New Data on Anti-Inflammatory and Wound Healing Potential of Transgenic Senna obtusifolia Hairy Roots: In Vitro Studies.

Asthma is an inflammatory disease whose etiology remains unclear. Its characteristics encompass a wide range of clinical symptoms, inflammatory processes, and reactions to standard therapies. Plants produce a range of constitutive products and secondary metabolites that may have therapeutic abilities. The aim of this study was to determine the effects of Senna obtusifolia transgenic hairy root extracts on virus-induced airway remodeling conditions. Three cell lines were incubated with extracts from transformed (SOA4) and transgenic (SOPSS2, with overexpression of the gene encoding squalene synthase 1) hairy roots of Senna obtusifolia in cell lines undergoing human rhinovirus-16 (HRV-16) infection. The effects of the extracts on the inflammatory process were determined based on the expression of inflammatory cytokines (IL-8, TNF-α, IL-1α and IFN-γ) and total thiol content. The transgenic Senna obtusifolia root extract reduced virus-induced expression of TNF, IL-8 and IL-1 in WI-38 and NHBE cells. The SOPSS2 extract reduced IL-1 expression only in lung epithelial cells. Both tested extracts significantly increased the concentration of thiol groups in epithelial lung cells. In addition, the SOPPS2 hairy root extract yielded a positive result in the scratch test. SOA4 and SOPPS2 Senna obtusifolia hairy root extracts demonstrated anti-inflammatory effects or wound healing activity. The SOPSS2 extract had stronger biological properties, which may result from a higher content of bioactive secondary metabolites.

Molecular and metabolic traits of some Egyptian species of Cassia L. and Senna Mill (Fabaceae-Caesalpinioideae).

The genus Cassia and Senna have been classified under subfamily Caesalpinioideae of family Fabaceae (Leguminosae) of order Fabales. There is a scarce taxonomical studies of the genus Cassia and Senna inhabiting Egyptian environments, thus, the main objective of the current was to revise and authenticate the phylogenetic relationship between studied taxa of the species of the genera Cassia and Senna in Egypt using the recent tools of ITS barcoding, RAPD analysis and metabolic profiling, in comparing to the traditional taxonomical features. From the cluster analysis of the traditional 27 morphological characters, the studied taxa were categorized into two major clades with an average taxonomic distance of 4.3. The clade I include Cassia fistula, C. renigera, C. javanica L subsp. nodosa and C. roughiia that belongs to series Obolospermae, and C. grandis that belongs to series Grandes. The clade (II) includes Senna surattensis and S. alata at taxonomic level 3.6. The taxonomical description of the studied taxa was confirmed from the molecular analysis of ITS sequences and RAPD analysis. The ITS sequences of the tested plants species C. fistula L, C. grandis MD4, C. javanica subsp. nodosa MD7, C. roxburghii MD5, C. renigera MD5 were deposited at genbank with accession numbers MW367973, MZ960447, MW386305, MW326753 and MW32685, respectively. While, the ITS sequences of the S. surrattensis and S. alata were deposited into genbank accession # MD14 MW367670 and MD20 MW412635, respectively. Thus, from the molecular analysis, two clades were clearly separated into Clade I of Cassia and Clade II of Senna. The cluster I represented by C. fistula, C. renigera, C. roxburghii, and C. javanica sub nodosa, and the cluster II represented by S. alata and S. surattensis. From the PCA of RAPD, a clearly discrimination between the two Taxa was observed revealing the characteristic grouping of Cassia and Senna. The species Senna alata and Senna surattensis were grouped together, but the species of C. renigera, C. javanica, C. roxburghii and C. grandis was grouped on a distinct group. The separation of Cassia and Senna species into two clusters verify the segregation of the genus Cassia L. senso lato into two distinct genera namely Senna P. and Cassia L. The morphological, molecular traits of the studied plants were authenticated from the metabolic profiling by GC-MS analysis. Among the 23 identified metabolites, four compounds namely hexadecanoic acid, methyl ester, 9-Octadecenoic acid (Z)-ethyl ester and Vitamin E were detected with fluctuated concentrations, among C. fistula, C. grandis, C. javanica subsp. nodosa and C. roxburghii. Conclusively, the traditional morphological features, molecular barcoding using ITS sequences, RAPD analysis and metabolic traits by GC-MS analysis, authenticates the taxonomical diversity of the genus Cassia and Senna.

