Evaluation of rohitukine-enriched fraction of Dysoxylum binectariferum Hook.f. (leaves) as anti-arthritic phytopharmaceutical candidate: Chemical standardization, in-vivo validation, formulation development and oral pharmacokinetics.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis is a chronic inflammatory disease of joints. Dysoxylum binectariferum Hook.f (Family: Meliaceae) is a Indian medicinal plant which is traditionally being used to heal inflammation of joints. AIM OF THE STUDY: This work was aimed to carry out chemical standardization, in-vitro/in-vivo validation, oral pharmacokinetics and formulation development of anti-arthritic botanical lead, the rohitukine-enriched fraction of D. binectariferum. MATERIALS AND METHODS: The rohitukine-enriched fraction of D. binectariferum was standardized using four chemical markers and was checked for microbial load, heavy metal content, aflatoxins and pesticides. Its in-vitro inhibitory effect on the lipopolysaccharide (LPS) induced production of pro-inflammatory cytokines TNF-α and IL-6 was studied in THP-1 cells. The in-vivo anti-arthritic activity was investigated in collagen-induced arthritis model in DBA/1J mice. The sustained release capsule formulation was developed and characterized for physicochemical and pharmacokinetic properties. RESULTS: Rohitukine and schumaniofioside A were found to be major chemical constituents of the botanical lead. The rohitukine-enriched fraction of D. binectariferum significantly reduced the production of both pro-inflammatory cytokines TNF-α and IL-6 (>50% inhibition at 3.12 µg/mL) in THP-1 cells. In LPS-treated wild-type mice model, the rohitukine-enriched fraction at 200 mg/kg (PO, QD) completely reduced serum TNF-α levels. In transgenic mice model (collagen-induced arthritis in DBA/1J mice), rohitukine-enriched fraction at 100 mg/kg (PO, QD) dose has resulted in >75% reduction of TNF-α/IL-6 serum levels, 68% reduction in anti-mouse type II collagen IgG1 antibody levels, decreased joint proteoglycan loss and reduced paw edema in DBA/1J mice. The sustained release capsule formulation of rohitukine-enriched fraction showed sustained-release of rohitukine over the period of 24 h, and resulted in an improved plasma-exposure of rohitukine in SD rats. CONCLUSIONS: The data presented herein demonstrated anti-arthritic potential of rohitukine-enriched fraction of D. binectariferum and this study will serve as the benchmark for further research on this botanical lead and developed sustained release capsule formulation.

Discovery and preclinical development of IIIM-160, a Bergenia ciliata-based anti-inflammatory and anti-arthritic botanical drug candidate.

OBJECTIVE: Bergenia ciliata (Haw.) Sternb. is used in the Indian traditional system of medicine to treat various ailments including rheumatism and to heal wounds. The objective of the present study was to perform a preclinical characterization of the B. ciliata-based botanical extract IIIM-160. METHODS: IIIM-160 was chemically standardized and analyzed for heavy metal content, aflatoxins, pesticides and microbial load. The in vitro and in vivo efficacies were determined in suitable models of inflammation, arthritis and nociception. An acute oral toxicity study was performed in Swiss albino mice. A suitable oral formulation was developed and characterized. RESULTS: Bergenin was found to be the major component (9.1% w/w) of IIIM-160. The botanical lead displayed inhibition of lipopolysaccharide-induced production of proinflammatory cytokines in THP-1 cells, with selectivity toward interleukin-6 (IL-6) and had an excellent safety-window. It showed anti-inflammatory, anti-arthritic and antinociceptive activity in animal models and was not toxic at oral doses up to 2 g/kg in Swiss-albino mice. The gastroretentive, sustained-release capsule formulation showed sustained-release of the bergenin over the period of 24 h, resulting in improved plasma-exposure of bergenin in Sprague-Dawley rats. CONCLUSION: The dual-activity of IL-6 inhibition and antinociception marks the suitability of IIIM-160 for treating rheumatoid arthritis. This study will serve as the benchmark for further research on this botanical formulation.

Identification and characterization of some putative genes involved in arabinoxylan biosynthesis in Plantago ovata.

