IN VITRO AND OMICS TECHNOLOGIES OPENS A NEW AVENUE FOR DECIPHERING WITHANOLIDE METABOLISM IN WITHANIA SOMNIFERA

Withania somnifera, commonly known as Ashwagandha or winter cherry, is a principal medicinal plant that has been used in Ayurvedic and native medicine. In view of its varied therapeutic prospective, it has also been the subject of considerable modern scientific attention. Ashwagandha roots are an integral of over 200 formulations in Ayurveda, Siddha and Unani medicine, which are used in the treatment of various physiological disorders. The major chemical constituents of this plant, the withanolides, are a group of naturally occurring C28-steroidal lactones. It has been extensively investigated in terms of chemistry and bioactivity profiling. However, there exists only very little fragmentary evidence about the dynamics of withanolide biosynthesis. This review examines different in vitro approaches that had been carried out over past decade of years and newly developed omics technologies for the large scale production of withanolides as well as for the analysis of genes associated with withanolide biosynthesis.Keywords: Withania somnifera, Withanolides, in vitro cultures, Genomics, Transcriptomics, In silico, Proteomics, Metabolomics

COMPARATIVE CHEMOMETRIC PROFILES BETWEEN LEAF TISSUES OF WITHANIA SOMNIFERA CULTURED IN VITRO AND FIELD

Objective: Metabolomic profiling of herbal extracts is indispensable to standardize drugs and to inaugurate the scientific basis of their therapeutic properties. The present study was attempted with an objective to investigate a comparative GC-MS (Gas chromatography–Mass spectrometry) analysis of in vitro and field grown leaf tissues of “Indian ginseng”. Methods: GC-MS often serves the methods of option for screening and quantitative metabolite profiling. In the present study, metabolic profiling of methanolic extracts of field and in vitro cultured Withania somnifera (Ashwagandha) leaf tissues were carried out using GC–MS technique. Results: A total number of 39 primary metabolites in leaf were identified. These include alcohols, organic acids, purine, pyrimidine, sugars and putrescine. Highly significant qualitative and quantitative differences were noticed between the leaf tissues cultured in vitro and from the field. Especially, significant elevation in the accumulation of GABA (γ amino butyric acid) and putrescine was recorded in in vitro cultured leaf samples. Conclusion: We conclude that in vitro cultures offers an intrinsic advantage to produce therapeutically valuable compounds, relatively in a short span of time and this principle determine its use as an alternative to field grown sample

Identification of Small-Molecule Inhibitors of the Salmonella FraB Deglycase Using a Live-Cell Assay

Nontyphoidal salmonellosis is one of the most significant foodborne diseases in the United States and globally. There are no vaccines available for human use to prevent this disease, and only broad-spectrum antibiotics are available to treat complicated cases of the disease. However, antibiotic resistance is on the rise and new therapeutics are needed. We previously identified the Salmonella fraB gene, that mutation of causes attenuation of fitness in the murine gastrointestinal tract. The FraB gene product is encoded in an operon responsible for the uptake and utilization of fructose-asparagine (F-Asn), an Amadori product found in several human foods. Mutations in fraB cause an accumulation of the FraB substrate, 6-phosphofructose-aspartate (6-P-F-Asp), which is toxic to Salmonella. The F-Asn catabolic pathway is found only in the nontyphoidal Salmonella serovars, a few Citrobacter and Klebsiella isolates, and a few species of Clostridium; it is not found in humans. Thus, targeting FraB with novel antimicrobials is expected to be Salmonella specific, leaving the normal microbiota largely intact and having no effect on the host. We performed high-throughput screening (HTS) to identify small-molecule inhibitors of FraB using growth-based assays comparing a wild-type Salmonella and a Δfra island mutant control. We screened 224,009 compounds in duplicate. After hit triage and validation, we found three compounds that inhibit Salmonella in an fra-dependent manner, with 50% inhibitory concentration (IC50) values ranging from 89 to 150 μM. Testing these compounds with recombinant FraB and synthetic 6-P-F-Asp confirmed that they are uncompetitive inhibitors of FraB with Ki′ (inhibitor constant) values ranging from 26 to 116 μM. IMPORTANCE Nontyphoidal salmonellosis is a serious threat in the United States and globally. We have recently identified an enzyme, FraB, that when mutated renders Salmonella growth defective in vitro and unfit in mouse models of gastroenteritis. FraB is quite rare in bacteria and is not found in humans or other animals. Here, we have identified small-molecule inhibitors of FraB that inhibit the growth of Salmonella. These could provide the foundation for a therapeutic to reduce the duration and severity of Salmonella infections

Metabolite Profiling of In Vitro Cultured and Field Grown Rhizomes of Acorus calamus from Mongolia Using GC-MS