Comparative FISH analysis of Senna tora tandem repeats revealed insights into the chromosome dynamics in Senna.

BACKGROUND: DNA tandem repeats (TRs) are often abundant and occupy discrete regions in eukaryotic genomes. These TRs often cause or generate chromosomal rearrangements, which, in turn, drive chromosome evolution and speciation. Tracing the chromosomal distribution of TRs could therefore provide insights into the chromosome dynamics and speciation among closely related taxa. The basic chromosome number in the genus Senna is 2n = 28, but dysploid species like Senna tora have also been observed. OBJECTIVE: To understand the dynamics of these TRs and their impact on S. tora dysploidization. METHODS: We performed a comparative fluorescence in situ hybridization (FISH) analysis among nine closely related Senna species and compared the chromosomal distribution of these repeats from a cytotaxonomic perspective by using the ITS1-5.8S-ITS2 sequence to infer phylogenetic relationships. RESULTS: Of the nine S. tora TRs, two did not show any FISH signal whereas seven TRs showed similar and contrasting patterns to other Senna species. StoTR01_86, which was localized in the pericentromeric regions in all S. tora, but not at the nucleolar organizer region (NOR) site, was colocalized at the NOR site in all species except in S. siamea. StoTR02_7_tel was mostly localized at chromosome termini, but some species had an interstitial telomeric repeat in a few chromosomes. StoTR05_180 was distributed in the subtelomeric region in most species and was highly amplified in the pericentromeric region in some species. StoTR06_159 was either absent or colocalized in the NOR site in some species, and StoIGS_463, which was localized at the NOR site in S. tora, was either absent or localized at the subtelomeric or pericentromeric regions in other species. CONCLUSIONS: These data suggest that TRs play important roles in S. tora dysploidy and suggest the involvement of 45S rDNA intergenic spacers in "carrying" repeats during genome reshuffling.

Genome-enabled discovery of anthraquinone biosynthesis in Senna tora.

Senna tora is a widely used medicinal plant. Its health benefits have been attributed to the large quantity of anthraquinones, but how they are made in plants remains a mystery. To identify the genes responsible for plant anthraquinone biosynthesis, we reveal the genome sequence of S. tora at the chromosome level with 526 Mb (96%) assembled into 13 chromosomes. Comparison among related plant species shows that a chalcone synthase-like (CHS-L) gene family has lineage-specifically and rapidly expanded in S. tora. Combining genomics, transcriptomics, metabolomics, and biochemistry, we identify a CHS-L gene contributing to the biosynthesis of anthraquinones. The S. tora reference genome will accelerate the discovery of biologically active anthraquinone biosynthesis pathways in medicinal plants.

De novo transcriptome sequence of Senna tora provides insights into anthraquinone biosynthesis.

Senna tora is an annual herb with rich source of anthraquinones that have tremendous pharmacological properties. However, there is little mention of genetic information for this species, especially regarding the biosynthetic pathways of anthraquinones. To understand the key genes and regulatory mechanism of anthraquinone biosynthesis pathways, we performed spatial and temporal transcriptome sequencing of S. tora using short RNA sequencing (RNA-Seq) and long-read isoform sequencing (Iso-Seq) technologies, and generated two unigene sets composed of 118,635 and 39,364, respectively. A comprehensive functional annotation and classification with multiple public databases identified array of genes involved in major secondary metabolite biosynthesis pathways and important transcription factor (TF) families (MYB, MYB-related, AP2/ERF, C2C2-YABBY, and bHLH). Differential expression analysis indicated that the expression level of genes involved in anthraquinone biosynthetic pathway regulates differently depending on the degree of tissues and seeds development. Furthermore, we identified that the amount of anthraquinone compounds were greater in late seeds than early ones. In conclusion, these results provide a rich resource for understanding the anthraquinone metabolism in S. tora.

