Photostability of sennosides and their aglycones in solution.

INTRODUCTION: Sennosides are the main active constituents of the dried leaves and/or pods of Senna alexandrina Mill. that are used as laxatives. A hypothesis is that aglycones are formed during the degradation of sennosides. However, it is unknown, whether this happens under visible light exposure and how photosensitive sennosides behave in solution. OBJECTIVES: Pure anthraquinone glycosides were tested on their behaviour during sample preparation in the lab under visible light exposure in dependence on the instability of the solvent. MATERIALS AND METHODS: Samples before and after exposure were analysed using UHPLC with UV/Vis and MS detection. RESULTS: Under visible light protection, the solutions were stable for 14 days at room temperature whereas a loss of 20%-60% was measured after 1 day of light exposure. The loss of sennosides due to degradation can be as fast as up to 2%-2.5% per hour, which might have a tremendous impact on phytochemical analysis results during the course of an analysis. The formation of aglycones was not observed in the degradation of sennosides and rhein-8-O-glucoside. CONCLUSION: Aglycones could not be found as a result of the forced degradation. The solutions of sennosides clearly need to be protected from light to obtain reliable analytical results, and light protection is a major point for the stability of liquid preparations.

Methyl Jasmonate Effect on Betulinic Acid Content and Biological Properties of Extract from Senna obtusifolia Transgenic Hairy Roots.

It is known that Senna obtusifolia has been used in medicine since ancient times due to the content of many valuable compounds with a pro-health effect. One of them is betulinic acid, which is a pentacyclic triterpene with antimalarial, antiviral, anti-inflammatory and anticancer properties. In this work, a continuation of our previous research, an attempt was made to increase the level of betulinic acid accumulation by the cultivation of transgenic hairy roots that overexpress the squalene synthase gene in a 10 L sprinkle bioreactor with methyl jasmonate elicitation. We present that the applied strategy allowed us to increase the content of betulinic acid in hairy root cultures to the level of 48 mg/g dry weight. The obtained plant extracts showed a stronger cytotoxic effect on the U87MG glioblastoma cell line than the roots grown without elicitors. Additionally, the induction of apoptosis, reduction of mitochondrial membrane potential, chromosomal DNA fragmentation and activation of caspase cascades are demonstrated. Moreover, the tested extract showed inhibition of topoisomerase I activity.

Enhanced Accumulation of Betulinic Acid in Transgenic Hairy Roots of Senna obtusifolia Growing in the Sprinkle Bioreactor and Evaluation of Their Biological Properties in Various Biological Models.

Betulinic acid, which is found in transgenic roots of Senna obtusifolia (L.) H.S.Irwin & Barneby, is a pentacyclic triterpene with distinctive pharmacological activities. In this study, we report the differences in the content of betulinic acid and selected anthraquinones in transgenic S. obtusifolia hairy roots with overexpression of the PgSS1 gene (SOPSS2 line) and in transformed hairy roots without this genetic construct (SOA41 line). Both hairy root lines grew in 10 L sprinkle bioreactor. Additionally, the extracts obtained from this plant material were used for biological tests. Our results demonstrated that the SOPSS2 hairy root cultures from the bioreactor showed an increase in the content of betulinic acid (38.125 mg/g DW), compared to the SOA41 hairy root line (4.213 mg/g DW). Biological studies have shown a cytotoxic and antiproliferative effect on U-87MG glioblastoma cells, and altering the level of apoptotic proteins (Bax, p53, Puma and Noxa). Antimicrobial properties were demonstrated for both tested extracts, with a stronger effect of SOPSS2 extract. Moreover, both extracts showed moderate antiviral properties on norovirus surrogates.

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.

Folium Sennae and emodin reverse airway smooth muscle contraction.

The objective of this project was to find a bronchodilatory compound from herbs and clarify the mechanism. We found that the ethanol extract of Folium Sennae (EEFS) can relax airway smooth muscle (ASM). EEFS inhibited ASM contraction, induced by acetylcholine, in mouse tracheal rings and lung slices. High-performance liquid chromatography assay showed that EEFS contained emodin. Emodin had a similar reversal action. Acetylcholine-evoked contraction was also partially reduced by nifedipine (a selective inhibitor of L-type voltage-dependent Ca2+ channels, LVDCCs), YM-58483 (a selective inhibitor of store-operated Ca2+ entry, SOCE), as well as Y-27632 (an inhibitor of Rho-associated protein kinase). In addition, LVDCC- and SOCE-mediated currents and cytosolic Ca2+ elevations were inhibited by emodin. Emodin reversed acetylcholine-caused increases in phosphorylation of myosin phosphatase target subunit 1. Furthermore, emodin, in vivo, inhibited acetylcholine-induced respiratory system resistance in mice. These results indicate that EEFS-induced relaxation results from emodin inhibiting LVDCC, SOCE, and Ca2+ sensitization. These findings suggest that Folium Sennae and emodin may be new sources of bronchodilators.

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.

Physiological, transcriptional, and metabolic alterations in spaceflight-subjected Senna obtusifolia.

