Comprehensive comparative analysis and development of molecular markers for Lasianthus species based on complete chloroplast genome sequences.

BACKGROUND: Lasianthus species are widely used in traditional Chinese folk medicine with high medicinal value. However, source materials and herbarium specimens are often misidentified due to morphological characteristics and commonly used DNA barcode fragments are not sufficient for accurately identifying Lasianthus species. To improve the molecular methods for distinguishing among Lasianthus species, we report the complete chloroplast (CP) genomes of Lasianthus attenuatus, Lasianthus henryi, Lasianthus hookeri, Lasianthus sikkimensis, obtained via high-throughput Illumina sequencing. RESULTS: These showed CP genomes size of 160164-160246 bp and a typical quadripartite structure, including a large single-copy region (86675-86848 bp), a small single-copy region (17177-17326 bp), and a pair of inverted repeats (28089-28135 bp). As a whole, the gene order, GC content and IR/SC boundary structure were remarkably similar among of the four Lasianthus CP genomes, the partial gene length and IR, LSC and SSC regions length are still different. The average GC content of the CP genomes was 36.71-36.75%, and a total of 129 genes were detected, including 83 different protein-coding genes, 8 different rRNA genes and 38 different tRNA genes. Furthermore, we compared our 4 complete CP genomes data with publicly available CP genome data from six other Lasianthus species, and we initially screened eleven highly variable region fragments were initially screened. We then evaluated the identification efficiency of eleven highly variable region fragments and 5 regular barcode fragments. Ultimately, we found that the optimal combination fragment' ITS2 + psaI-ycf4' could authenticated the Lasianthus species well. Additionally, the results of genome comparison of Rubiaceae species showed that the coding region is more conservative than the non-coding region, and the ycf1 gene shows the most significant variation. Finally, 49 species of CP genome sequences belonging to 16 genera of the Rubiaceae family were used to construct phylogenetic trees. CONCLUSIONS: Our research is the first to analyze the chloroplast genomes of four species of Lasianthus in detail and we ultimately determined that the combination fragment' ITS2 + psaI-ycf4' is the optimal barcode combination for identifying the genus of Lasianthus. Meanwhile, we gathered the available CP genome sequences from the Rubiaceae and used them to construct the most comprehensive phylogenetic tree for the Rubiaceae family. These investigations provide an important reference point for further studies in the species identification, genetic diversity, and phylogenetic analyses of Rubiaceae species.

Six new iridoid glycosides from the whole plants of Hedyotis diffusa.

Six new iridoid glycosides were isolated from the ethyl acetate fraction of the whole plants of Hedyotis diffusa Willd. They were identified as E-6-O-p-methoxycinnamoyl-10-O-acetyl scandoside acid methyl ester (1), Z-6-O-p-methoxycinnamoyl-10-O-acetyl scandoside acid methyl ester (2), E-6-O-caffeoyl scandoside methyl ester (3), E-6-O-p-coumaroyl-6'-O-acetyl scandoside methyl ester (4), Z-6-O-p-coumaroyl-6'-O-acetyl scandoside methyl ester (5), and E-6-O-p-coumaroyl-4'-O-acetyl scandoside methyl ester (6). The structures of them were elucidated based on unambiguous spectroscopic data (UV, IR, HRESIMS, and NMR). They were screened for anti-inflammatory effect, antioxidant effect, antitumor effect, and neuroprotective effect and did not show potent activities.

Triterpenoids from the hook-bearing stems of Uncaria rhynchophylla.

An unprescribed nortriterpenoid with an aromatic E ring, uncanortriterpenoid A (1), together with fourteen known triterpenoids (2-15), were isolated from the hook-bearing stems of Uncaria rhynchophylla Miq. Based on extensive spectroscopic analyses, the NMR data of 2, 5, and 10 in CD3OD were assigned for the first time, and the wrongly assigned δC of C-27 and C-29 of 2 were revised. Among the known compounds, 7, 13, and 15 were isolated from this species for the first time, and 15 represents the first lanostane triterpenoid bearing an extra methylidene at C-24 for the Rubiaceae family. Additionally, compounds 6 and 14 exhibited moderate ferroptosis inhibitory activity, with an EC50 value of 14.74 ± 0.20 µM for 6 and 23.11 ± 1.31 µM for 14.