An Extract of Transgenic Senna obtusifolia L. Hairy Roots with Overexpression of PgSS1 Gene in Combination with Chemotherapeutic Agent Induces Apoptosis in the Leukemia Cell Line.

Many biologically-active plant-derived compounds have therapeutic or chemopreventive effects. The use of plant in vitro cultures in conjunction with modern genetic engineering techniques allows greater amounts of valuable secondary metabolites to be obtained without interfering with the natural environment. This work presents the first findings concerning the acquisition of transgenic hairy roots of Senna obtusifolia overexpressing the gene encoding squalene synthase 1 from Panax ginseng (PgSS1) (SOPSS hairy loot lines) involved in terpenoid biosynthesis. Our results confirm that one of PgSS1-overexpressing hairy root line extracts (SOPSS2) possess a high cytotoxic effect against a human acute lymphoblastic leukemia (NALM6) cell line. Further analysis of the cell cycle, the expression of apoptosis-related genes (TP53, PUMA, NOXA, BAX) and the observed decrease in mitochondrial membrane potential also confirmed that the SOPSS2 hairy root extract displays the highest effects; similar results were also obtained for this extract combined with doxorubicin. The high cytotoxic activity, observed both alone or in combination with doxorubicin, may be due to the higher content of betulinic acid as determined by HPLC analysis. Our results suggest synergistic effects of tested extract (betulinic acid in greater amount) with doxorubicin which may be used in the future to develop new effective strategies of cancer chemosensitization.

Authentication of the market samples of Ashwagandha by DNA barcoding reveals that powders are significantly more adulterated than roots.

ETHNOPHARMACOLOGICAL RELEVANCE: Ashwagandha, also known as Indian Ginseng, is a highly traded medicinal plant, which is used in Ayurveda, Siddha and Unani systems of medicine to improve cognitive function, decrease inflammation, and to counter the ill-effects of aging. Withanolide A and Withaferin A from Ashwagandha were shown to improve immunity and have anti-cancer property, respectively. AIM OF THE STUDY: Here, we aimed to create reference DNA barcodes for W. somnifera and to authenticate root and powder samples of Ashwagandha collected from markets. MATERIALS AND METHODS: Three plant specimen of W. somnifera were collected, and reference DNA barcodes were generated using rbcL, matK, trnH-psbA, and ITS2 DNA barcode markers. Market samples in the form of root (n = 33) and powder (n = 70) were collected and authenticated using ITS2 and trnH-psbA DNA barcodes. RESULTS: Genomic DNA was successfully isolated from all plant specimens and market samples. DNA barcoding showed that 77% of samples were authentic. About 22% of non-authentic samples were powder samples and only 1% were root samples. Among the non-authentic samples, 18% were completely substituted with single species (Mucuna pruriens (L.) DC., Trigonella foenum-graceum L., or Senna auriculata (L.) Roxb.) and 82% were mixed samples containing more than one species. About 63% of the mixed samples contained Ashwagandha as the major ingredient. Furthermore, we identified that six taxonomically divergent plant species from four families were present as adulterants in the mixed samples. CONCLUSION: DNA barcoding revealed that botanical adulteration in the market samples of Ashwagandha is significant. Powder samples are more prone to adulteration than root samples. The adulterated samples contained plant material that is not related to Ashwagandha, which warrants strict quantity control and market surveillance to derive the true medicinal benefits of this medicinal plant.

In vitro chondroprotective potential of Senna alata and Senna tora in porcine cartilage explants and their species differentiation by DNA barcoding-high resolution melting (Bar-HRM) analysis.