Senna obtusifolia is a widely used medicinal herb in Asian countries. To select elite cultivars, S. obtusifolia seeds were carried by "ShenZhou Ⅷ" recoverable satellite to space. Three spaceflight-subjected lines (SP-lines), namely QC10, QC29, QC46, and their ground control line (GC-line) were cultivated on the ground. Previous studies demonstrated that biological traits and secondary metabolites are different between SP-lines and GC-line. Here, we combined physiological, transcriptional, and metabolic studies to compare the differences between SP-lines and GC-line. The results showed that activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and monodehydroascorbate reductase (MDHAR) were dramatically increased in SP-lines as compared to that of GC-line. Transcript levels of SOD, POD, CAT, APX, and MDHAR were significantly up-regulated in SP-lines. Malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents decreased in SP-lines. Seed yields of QC29 and QC46 were considerably higher than that of GC-line. Besides, QC29 had significantly higher aurantio-obtusin content. Pearson correlation coefficient analysis revealed positive relationships between POD and aurantio-obtusin, as well as APX and aurantio-obtusin. In conclusion, SP-lines have higher antioxidant gene expression level and antioxidant enzyme activity as compared to that of GC-line. With higher seed yield and aurantio-obtusin content, QC29 can be used to breed elite S. obtusifolia cultivars. This study provides a new insight in SP-lines and paves the way to breed elite S. obtusifolia cultivars in the future.

High CO2 effects on growth and biometal contents in the pioneer species Senna reticulata: climate change predictions.

The aim of the present study consisted in evaluating the effects of CO2 enrichment on the growth and biometal/nutrient content and accumulation in Senna reticulata germinated under two different carbon dioxide concentrations: atmospheric (360 mg L-1) and elevated (720 mg L-1). Biometal/nutrient determinations were performed on three different plant portions (leaflets, stem and root) using flame atomic absorption spectrometry. In general, the biometal and nutrient stoichiometries in roots were increased, probably due to reduced transpiration, and consequent biometal accumulation. An Artifical Neural Network analysis suggests that Mg, Na and Fe display the most different behavior when comparing plants germinated at atmospheric and elevated CO2 conditions. Biomass and growth increases and certain elemental levels indicate that S. reticulata benefits from increased CO2 levels, however some results indicate the contrary, making further studies in this context necessary, as these changes may lead to direct effects on food safety, crop yields, and phytoremediation efficiency.

Piriformospora indica, an excellent system for heavy metal sequestration and amelioration of oxidative stress and DNA damage in Cassia angustifolia Vahl under copper stress.

Present investigation reveals copper induced phytotoxicity, oxidative stress and DNA damage in Cassia angustifolia Vahl and its amelioration by employing a symbiotic fungus, Piriformospora indica. Seeds were germinated on Knop's medium containing five Cu levels (0, 1, 10, 50, 100 and 200 mg L-1), with and without P. indica. Colonization with P. indica significantly (P < 0.05) ameliorated Cu induced oxidative stress. However, maximum amelioration was observed at 50 mg L-1 Cu with P. indica. Atomic absorption spectroscopy revealed that P. indica colonization significantly inhibited Cu accumulation in shoots. Maximum decline in Cu accumulation in shoots was observed at 50 mg L-1 (27.27%) with P. indica over Cu alone. Besides, P. indica colonized seedlings stored 16.86% higher Cu in roots as compared to Cu alone at 200 mg L-1. Similarly, maximum proline accumulation increased up to 19.32% over Cu alone at 50 mg L-1 Cu with P. indica. Significant elevation in antioxidant enzyme levels of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase and glutathione reductase was seen with P. indica. Contrary to increase in antioxidant level, toxic parameters such as lipid peroxidation and hydrogen peroxide decreased significantly with P. indica. Maximum decline in lipid peroxidation (13.76%) and hydrogen peroxide (18.58%) was observed at 50 mg L-1 with P. indica over Cu alone. P. indica significantly reduced DNA damage as well as changed the protein profile in C. angustifolia seedlings. Thus, P. indica proved to be an excellent system to alleviate Cu induced oxidative stress and might be useful as a phytostabilization tool.

Measuring metabolic rates of small terrestrial organisms by fluorescence-based closed-system respirometry.

We explore a recent, innovative variation of closed-system respirometry for terrestrial organisms, whereby oxygen partial pressure (PO2 ) is repeatedly measured fluorometrically in a constant-volume chamber over multiple time points. We outline a protocol that aligns this technology with the broader literature on aerial respirometry, including the calculations required to accurately convert O2 depletion to metabolic rate (MR). We identify a series of assumptions, and sources of error associated with this technique, including thresholds where O2 depletion becomes limiting, that impart errors to the calculation and interpretation of MR. Using these adjusted calculations, we found that the resting MR of five species of angiosperm seeds ranged from 0.011 to 0.640 ml g-1 h-1, consistent with published seed MR values. This innovative methodology greatly expands the lower size limit of terrestrial organisms that can be measured, and offers the potential for measuring MR changes over time as a result of physiological processes of the organism.