Acorus calamus (sweet flag) is used in the traditional Chinese and Indian medicines for various ailments. Due to its extensive use in herbal medicine, natural resources from the world's forests are being depleted at an alarming rate. In the present study, an in vitro cell culture technique is being explored as an alternative to field grown A. calamus with respect to the metabolite profile, antioxidant properties, total phenol, and total flavonoid content. Gas chromatography mass spectrometry (GC-MS) was utilized to compare the metabolite profiling between methanolic extracts of in vitro and field grown rhizome tissues of A. calamus. A statistical analysis indicated an upregulation of a-selinene, which is representative of sesquiterpene ketones, and a cyclic polyol, D-pinitol, which has an insulin mimicking effect in the in vitro cultivated rhizome tissue when compared to field grown rhizomes. Significantly higher free-radical scavenging activity (IC50 69.32 mu g mL(-1)), total phenolic content (71.60 mg GAE g(-1)), and total flavonoid content (42.34 mg CE g(-1)) were observed in in vitro rhizome tissues compared with those from field grown rhizomes. These observations suggest that the in vitro cultivation of Acorus rhizomes could be exploited as an alternative to field grown A. calamus, as it is an endangered medicinal plant. The production of useful metabolites by the in vitro cultured rhizomes can be explored successfully for utilization by various food and drug industries

Free radical scavenging activity (FRSA), total phenolic contents (TPC) and total flavonoid content (TFC) of <i>W</i>. <i>somnifera</i> roots grown <i>in vitro</i> and in the field.

<p>Each value is shown as the mean ± SD (N = 7). Different letters in the same column indicate a significant difference (<i>p</i><0.05). GAE: gallic acid equivalent, CE: catechin equivalent.</p><p>^a2MFR, 2 months field-grown root; 5MFR, 5 months field-grown root; 1MIR, 1MIR, 1 month <i>in vitro</i> root; 1.5 months <i>in vitro</i> root.</p><p>Free radical scavenging activity (FRSA), total phenolic contents (TPC) and total flavonoid content (TFC) of <i>W</i>. <i>somnifera</i> roots grown <i>in vitro</i> and in the field.</p

Pearson’s correlation coefficients (<i>p</i><0.01) between antioxidant activity (FRSA), total phenolic content (TPC), and total flavonoid content (TFC) of the extracts from roots of <i>W</i>. <i>somnifera</i>.

<p>Pearson’s correlation coefficients (<i>p</i><0.01) between antioxidant activity (FRSA), total phenolic content (TPC), and total flavonoid content (TFC) of the extracts from roots of <i>W</i>. <i>somnifera</i>.</p

Schematic diagram of the metabolic pathway and relative levels of major compounds detected in <i>W</i>. <i>somnifera</i> root extracts.

<p>This was modified from the pathways presented in the KEGG database (<a href="http://www.genome.jp/kegg/" target="_blank">http://www.genome.jp/kegg/</a>). ANOVA was performed to assess the statistical significance of differences between samples (<i>p</i>-value < 0.05). Data are presented as mean values and error bars represent the standard deviation. Different letters represent the statistical significance of metabolite levels.</p

Free Radical Scavenging Activity and Comparative Metabolic Profiling of <i>In Vitro</i> Cultured and Field Grown <i>Withania somnifera</i> Roots

<div><p>Free radical scavenging activity (FRSA), total phenolic content (TPC), and total flavonoid content (TFC) of <i>in vitro</i> cultured and field grown <i>Withania somnifera</i> (Ashwagandha) roots were investigated. Withanolides analysis and comprehensive metabolic profiling between 100% methanol extracts of <i>in vitro</i> and field grown root tissues was performed using high performance thin layer chromatography (HPTLC) and gas chromatography-mass spectrometry (GC-MS), respectively. Significantly higher levels of FRSA, TPC, and TFC were observed in <i>in-vitro</i> cultured roots compared with field grown samples. In addition, 30 day-cultured <i>in vitro</i> root samples (1MIR) exhibited a significantly higher FRSA (IC₅₀ 81.01 μg/mL), TPC (118.91 mg GAE/g), and TFC (32.68 mg CE/g) compared with those in 45 day-cultured samples (1.5MIR). Total of 29 metabolites were identified in <i>in vitro</i> cultured and field grown roots by GC-MS analysis. The metabolites included alcohols, organic acids, purine, pyrimidine, sugars, and putrescine. Vanillic acid was only observed in the <i>in vitro</i> cultured root samples, and higher level of the vanillic acid was observed in 1MIR when compared to 1.5MIR. Therefore, it is suggested that 1MIR might serve as an alternative to field grown roots for the development of medicinal and functional food products.</p></div

Pattern of withanolide accumulation in <i>in vitro</i> and field grown tissues quantified using HPTLC.

<p>The data presented in the figure are the mean ± standard deviation of three replicates obtained from three independent experiments. Different letters in the same column indicate a significant difference (<i>p</i><0.05). Data represents Mean ± Standard deviation of three replicates obtained from three independent experiments.</p><p>^a2MFR, 2 months field-grown root; 5MFR, 5 months field-grown root; 1MIR, 1MIR, 1 month <i>in vitro</i> root; 1.5 months <i>in vitro</i> root.</p><p>Pattern of withanolide accumulation in <i>in vitro</i> and field grown tissues quantified using HPTLC.</p