Plantago ovata is an important source of Psyllium (Isabgol), which swells upon contact with water forming mucilaginous mass, largely composed of arabinoxylans. In this study, we analyzed the expression pattern of arabinoxylan biosynthetic pathway genes at different stages of seed development in P. ovata. Besides, arabinoxylans were quantified at different stages of seed development in water extractable and water unextractable fractions. The expression analysis revealed 5-8 fold increase in the levels of expression of some genes involved in arabinoxylan biosynthetic pathway such as UDP-arabinopyranose mutase, UDP-xylosyltransferase 2 and xylan glucuronosyltransferase at 15 days after pollination stage in seed. The xylose and arabinose units were analyzed at different stages of seed development and also in water-soluble (cold water and hot water), alkali and ethanolic fractions. The concentration of xylose and arabinose units increased steadily after pollination. Overall, alkali extract had high concentration of xylose (0.70 ± 0.022 mg/g) and arabinose units (0.10 ± 0.01 mg/g) at 15 days after pollination stage.

Preclinical Development of Crocus sativus-Based Botanical Lead IIIM-141 for Alzheimer's Disease: Chemical Standardization, Efficacy, Formulation Development, Pharmacokinetics, and Safety Pharmacology.

Crocus sativus L. (family: Iridaceae) has been documented in traditional medicine with numerous medicinal properties. Recently, we have shown that C. sativus extract (IIIM-141) displays promising efficacy in a genetic mice (5XFAD) model of Alzheimer's disease (AD) (ACS Chem. Neurosci. 2017, 16, 1756). To translate the available traditional knowledge and the scientifically validated results into modern medicine, herein we aimed to carry out its preclinical development. IIIM-141 is primarily a mixture of crocins containing trans-4-GG-crocin (36 % w/w) as the principal component. The in vitro studies show that IIIM-141 has protective as well as therapeutic properties in assays related to AD. It induces the expression of P-gp, thereby enhancing the amyloid-ß clearance from an AD brain. It also inhibits NLRP3 inflammasome and protects SH-SY5Y cells against amyloid-ß- and glutamate-induced neurotoxicities. In behavioral models, it decreased the streptozotocin-induced memory impairment in rats and recovered the scopolamine-induced memory deficit in Swiss albino mice at 100 mg/kg dose. The acute oral toxicity study shows that IIIM-141 is safe up to the dose of 2000 mg/kg, with no effect on the body weight and on the biochemical/hematological parameters of the rats. The repeated oral administration of IIIM-141 for 28 days at 100 mg/kg dose did not cause any preterminal deaths and abnormalities in Wistar rats. The pharmacokinetic analysis indicated that after oral administration of IIIM-141, the majority of crocin gets hydrolyzed to its aglycone crocetin. The sustained release (SR) capsule formulation was developed, which showed an improved in vitro dissolution profile and a significantly enhanced plasma exposure in the pharmacokinetic study. The SR formulation resulted in 3.3-fold enhancement in the area under the curve of crocetin and doubling of the crocetin/crocin ratio in plasma compared with the extract. The data presented herein will serve as the benchmark for further research on this botanical candidate.

Mapping resistance responses to Sclerotinia infestation in introgression lines of Brassica juncea carrying genomic segments from wild Brassicaceae B. fruticulosa.

Sclerotinia stem rot (Sclerotinia sclerotiorum) is a major disease of Brassica oilseeds. As suitable donors to develop resistant cultivars are not available in crop Brassicas, we introgressed resistance from a wild Brassicaceae species, B. fruticulosa. We produced 206 B. juncea-B. fruticulosa introgression lines (ILs). These were assessed for pollen grain fertility, genome size variations and resistance responses to Sclerotinia following stem inoculations under disease-conducive conditions. Of these, 115 ILs showing normal fertility and genome size were selected for cytogenetic characterization using florescent genomic in situ hybridization (Fl-GISH). B. fruticulosa segment substitutions were indicated in 28 ILs. These were predominantly terminal and located on B-genome chromosomes. A final set of 93 highly fertile and euploid (2n = 36) ILs were repeat-evaluated for their resistance responses during 2014-15. They were also genotyped with 202 transferable and 60 candidate gene SSRs. Association mapping allowed detection of ten significant marker trait associations (MTAs) after Bonferroni correction. These were: CNU-m157-2, RA2G05, CNU-m353-3, CNU-m442-5, ACMP00454-2, ACMP00454-3, EIN2-3-1, M641-1, Na10D09-1 and Na10D11-1. This is the first time such a molecular mapping technique has been deployed with introgression lines carrying genomic segments from B. fruticulosa, and the first to show that they possess high levels of resistance against S. sclerotiorum.

