamiaceae based on plastome phylogenomics.

BACKGROUND: A robust molecular phylogeny is fundamental for developing a stable classification and providing a solid framework to understand patterns of diversification, historical biogeography, and character evolution. As the sixth largest angiosperm family, Lamiaceae, or the mint family, consitutes a major source of aromatic oil, wood, ornamentals, and culinary and medicinal herbs, making it an exceptionally important group ecologically, ethnobotanically, and floristically. The lack of a reliable phylogenetic framework for this family has thus far hindered broad-scale biogeographic studies and our comprehension of diversification. Although significant progress has been made towards clarifying Lamiaceae relationships during the past three decades, the resolution of a phylogenetic backbone at the tribal level has remained one of the greatest challenges due to limited availability of genetic data. RESULTS: We performed phylogenetic analyses of Lamiaceae to infer relationships at the tribal level using 79 protein-coding plastid genes from 175 accessions representing 170 taxa, 79 genera, and all 12 subfamilies. Both maximum likelihood and Bayesian analyses yielded a more robust phylogenetic hypothesis relative to previous studies and supported the monophyly of all 12 subfamilies, and a classification for 22 tribes, three of which are newly recognized in this study. As a consequence, we propose an updated phylogenetically informed tribal classification for Lamiaceae that is supplemented with a detailed summary of taxonomic history, generic and species diversity, morphology, synapomorphies, and distribution for each subfamily and tribe. CONCLUSIONS: Increased taxon sampling conjoined with phylogenetic analyses based on plastome sequences has provided robust support at both deep and shallow nodes and offers new insights into the phylogenetic relationships among tribes and subfamilies of Lamiaceae. This robust phylogenetic backbone of Lamiaceae will serve as a framework for future studies on mint classification, biogeography, character evolution, and diversification.

Complete Chloroplast Genome Sequencing and Phylogenetic Analysis of Two Dracocephalum Plants.

Dracocephalum tanguticum and Dracocephalum moldavica are important herbs from Lamiaceae and have great medicinal value. We used the Illumina sequencing technology to sequence the complete chloroplast genome of D. tanguticum and D. moldavica and then conducted de novo assembly. The two chloroplast genomes have a typical quadripartite structure, with the gene's lengths of 82,221 bp and 81,450 bp, large single-copy region's (LSC) lengths of 82,221 bp and 81,450 bp, and small single-copy region's (SSC) lengths of 17,363 bp and 17,066 bp, inverted repeat region's (IR) lengths of 51,370 bp and 51,352 bp, respectively. The GC content of the two chloroplast genomes was 37.80% and 37.83%, respectively. The chloroplast genomes of the two plants encode 133 and 132 genes, respectively, among which there are 88 and 87 protein-coding genes, respectively, as well as 37 tRNA genes and 8 rRNA genes. Among them, the rps2 gene is unique to D. tanguticum, which is not found in D. moldavica. Through SSR analysis, we also found 6 mutation hotspot regions, which can be used as molecular markers for taxonomic studies. Phylogenetic analysis showed that Dracocephalum was more closely related to Mentha.

Poejo (Cunila galioides Benth. ) Production in Five Agroecological Regions of Rio Grande do Sul

Abstract The objective of this study was to evaluate the biomass and essential oil production of nine populations of poejo (Cunila galioides) cultivated in five agroecological regions of the state of Rio Grande do Sul, under different edaphoclimatic conditions. The experiments were performed in field conditions in Erechim, Caxias do Sul, Pelotas, São Francisco de Paula, and Santa Vitoria do Palmar. The experimental design was completely randomized, with nine populations, eight plants per plot and four repetitions. The following were evaluated: biomass production and essential oil chemical composition and yield. The data underwent ANOVA, followed by Tukey's multiple range test. The adaptability and stability of the populations in the different environments were also evaluated by regression analysis. The results showed great differences between the populations and cultivation sites, with genotype vs. environment interaction. Most populations presented the best biomass production results at Erechim. Pelotas and Santa Vitória do Palmar were the worst locations for poejo production, mainly due to a water deficit occurred during the experiment. The Santa Lucia population presented broad stability and the greatest adaptability to the environments for biomass and essential oil production, but its average production was not satisfactory. The André da Rocha population presented the highest average production of essential oil, and was favored in favorable environments. Regarding essential oil chemical composition, the populations kept stable contents of the major compounds at all locations, with a few variations. In some populations, a higher concentration of sesquiterpenes was observed, which can be attributed to environmental stress.