The Activity of the Antioxidant Defense System of the Weed Species Senna obtusifolia L. and its Resistance to Allelochemical Stress.

Senna obtusifolia L., a common weed in the tropical and subtropical regions of the world, is able to germinate under adverse environmental conditions, suggesting that this species has efficient stress-adaptation strategies. The aims of the present work were to examine the energy metabolism and the antioxidant defense system of the Senna obtusifolia L. during seed germination and initial growth, and the responses to allelochemical-induced stress. Respiratory activity, the activities of alcohol dehydrogenase (ADH), superoxide dismutase (SOD), catalase (CAT),guaicol peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), lipoxygenase (LOX) and the content of malondialdehyde (MDA) and glutathione (GSSG and GSH) were measured. Shortly after seed imbibition, mitochondrial respiratory activity was active and the presence of SOD, CAT, GR and LOX activity in embryos, along with significant KCN-insensitive respiration, indicated that the production of reactive oxygen species (ROS) is initiated as soon as mitochondrial respiration resumes. Among the fourteen allelochemicals assayed, only coumarin significantly supressed the growth of S. obtusifolia seedlings. Although coumarin reduced the activities of CAT, POD and APX, the GSH, GSSG and MDA levels were not altered. Alpha-pinene, quercetin and ferulic acid did not modify the activity of the antioxidant enzymes or the contents of GSH, GSSH and MDA. Thus the antioxidant defense system of S. obstusifolia may be effective in counteracting the harmful effects of ROS generated during seed germination and initial growth in the presence of toxic allelochemicals.

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.

Synthetic seed production and physio-biochemical studies in Cassia angustifolia Vahl. - a medicinal plant.

Synthetic seed technology is an alternative to traditional micropropagation for production and delivery of cloned plantlets. Synthetic seeds were produced by encapsulating nodal segments of C. angustifolia in calcium alginate gel. 3% (w/v) sodium alginate and 100 mM CaCl2 · 2H2O were found most suitable for encapsulation of nodal segments. Synthetic seeds cultured on half strength Murashige and Skoog medium supplemented with thidiazuron (5.0 µM) + indole-3-acetic acid (1.0 µM) produced maximum number of shoots (10.9 ± 0.78) after 8 weeks of culture exhibiting (78%) in vitro conversion response. Encapsulated nodal segments demonstrated successful regeneration after different period (1-6 weeks) of cold storage at 4 °C. The synthetic seeds stored at 4 °C for a period of 4 weeks resulted in maximum conversion frequency (93%) after 8 weeks when placed back to regeneration medium. The isolated shoots when cultured on half strength Murashige and Skoog medium supplemented with 1.0 µM indole-3-butyric acid (IBA), produced healthy roots and plantlets with well-developed shoot and roots were successfully hardened off in plastic pots containing sterile soilrite inside the growth chamber and gradually transferred to greenhouse where they grew well with 85% survival rate. Growth performance of 2 months old in vitro-raised plant was compared with in vivo seedlings of the same age. Changes in the content of photosynthetic pigments, net photosynthetic rate (PN), superoxide dismutase and catalase activity in C. angustifolia indicated the adaptation of micropropagated plants to ex vitro conditions.

Liquid chromatographic determination of sennosides in Cassia angustifolia leaves.

A simple liquid chromatographic method was developed for the determination of sennosides B and A in leaves of Cassia angustifolia. These compounds were extracted from leaves with a mixture of methanol-water (70 + 30, v/v) after defatting with hexane. Analyte separation and quantitation were achieved by gradient reversed-phase liquid chromatography and UV absorbance at 270 nm using a photodiode array detector. The method involves the use of an RP-18 Lichrocart reversed-phase column (5 microm, 125 x 4.0 mm id) and a binary gradient mobile-phase profile. The various other aspects of analysis, namely, peak purity, similarity, recovery, repeatability, and robustness, were validated. Average recoveries of 98.5 and 98.6%, with a coefficient of variation of 0.8 and 0.3%, were obtained by spiking sample solution with 3 different concentration solutions of standards (60, 100, and 200 microg/mL). Detection limits were 10 microg/mL for sennoside B and 35 microg/mL for sennoside A, present in the sample solution. The quantitation limits were 28 and 100 microg/mL. The analytical method was applied to a large number of senna leaf samples. The new method provides a reliable tool for rapid screening of C. angustifolia samples in large numbers, which is needed in breeding/genetic engineering and genetic mapping experiments.

Sennosides A and B production by hairy roots of Senna alata (L.) Roxb.

Hairy roots of Senna alata transformed with Agrobacterium rhizogenes, strain ATCC 15834 were induced and grown in half-strength Murashige and Skoog (MS) medium. Effects of sucrose contents and hormones on the growth and sennosides A, B production were investigated. Hairy roots cultured on hormone-free half-strength MS medium containing 5% sucrose under dark condition mostly stimulated the growth of hairy roots and increased the content of sennosides A and B yielding (169 +/- 4) and (34 +/- 3) microg g(-1) dry wt, respectively.