Antinociceptive iridoid glycosides from the aerial parts of Paederia foetida.

Two previously undescribed iridoid glycosides, 6'-O-trans-feruloyl-(4S,6R)-3,4-dihydro-3ß-ethoxypaederoside (1) and 6'-O-trans-caffeoyl-(4S,6R)-3,4-dihydro-2'-O-3α-paederoside (2), were isolated from the 90% EtOH extract of the air dried aerial parts of Paederia Foetida. Structural elucidation of all the compounds was performed by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy. The two isolated iridoid glycosides were tested in vivo for their antinociceptive properties. As a result, 2 showed potent antinociceptive effect and its ID50 value (53.4 µmol/kg) was 2-fold less than those of the positive control drugs aspirin and acetaminophen.

Cytotoxic iridoid glycosides from the leaves of Paederia scandens.

A phytochemical investigation on the 80% EtOH extract of the leaves of Paederia scandens (Lour.) Merr. resulted into the isolation of three undescribed iridoid glycosides, 10-O-trans-p-coumaroyl-(4R,6R)-3,4-dihydro-3α-methylthiopaederoside (1), 10-O-trans-feruloyl-(4S,6R)-3,4-dihydro-2'-O-3α-paederoside (2), and 10-O-trans-caffeoyl-paederosidic acid ethyl ester (3). The structures of the new compounds were elucidated by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy, as well as high resolution mass spectrometry. The isolated compounds were tested in vitro for cytotoxic activity against five endocrine tumor cell lines. As a result, compound 1 exhibited some cytotoxicities against all the tested tumor cell lines with IC50 value less than 20.0 µM.

Minor alkaloids from an aqueous extract of the hook-bearing stem of Uncaria rhynchophylla.

Seven new monoterpene alkaloids (1 - 7), along with 16 known analogues, were isolated from an aqueous decoction of the hook-bearing stems of Uncaria rhynchophylla (Gou-teng). Their structures were determined by spectroscopic data analysis, single crystal X-ray diffraction, and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 are stereoisomers belonging to a novel type of pseudoindoxyl monoterpene alkaloids, 3 is the first monoterpene furoindole alkaloid from nature, and 4 - 7 are derivatives of the known monoterpene alkaloids featuring different structures.

Compartmentalization of specialized metabolites across vegetative and reproductive tissues in two sympatric Psychotria species.

PREMISE: The specialized metabolites of plants are recognized as key chemical traits in mediating the ecology and evolution of sundry plant-biotic interactions, from pollination to seed predation. Intra- and interspecific patterns of specialized metabolite diversity have been studied extensively in leaves, but the diverse biotic interactions that contribute to specialized metabolite diversity encompass all plant organs. Focusing on two species of Psychotria shrubs, we investigated and compared patterns of specialized metabolite diversity in leaves and fruit with respect to each organ's diversity of biotic interactions. METHODS: To evaluate associations between biotic interaction diversity and specialized metabolite diversity, we combined UPLC-MS metabolomic analysis of foliar and fruit specialized metabolites with existing surveys of leaf- and fruit-centered biotic interactions. We compared patterns of specialized metabolite richness and variance among vegetative and reproductive tissues, among plants, and between species. RESULTS: In our study system, leaves interact with a far larger number of consumer species than do fruit, while fruit-centric interactions are more ecologically diverse in that they involve antagonistic and mutualistic consumers. This aspect of fruit-centric interactions was reflected in specialized metabolite richness-leaves contained more than fruit, while each organ contained over 200 organ-specific specialized metabolites. Within each species, leaf- and fruit-specialized metabolite composition varied independently of one another across individual plants. Contrasts in specialized metabolite composition were stronger between organs than between species. CONCLUSIONS: As ecologically disparate plant organs with organ-specific specialized metabolite traits, leaves and fruit can each contribute to the tremendous overall diversity of plant specialized metabolites.