The effect of purine-type cytokinin on the proliferation and production of phenolic compounds in transformed shoots of Dracocephalum forrestii.

Dracocephalum forrestii is a perennial, endemic to China plant with a number of pharmaceutical properties. Transformed shoots of the species spontaneously regenerated from hairy roots induced by Agrobacterium rhizogenes. The transgenic nature of the shoots was confirmed by polymerase chain reaction (PCR). The shoot culture was multiplied on Murashige and Skoog (MS) medium with 0.2 mg/l IAA and 0.2, 0.5, 1.0, 2.0 or 5.0 mg/l purine-type cytokinins (mT, BAR, BPA or BAP). The highest multiplication rate (about thirteen shoot or buds per explant) was obtained on MS medium with 0.2 mg/l mT after four weeks of culture. The phenolic compounds present in the hydromethanolic extracts from the D. forrestii transgenic shoots were characterized using UPLC-PDA-ESI-MS. The shoots were found to biosynthesize three phenolic acids and five flavonoid glycosides. UHPLC analysis of the hydromethanolic extracts found the predominant phenolic acid to be rosmarinic acid, with its highest content observed in shoots cultivated with 5.0 mg/l BPA. In contrast, the greatest production of flavonoid derivatives (especially acacetin derivatives) was observed in the medium supplemented with 2 mg/l BPA.

Authentication of Herbal Medicines Dipsacus asper and Phlomoides umbrosa Using DNA Barcodes, Chloroplast Genome, and Sequence Characterized Amplified Region (SCAR) Marker.

Dried roots of Dipsacus asper (Caprifoliaceae) are used as important traditional herbal medicines in Korea. However, the roots are often used as a mixture or contaminated with Dipsacus japonicus in Korean herbal markets. Furthermore, the dried roots of Phlomoides umbrosa (Lamiaceae) are used indiscriminately with those of D. asper, with the confusing Korean names of Sok-Dan and Han-Sok-Dan for D. asper and P. umbrosa, respectively. Although D. asper and P. umbrosa are important herbal medicines, the molecular marker and genomic information available for these species are limited. In this study, we analysed DNA barcodes to distinguish among D. asper, D. japonicus, and P. umbrosa and sequenced the chloroplast (CP) genomes of D. asper and D. japonicus. The CP genomes of D. asper and D. japonicus were 160,530 and 160,371 bp in length, respectively, and were highly divergent from those of the other Caprifoliaceae species. Phylogenetic analysis revealed a monophyletic group within Caprifoliaceae. We also developed a novel sequence characterised amplified region (SCAR) markers to distinguish among D. asper, D. japonicus, and P. umbrosa. Our results provide important taxonomic, phylogenetic, and evolutionary information on the Dipsacus species. The SCAR markers developed here will be useful for the authentication of herbal medicines.

[Bioinformatics analysis of geranylgeranyl pyrophosphate synthase coding gene and amino acid sequence in Lamiaceae].

Geranylgeranyl pyrophosphate synthase enzyme is one of the key enzymes in the synthesis pathway of diterpenoid. Nine Lamiaceae genus GGPS synthase in Genebank was analyzed in this article. GGPS synthase the nucleic acid sequences and amino acid sequences, physicochemical properties, the signal peptide, leader peptides, transmembrane topological structure, hydrophobic, hydrophilic, subcellular localization, secondary structure, function domain, tertiary structure and evolutional relationship were predicted by using bioinformatics methods.Phylogenetic tree was constructed for the geranylgeranyl pyrophosphate synthase enzyme protein family. The results showed that GGPS amino acid sequence of the physical and chemical properties were basically identical, mainly hydrophilic protein, there existed chloroplast transit peptide, and no signal peptide and membrane structure domain, which mainly located in the chloroplast, the minor part located in mitochondria. The main secondary structures of the proteins are alpha helix and random coil. All these proteins have catalytic residues, aspartate-rich region, active site lid residues, substrate-Mg2+ binding site. The results provide theoretical reference for study on both the enzymatic characteristics of GGPS and the biosynthesis pathway of diterpenoid.