Senna species and anthraquinone derivatives generated by these organisms, rhein and aloe-emodin, exert anti-inflammatory effects. These species present a similar morphology but produce different ingredients when they are used as medicinal products. In this study, a DNA barcoding- (Bar-) high-resolution melting (HRM) technique was developed using internal transcribed sequence 2 (ITS2) to differentiate between Senna alata and Senna tora as a result of significant differences in their melting profiles. We used this approach for confirmation of S. alata and S. tora raw materials, and we examined the chondroprotective properties of the ethanolic extracts of S. alata and S. tora using a porcine model of cartilage degradation induced by a combination of interleukin-17A (IL-17A) and IL-1ß. We found that both Senna ethanolic extracts, at a concentration of 25 µg/mL, effectively prevented cartilage degradation. Rhein and aloe-emodin were present in the extract of S. alata but not in that of S. tora. We observed a reduction in the release of sulfated glycosaminoglycans (S-GAGs) and hyaluronic acid (HA) into media in both treatments of Senna extracts, which indicated proteoglycan preservation in explant tissues. These results suggest that neither rhein nor aloe-emodin are the main factors responsible for cartilage-protecting properties. Taken together, results show that both S. alata and S. tora are promising for further development as anti-osteoarthritic agents and that Bar-HRM using ITS2 could be applied for species confirmation with Senna products.

Mitochondrial cytochrome oxidase (COI) sequence-based identification of pod borer of Thylacoptila sp. AA isolated from Senna alata (L.) Roxb. for the first time in India.

In the present study, two pod borer larvae were found to damage pods of the host plant Senna alata. As pods (seeds) are commercially important, it prompted the investigators to find out the adult of pod borer larvae by molecular sequencing and reported in this article for the first time from S. alata. Successful amplification was achieved for chloroplast DNA isolated from leaves of host plant S. alata and tissue DNA from unknown larvae and sequenced. The sequences were submitted to NCBI domain, and the taxonomic position of host plant of pod borer larvae was confirmed as S. alata (L.) Roxb. Of the two sequences belonging to pod borer larvae, specimen 1 (S1) matched with already available three sequences of Thylacoptila sp. AA. But, specimen 2 (S2) showed significant variation in its genetic distance in Neighborhood Joining tree and maximum likelihood tree; these factors imply that specimen 2 (S2) is distinct from Thylacoptila sp. AA. Therefore, it is reported that specimen 2 may be represented as Thylacoptila sp. BB where in BB indicates variant from other available sequences. Nonetheless, reporting species of Thylacoptila as insect pest in pods of S. alata is an important contribution to the annals of insect-pest-plant interaction more importantly in medicinally important plant species S. alata.

Exploring drought stress-regulated genes in senna (Cassia angustifolia Vahl.): a transcriptomic approach.

De novo assembly of reads produced by next-generation sequencing (NGS) technologies offers a rapid approach to obtain expressed gene sequences for non-model organisms. Senna (Cassia angustifolia Vahl.) is a drought-tolerant annual undershrub of Caesalpiniaceae, a subfamily of Fabaceae. There are insufficient transcriptomic and genomic data in public databases for understanding the molecular mechanism underlying the drought tolerance of senna. Therefore, the main purpose of this study was to know the transcriptome profile of senna, with special reference to drought stress. RNA from two different stages of leaf development was extracted and sequenced separately using the Illumina technology. A total of 200 million reads were generated, and a de novo assembly of processed reads in the pooled transcriptome using Trinity yielded 43,413 transcripts which were further annotated using NCBI BLAST with "green plant database (txid 33090)," Swiss Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), and Gene Ontology (GO). Out of the total transcripts, 42,280 (95.0 %) were annotated by BLASTX against the green plant database of NCBI. Senna transcriptome showed the highest similarity to Glycine max (41 %), followed by Phaseolus vulgaris (16 %), Cicer arietinum (15 %), and Medicago trancatula (5 %). The highest number of GO terms were enriched for the molecular functions category; of these "catalytic activity" (GO: 0003824) (25.10 %) and "binding activity" (GO: 0005488) (20.10 %) were most abundantly represented. We used InterProscan to see protein similarity at domain level; a total of 33,256 transcripts were annotated against the Pfam domains. The transcripts were assigned with various KEGG pathways. Coding DNA sequences (CDS) encoding various drought stress-regulated pathways such as signaling factors, protein-modifying/degrading enzymes, biosynthesis of phytohormone, phytohormone signaling, osmotically active compounds, free radical scavengers, chlorophyll metabolism, leaf cuticular wax, polyamines, and protective proteins were identified through BLASTX search. The lucine-rich repeat kinase family was the most abundantly found group of protein kinases. Orphan, bHLH, and bZIP family TFs were the most abundantly found in senna. Six genes encoding MYC2 transcription factor, 9-cis-epoxycarotenoid dioxygenase (NCED), l -ascorbate peroxidase (APX), aminocyclopropane carboxylate oxidase (ACO), abscisic acid 8'-hydroxylase (ABA), and WRKY transcription factor were confirmed through reverse transcriptase-PCR (RT-PCR) and Sanger sequencing for the first time in senna. The potential drought stress-related transcripts identified in this study provide a good start for further investigation into the drought adaptation in senna. Additionally, our transcriptome sequences are the valuable resource for accelerated genomics-assisted genetic improvement programs and facilitate manipulation of biochemical pathways for developing drought-tolerant genotypes of crop plants.