Complementary roles of hummingbirds and bees: Pollen pickup, pollen deposition, and fruit production in the distylous Palicourea rigida.

PREMISE: Distyly is a condition in which individual plants in a population express two floral morphs, L- and S-morph, characterized by reciprocal placements of anthers and stigmas between morphs. The function of distyly requires that pollinators collect pollen from L- and S-morphs on different parts along their bodies to then deposit it on the stigmas of the opposite morph, known as legitimate pollination. However, different pollinator groups might differ in the ability to transfer pollen legitimately. METHODS: We investigated patterns of pollen pickup along the body of different functional groups (hummingbirds and bees) using preserved specimens to analyze their role in the reproductive success of Palicourea rigida. We measured pollen deposition on the body of pollinators, on stigmas, and fruit production after a single visit. RESULTS: Pollen from L- and S-flowers appeared segregated on different body parts of the hummingbird and bee used in the study. S-pollen was deposited primarily on the proximal regions (near the head), and L-pollen was placed in the distal regions (tip of the proboscis and bill). Hummingbirds were more efficient at legitimate pollination than bees, particularly to S-stigmas. However, fruit formation after single visits by both pollinators was similar. CONCLUSIONS: The morphology of distylous flowers allows the segregated placement of L-and S-pollen on different body parts of the animal specimens used, facilitating the promotion of legitimate pollen transfer, an observation consistent between the two functional pollinator groups. Also, the results show that full fruit set requires more than one visit.

Uncariphyllin A-J, indole alkaloids from Uncaria rhynchophylla as antagonists of dopamine D2 and Mu opioid receptors.

Ten new indole alkaloids (1-10) as well as eleven known analogs (11-21) were isolated from the stems and hooks of Uncaria rhynchophylla. Their structure elucidation was based on extensive NMR studies, MS and ECD data, with the essential aid of DFT prediction of ECD spectra. Compound 1 was determined as a 17,19-seco-cadambine-type alkaloid, and compound 3 was confirmed to be a 3,4-seco-tricyclic monoterpene indole alkaloid, which are the first seco-alkaloids possessing such cleavage positions from U. rhynchophylla. All the isolated compounds were evaluated for their bioactivities on dopamine D2 and Mu opioid receptors for discovering natural therapeutic drugs targeting central nervous system (CNS) diseases. Compounds 1, 2, 4, 5, 20 and 21 showed antagonistic bioactivities on the D2 receptor (IC50 0.678-15.200 µM), and compounds 1, 3, 6, 9, 10, 13, 18, 19 and 21 exhibited antagonistic effects on the Mu receptor (IC50 2.243-32.200 µM). Among them, compounds 1 and 21 displayed dual-target activities. Compound 1 showed conspicuous antagonistic activity on D2 and Mu receptors with the IC50 values of 0.678 ± 0.182 µM and 13.520 ± 2.480 µM, respectively. Compound 21 displayed moderate antagonistic activity on the two receptors with the IC50 values at 15.200 ± 1.764 µM and 32.200 ± 5.695 µM, respectively.

A new iridoid glucoside from the roots of Morinda officinalis.

A new iridoid glucoside, moridoside (1), and nine known compounds, asperulosidic acid (2), 6-O-epi-acetylscandoside (3), geniposidic acid (4), 2-hydroxymethylanthraquinone (5), 2-hydroxymethyl-3-hydroxyanthraquinone (6), damnacanthol (7), lucidine-ω-methyl ether (8), 2-hydroxy-1-methoxyanthraquinone (9), and 3,8-dihydroxy-1,2-dimethoxyanthraquinone (10) were isolated from the methanol extract of Morinda officinalis How. roots. Their structural identification was carried out based on the spectroscopic evidence. All compounds were evaluated for their nitric oxide (NO) production inhibitory activities in LPS-stimulated RAW264.7 macrophages. Compounds 5-7 significantly inhibited the production of NO with IC50 values of 28.4, 33.6, and 30.5 µM, respectively.

Characterization and antimicrobial evaluation of anthraquinones and triterpenes from Rubia cordifolia.