Assessment of genetic diversity of a native population of Eplingiella fruticosa: a plant with therapeutic potential.

Eplingiella fruticosa (Salzm. ex Benth.) Harley & J.F.B. Pastore, ex Hyptis fruticosa Salzm. ex Benth. is an aromatic and medicinal plant of the family Lamiaceae, found mainly in regions with intense anthropic activity. Information on the genetic diversity of this species is scarce. However, it can be assessed using molecular markers that identify the level of diversity among phenotypically identical individuals. The present study aimed to characterize the genetic diversity of a native population of E. fruticosa from the State of Sergipe using ISSR molecular markers. Samples of 100 plants were collected in 11 municipalities of the State of Sergipe and analyzed using eight ISSR primers, resulting in 72 informative bands. The cluster analysis obtained using the neighbor joining method resulted in three groups: Group I consisted of 50 plants, mainly from the municipalities of Areia Branca, Estância, Japaratuba, Moita Bonita, Pirambu, and Salgado; Group II was formed by 21 plants, with nine representatives from the municipality of Itaporanga d'Ajuda and 13 representatives from other municipalities; Group III was composed by 29 plants, being represented mainly by the municipalities of Malhada dos Bois and São Cristóvão. The smallest genetic distance occurred between plants EPF94 and EPF96 (0.250), and the greatest distance occurred between plants EPF50 and EPF96 (0.9778). The Shannon index had a mean value of 0.42, and diversity was considered moderate. Heterozygosity had a mean value of 0.267 and was considered low. Polymorphic information content (0.253) was considered moderately informative. Genetic diversity of E. fruticosa plants was intermediate, and the results of the present study can assist in the conservation and use of the genetic resources of this species.

Selective in vitro and in silico butyrylcholinesterase inhibitory activity of diterpenes and rosmarinic acid isolated from Perovskia atriplicifolia Benth. and Salvia glutinosa L.

Cholinesterase inhibition is one of the most treatment strategies against Alzheimer's disease (AD) where metal accumulation is also strongly associated with pathology of the disease. In the current study, we assessed inhibitory effect against acetyl- (AChE) and butyrylcholinesterase (BChE) and metal-chelating capacity of twelve diterpenes: arucadiol, miltirone, tanshinone IIa, 1-oxomiltirone, cryptotanshinone, 1,2-didehydromiltirone, 1,2-didehydrotanshinone IIa, 1ß-hydroxycryptotanshinone, 15,16-dihydrotanshinone, tanshinone I, isotanshinone II, 1(S)-hydroxytanshinone IIa, and rosmarinic acid, isolated from Perovskia atriplicifolia and Salvia glutinosa. The compounds were tested at 10 µg/mL using ELISA microtiter assays against AChE and BChE. QSAR and molecular docking studies have been also performed on the active compounds. All of the compounds showed higher [e.g., IC50 = 1.12 ± 0.07 µg/mL for 1,2-didehydromiltirone, IC50 = 1.15 ± 0.07 µg/mL for cryptotanshinone, IC50 = 1.20 ± 0.03 µg/mL for arucadiol, etc.)] or closer [1,2-didehydrotanshinone IIa (IC50 = 5.98 ± 0.49 µg/mL) and 1(S)-hydroxytanshinone IIa (IC50 = 5.71 ± 0.27 µg/mL)] inhibition against BChE as compared to that of galanthamine (IC50 = 12.56 ± 0.37 µg/mL), whereas only 15,16-dihydrotanshinone moderately inhibited AChE (65.17 ± 1.39%). 1,2-Didehydrotanshinone IIa (48.94 ± 0.26%) and 1(S)-hydroxytanshinone IIa (47.18 ± 5.10%) possessed the highest metal-chelation capacity. The present study affords an evidence for the fact that selective BChE inhibitors should be further investigated as promising candidate molecules for AD therapy.