Next Generation Sequencing and Transcriptome Analysis Predicts Biosynthetic Pathway of Sennosides from Senna (Cassia angustifolia Vahl.), a Non-Model Plant with Potent Laxative Properties.

Senna (Cassia angustifolia Vahl.) is a world's natural laxative medicinal plant. Laxative properties are due to sennosides (anthraquinone glycosides) natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6.34 Gb of raw nucleotide sequence. The sequence assembly resulted in 42230 and 37174 transcripts with an average length of 1119 bp and 1467 bp for young and mature leaf, respectively. The transcripts were annotated using NCBI BLAST with 'green plant database (txid 33090)', Swiss Prot, Kyoto Encylcopedia of Genes & Genomes (KEGG), Cluster of Orthologous Gene (COG) and Gene Ontology (GO). Out of the total transcripts, 40138 (95.0%) and 36349 (97.7%) from young and mature leaf, respectively, were annotated by BLASTX against green plant database of NCBI. We used InterProscan to see protein similarity at domain level, a total of 34031 (young leaf) and 32077 (mature leaf) transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to 191 KEGG pathways. There were 166 and 159 CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides. Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7%) CDS were up-regulated in young compared to mature leaf. Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites. We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in various secondary metabolite pathways, especially those related to the synthesis of sennosides which will serve as an important platform for public information about gene expression, genomics, and functional genomics in senna.

Assessing product adulteration in natural health products for laxative yielding plants, Cassia, Senna, and Chamaecrista, in Southern India using DNA barcoding.

Medicinal plants such as Cassia, Senna, and Chamaecrista (belonging to the family Fabaceae) are well known for their laxative properties. They are extensively used within indigenous health care systems in India and several other countries. India exports over 5000 metric tonnes per year of these specific herbal products, and the demand for natural health product market is growing at approximately 10-15% annually. The raw plant material used as active ingredients is almost exclusively sourced from wild populations. Consequently, it is widely suspected that the commercial herbal products claiming to contain these species may be adulterated or contaminated. In this study, we have attempted to assess product authentication and the extent of adulteration in the herbal trade of these species using DNA barcoding. Our method includes four common DNA barcode regions: ITS, matK, rbcL, and psbA-trnH. Analysis of market samples revealed considerable adulteration of herbal products: 50% in the case of Senna auriculata, 37% in Senna tora, and 8% in Senna alexandrina. All herbal products containing Cassia fistula were authentic, while the species under the genus Chamaecrista were not in trade. Our results confirm the suspicion that there is rampant herbal product adulteration in Indian markets. DNA barcodes such as that demonstrated in this study could be effectively used as a regulatory tool to control the adulteration of herbal products and contribute to restoring quality assurance and consumer confidence in natural health products.

Diversity and evolution of a trait mediating ant-plant interactions: insights from extrafloral nectaries in Senna (Leguminosae).