Chemical investigation of roots of the plant, Rubia cordifolia Linn, led to the isolation of an undescribed anthraquinone, cordifoquinone R, determined as 1,2-dihydroxy-6-methoxyanthracene-9,10-dione (6) based on the 1D and 2D NMR analyses and HRESIMS. Ten other known compounds viz.1,4-dihydroxy-2-methoxyanthracene-9,10-dione (1), rubiadin (2), xanthopurpurin (3), 1-methoxy-3-hydroxy-2-carbomethoxy-9,10-anthraquinone (4), alizarin (5), ß-sitosterol glucoside (7), scopoletin (8), oleanolic acid, (9), pomolic acid (10), queretaroic acid (11) were also isolated. Out of these compounds, 4, 10, and 11 are first reported from this plant species. Compounds 2, 3, 6, 7, and 10 showed activity in the range of 16-32 µg/ml against S. aureus ATCC 29213.

Two new iridoid glycosides from Hedyotis diffusa.

Two new iridoid glycosides, named productasperulosidic acid butyl ester (1) and E-6-O-3-hydroxy-p-methoxycinnamoyl scandoside methyl ester (2), along with nine known ones (3-11), were isolated from Hedyotis diffusa Willd. The structures of them were elucidated by extensive 1D, 2D NMR and HR-ESI-MS spectral data. Compounds 1-11 showed no significant cytotoxic activity against HeLa cells.

A complete chloroplast genome of a traditional Chinese medicine herb, Rubia podantha, and phylogenomics of Rubiaceae.

Rubia podantha Diels is endemic to southwestern China and belongs to the family Rubiaceae. It is used in traditional Chinese medicines. To enrich the genetic data and resolve Rubiaceae's phylogeny, we assembled a complete chloroplast (cp) genome of R. podantha using Illumina HiSeq reads. The whole length of the cp genome was 154,866 bp. Annotation using PGA software found 113 genes, including 79 protein coding genes, 30 tRNA genes, and four rRNA genes. The large single-copy region was 84,717 bp, the inverted repeat B (IRa) region was 26,516 bp, the small single copy was 17,117 bp, and the inverted repeats B (IRb) region was 26,516 bp. Moreover, 64 SSRs were identified. Phylogenomic analysis using cp genomes of 109 Rubiaceae species found that R. podantha is closely related to R. cordifola. Rubiaceae was separated into three subfamilies: Ixoroideae, Cinchonoideae, and Rubiodeae. The genus Saprosma was not imbedded within the Spermacoceae alliance as reported in previous studies. Instead, it was imbedded within the Psychotrieae alliance. Divergence time estimation indicated that R. podantha split from its relative R. cordifolia around 1.25 million years ago. The assembled chloroplast genome in this study provided useful molecular information about the evolution of R. podantha and was a basis for phylogenetic analyses of Rubiaceae. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01302-y.

Phylogenomic and comparative analyses of Coffeeae alliance (Rubiaceae): deep insights into phylogenetic relationships and plastome evolution.

BACKGROUND: The large and diverse Coffeeae alliance clade of subfamily Ixoroideae (Rubiaceae) consists of 10 tribes, > 90 genera, and > 2000 species. Previous molecular phylogenetics using limited numbers of markers were often unable to fully resolve the phylogenetic relationships at tribal and generic levels. Also, the structural variations of plastomes (PSVs) within the Coffeeae alliance tribes have been poorly investigated in previous studies. To fully understand the phylogenetic relationships and PSVs within the clade, highly reliable and sufficient sampling with superior next-generation analysis techniques is required. In this study, 71 plastomes (40 newly sequenced and assembled and the rest from the GenBank) were comparatively analyzed to decipher the PSVs and resolve the phylogenetic relationships of the Coffeeae alliance using four molecular data matrices. RESULTS: All plastomes are typically quadripartite with the size ranging from 153,055 to 155,908 bp and contained 111 unique genes. The inverted repeat (IR) regions experienced multiple contraction and expansion; five repeat types were detected but the most abundant was SSR. The size of the Coffeeae alliance clade plastomes and its elements are affected by the IR boundary shifts and the repeat types. However, the emerging PSVs had no taxonomic and phylogenetic implications. Eight highly divergent regions were identified within the plastome regions ndhF, ccsA, ndhD, ndhA, ndhH, ycf1, rps16-trnQ-UUG, and psbM-trnD. These highly variable regions may be potential molecular markers for further species delimitation and population genetic analyses for the clade. Our plastome phylogenomic analyses yielded a well-resolved phylogeny tree with well-support at the tribal and generic levels within the Coffeeae alliance. CONCLUSIONS: Plastome data could be indispensable in resolving the phylogenetic relationships of the Coffeeae alliance tribes. Therefore, this study provides deep insights into the PSVs and phylogenetic relationships of the Coffeeae alliance and the Rubiaceae family as a whole.