Segregation for fertility and meiotic stability in novel Brassica allohexaploids.

KEY MESSAGE: Allohexaploid Brassica populations reveal ongoing segregation for fertility, while genotype influences fertility and meiotic stability. Creation of a new Brassica allohexaploid species is of interest for the development of a crop type with increased heterosis and adaptability. At present, no naturally occurring, meiotically stable Brassica allohexaploid exists, with little data available on chromosome behaviour and meiotic control in allohexaploid germplasm. In this study, 100 plants from the cross B. carinata × B. rapa (A2 allohexaploid population) and 69 plants from the cross (B. napus × B. carinata) × B. juncea (H2 allohexaploid population) were assessed for fertility and meiotic behaviour. Estimated pollen viability, self-pollinated seed set, number of seeds on the main shoot, number of pods on the main shoot, seeds per ten pods and plant height were measured for both the A2 and H2 populations and for a set of reference control cultivars. The H2 population had high segregation for pollen viability and meiotic stability, while the A2 population was characterised by low pollen fertility and a high level of chromosome loss. Both populations were taller, but had lower average fertility trait values than the control cultivar samples. The study also characterises fertility and meiotic chromosome behaviour in genotypes and progeny sets in heterozygous allotetraploid Brassica derived lines, and indicates that genotypes of the parents and H1 hybrids are affecting chromosome pairing and fertility phenotypes in the H2 population. The identification and characterisation of factors influencing stability in novel allohexaploid Brassica populations will assist in the development of this as a new crop species for food and agricultural benefit.

Development and molecular-genetic characterization of a stable Brassica allohexaploid.

KEY MESSAGE: We report first-time synthesis of a stable Brassica allohexaploid. It may evolve into a new species and also advance our understanding of pairing regulation and genome evolution in complex allopolyploids. Crop Brassicas include both monogenomic and digenomic species. A trigenomic Brassica (AABBCC) is not known to exist in nature. Past attempts to synthesize a stable allohexaploid were not successful due to aberrant meiosis and very high proportion of aneuploid plants in the selfed progenies. We report the development of a stable allohexaploid Brassica (2n = 54; AABBCC). Genomic in situ hybridization confirmed the complete assemblage of three genomes. Only allohexaploids involving B. rapa cv. R01 (2n = 20; AA) as pollinator with a set of B. carinata (2n = 34; BBCC) were stable. These exhibited a high proportion (0.78-0.94) of pollen mother cells with normal meiosis and an excellent hexaploid ratio (0.80-0.94) in the selfed progenies. Stability of two allohexaploid combinations was demonstrated from H1 to H4 generations at two very diverse locations in India. Graphical genotyping of allohexaploids allowed detection of chromosome fragment exchanges among three genomes. These were much smaller for meiotically stable allohexaploids as compared to unstable ones. The putative hexaploids were morphologically closer to the female donor, B. carinata, for leaf morphology, inflorescence structure and flower shape. The newly formed allohexaploid may also provide unique opportunities to investigate the immediate genetic and genomic consequences of a Brassica allohexaploid with three resident genomes.

De Novo Transcriptome Analysis of Medicinally Important Plantago ovata Using RNA-Seq.

Plantago ovata is an economically and medicinally important plant of the family Plantaginaceae. It is used extensively for the production of seed husk for its application in pharmaceutical, food and cosmetic industries. In the present study, the transcriptome of P. ovata ovary was sequenced using Illumina Genome Analyzer platform to characterize the mucilage biosynthesis pathway in the plant. De novo assembly was carried out using Oases followed by velvet. A total of 46,955 non-redundant transcripts (≥100 bp) using ~29 million high-quality paired end reads were generated. Functional categorization of these transcripts revealed the presence of several genes involved in various biological processes like metabolic pathways, mucilage biosynthesis, biosynthesis of secondary metabolites and antioxidants. In addition, simple sequence-repeat motifs, non-coding RNAs and transcription factors were also identified. Expression profiling of some genes involved in mucilage biosynthetic pathway was performed in different tissues of P. ovata using Real time PCR analysis. The study has resulted in a valuable resource for further studies on gene expression, genomics and functional genomics in P. ovata.