BACKGROUND AND AIMS: Plants display a wide range of traits that allow them to use animals for vital tasks. To attract and reward aggressive ants that protect developing leaves and flowers from consumers, many plants bear extrafloral nectaries (EFNs). EFNs are exceptionally diverse in morphology and locations on a plant. In this study the evolution of EFN diversity is explored by focusing on the legume genus Senna, in which EFNs underwent remarkable morphological diversification and occur in over 80 % of the approx. 350 species. METHODS: EFN diversity in location, morphology and plant ontogeny was characterized in wild and cultivated plants, using scanning electron microscopy and microtome sectioning. From these data EFN evolution was reconstructed in a phylogenetic framework comprising 83 Senna species. KEY RESULTS: Two distinct kinds of EFNs exist in two unrelated clades within Senna. 'Individualized' EFNs (iEFNs), located on the compound leaves and sometimes at the base of pedicels, display a conspicuous, gland-like nectary structure, are highly diverse in shape and characterize the species-rich EFN clade. Previously overlooked 'non-individualized' EFNs (non-iEFNs) embedded within stipules, bracts, and sepals are cryptic and may represent a new synapomorphy for clade II. Leaves bear EFNs consistently throughout plant ontogeny. In one species, however, early seedlings develop iEFNs between the first pair of leaflets, but later leaves produce them at the leaf base. This ontogenetic shift reflects our inferred diversification history of iEFN location: ancestral leaves bore EFNs between the first pair of leaflets, while leaves derived from them bore EFNs either between multiple pairs of leaflets or at the leaf base. CONCLUSIONS: EFNs are more diverse than previously thought. EFN-bearing plant parts provide different opportunities for EFN presentation (i.e. location) and individualization (i.e. morphology), with implications for EFN morphological evolution, EFN-ant protective mutualisms and the evolutionary role of EFNs in plant diversification.

Large-scale patterns of diversification in the widespread legume genus Senna and the evolutionary role of extrafloral nectaries.

Unraveling the diversification history of old, species-rich and widespread clades is difficult because of extinction, undersampling, and taxonomic uncertainty. In the context of these challenges, we investigated the timing and mode of lineage diversification in Senna (Leguminosae) to gain insights into the evolutionary role of extrafloral nectaries (EFNs). EFNs secrete nectar, attracting ants and forming ecologically important ant-plant mutualisms. In Senna, EFNs characterize one large clade (EFN clade), including 80% of its 350 species. Taxonomic accounts make Senna the largest caesalpinioid genus, but quantitative comparisons to other taxa require inferences about rates. Molecular dating analyses suggest that Senna originated in the early Eocene, and its major lineages appeared during early/mid Eocene to early Oligocene. EFNs evolved in the late Eocene, after the main radiation of ants. The EFN clade diversified faster, becoming significantly more species-rich than non-EFN clades. The shift in diversification rates associated with EFN evolution supports the hypothesis that EFNs represent a (relatively old) key innovation in Senna. EFNs may have promoted the colonization of new habitats appearing with the early uplift of the Andes. This would explain the distinctive geographic concentration of the EFN clade in South America.

Purification and characterization of a novel cholesterol-lowering protein from the seeds of Senna obtusifolia.

"Juemingzi", a source of traditional Chinese herbal medicine, has been demonstrated to play a role in decreasing serum cholesterol concentration. In this study, a novel protein, which has shown an inhibitory effect on cholesterol biosynthesis, was isolated from Senna obtusifolia L. seed by gel filtration and ion exchange chromatography. The novel protein's molecular mass was 19.7 kD and its pI was 4.80. Both SDS-PAGE and isoelectric-focusing (IEF) revealed a single Coomassie brilliant blue stained band, indicating that the novel protein was a single peptide. The N-terminal amino acid sequence of the protein was IPYISASFPLNIEFLPSE, which had no similarity with any other protein sequences in the NCBI protein database. Circular dichroism (CD) signals indicated that S. obtusifolia seed protein contained 12.5% alpha-helix, 55.6% beta-sheet, and 31.9% random coil.