Phylogeny of the Madagascar-centred tribe Danaideae (Rubiaceae) as a precursor to taxonomic revision: insights into its generic and species limits, affinities and distribution.

BACKGROUND AND AIMS: The tribe Danaideae (Rubiaceae) is almost exclusively endemic to the Western Indian Ocean Region (WIOR), and encompasses the genera Danais, Payera and Schismatoclada that occur in humid, sub-humid and mountain and mountain bio climate zones. Much of the species diversity is endemic to restricted, remote and/or mountainous areas of Madagascar and recent field work on the island indicates substantial unknown diversity of the Danaideae. Furthermore, the monophyly of the Malagasy genera Payera and Schismatoclada has been questioned in previous work, species delimitations and phylogenetic relationships within the genera are poorly understood, and the distribution and evolution of gross morphological features have not been assessed. METHODS: We conducted morphological investigations, and produced robust phylogenies of Danaideae based on nuclear and plastid sequence data from 193 terminals. Ample plant material has been newly collected in the WIOR for the purpose of the present study, including potentially new species unknown to science. We performed Bayesian non-clock and relaxed-clock analyses employing three alternative clock models of a dataset with a dense sample of taxa from the entire geographical ranges of Danaideae. Based on the results, we discuss species diversity and distribution, relationships, and morphology in Danaideae. KEY RESULTS: Our results demonstrate the monophyly of Danaideae, its three genera and 42 species. Nine species are resolved as non-monophyletic. Many geographically distinct but morphologically heterogeneous lineages were identified, and morphological features traditionally considered diagnostic of subgroups of the genera, used for example in species identification keys, are not clade-specific. CONCLUSIONS: Our results demonstrate that Madagascar contains ample previously undocumented morphological and species diversity of Danaideae. Our novel approach to molecular phylogenetic analyses as a precursor to taxonomic revisions provides numerous benefits for the latter. There are tentative indications of parallel northward diversification in Payera and Schismatoclada on Madagascar, and of geographical phylogenetic clustering despite the anemochorous condition of Danaideae.

Richardia brasiliensis Gomes: phytochemical characterization, antiproliferative capacity and in vitro and in vivo toxicity.

Richardia brasiliensis, known as poaia branca, is a medicinal species widely distributed throughout Brazil and used in folk medicine. However, studies on its toxicity are practically non-existent, and little is known about its biological activity. This study aimed to investigate its phytochemical compounds, assess its in vitro and in vivo toxicities, and determine its antiproliferative activity. UHPLC-ESI-HRFTMS performed the phytochemical characterization, and the antiproliferative activity was analyzed in different tumor cell lines. In vitro toxicity was evaluated in PBMC cells, and in vivo acute and repeated dose toxicity was evaluated according to OECD guidelines. It was identified alkaloids and terpenes as significant compounds. Regarding its antiproliferative activity, the human melanoma strain decreased its viability by about 95%. In vitro toxicity showed that the extracts maintained the viability of PBMCs; however, higher concentrations were able to increase the production of dsDNA quantity. In vivo tests showed no mortality nor signs of toxicity; the alterations found in hematological and biochemical parameters are within the standards for the species. The results indicate that R. brasiliensis has a good effect against the tumor cell line; still, more studies on its toxicity at higher concentrations are needed.

Notes on the Newly Identified Asperula cankiriense B. Sahin & Sagiroglu (Rubiaceae) from Turkey Based on a Light and Scanning Electron Microscopic Study.

Micromorphological features of vegetative and reproductive organs in plants are considered important because they can be used as taxonomic characters. Asperula cankiriense B. Sahin & Sagiroglu, commonly known as "Çankiri belumu", belongs to the family Rubiaceae and generally occurs in gypsum steppes. This research reports for the first time the micromorphological characteristics of the vegetative and reproductive organs of A. cankiriense using light and scanning electron microscopy. Taxonomically critical diagnostic features, such as raphide crystals, presence and shape of trichomes, leaf cross-section shape, flower structure, and epidermal ornamentation, were described in detail. This study provided up-to-date information on the micromorphology of this newly described species and provided additional systematic information on A. cankiriense. New data about the species are reported here and will contribute to the knowledge about the family Rubiaceae.

Chromosomal-level assembly of the Leptodermis oblonga (Rubiaceae) genome and its phylogenetic implications.

Rubiaceae is the fourth largest and a taxonomically complex family of angiosperms. Many species in this family harbor low reproductive isolation and frequently exhibit inconsistent phenotypic characteristics. Therefore, taxonomic classification and their phylogenetic relationships in the Rubiaceae family is challenging, especially in the genus Leptodermis. Considering the low taxonomic confusion and wide distribution, Leptodermis oblonga is selected as a representative Leptodermis for genome sequencing. The assemblies resulted in 497 Mbp nuclear and 155,100 bp chloroplast genomes, respectively. Using the nuclear genome as a reference, SNPs were called from 37 Leptodermis species or varieties. The phylogenetic tree based on SNPs exhibited high resolution for species delimitation of the complex and well-resolved phylogenetic relationships in the genus. Moreover, 28,987 genes were predicted in the nuclear genome and used for comparative genomics study. As the first chromosomal-level genome of the subfamily Rubioideae in Rubiaceae, it will provide fruitfully evolutionary understanding in the family.

Minor monoterpene derivatives from an aqueous extract of the hook-bearing stem of Uncaria rhynchophylla.

Seven new minor monoterpene derivatives (1-7), together with six known analogues, were isolated from an aqueous decoction of the hook-bearing stems of Uncaria rhynchophylla (Gou-teng). Their structures were determined by spectroscopic data analysis and electronic circular dichroism (ECD) calculations, of which 1 was confirmed by single crystal X-ray diffraction.

Bioassay-Guided Isolation of Iridoid Glucosides from Stenaria nigricans, Their Biting Deterrence against Aedes aegypti (Diptera: Culicidae), and Repellency Assessment against Imported Fire Ants (Hymenoptera: Formicidae).

In our natural product screening program, we screened natural products for their repellency and toxicity against insect vectors. Methanolic extract of aerial parts of Stenaria nigricans (Lam.), with no published chemistry, was tested for repellency against mosquitoes and imported hybrid fire ants. Methanolic extracts showed biting deterrence similar to DEET (N,N-diethyl-3-methylbenzamide) against Aedes aegypti L. Based on this activity, the crude extract was fractionated into chloroform, ethyl acetate, and methanol subfractions. The active methanolic subfraction was further fractionated into 13 subfractions. These fractions were tested for their biting deterrence against Ae. Aegypti. Active subfractions were further characterized to identify the compounds responsible for this activity. Four undescribed iridoid glucosides (1-4) and three previously reported compounds (5-7) were isolated from active subfractions and tested for their biting deterrent activity. Based on BDI values, compounds 2, 3, 6, and 7, with biting deterrence similar to DEET, showed the potential to be used as repellents against mosquitoes. In an in vitro digging bioassay, none of these compounds showed any repellency against hybrid imported fire ants at a dose of 125 µg/g. This is the first report of biting deterrence and repellency of S. nigricans extract and its pure compounds, iridoid glucosides against mosquitoes and imported fire ants. Further studies will be conducted to explore the repellent potential of these compounds in different formulations under field conditions.