Rapid metabolic fingerprinting with the aid of chemometric models to identify authenticity of natural medicines: Turmeric, Ocimum, and Withania somnifera study.

Herbal medicines are popular natural medicines that have been used for decades. The use of alternative medicines continues to expand rapidly across the world. The World Health Organization suggests that quality assessment of natural medicines is essential for any therapeutic or health care applications, as their therapeutic potential varies between different geographic origins, plant species, and varieties. Classification of herbal medicines based on a limited number of secondary metabolites is not an ideal approach. Their quality should be considered based on a complete metabolic profile, as their pharmacological activity is not due to a few specific secondary metabolites but rather a larger group of bioactive compounds. A holistic and integrative approach using rapid and nondestructive analytical strategies for the screening of herbal medicines is required for robust characterization. In this study, a rapid and effective quality assessment system for geographical traceability, species, and variety-specific authenticity of the widely used natural medicines turmeric, Ocimum, and Withania somnifera was investigated using Fourier transform near-infrared (FT-NIR) spectroscopy-based metabolic fingerprinting. Four different geographical origins of turmeric, five different Ocimum species, and three different varieties of roots and leaves of Withania somnifera were studied with the aid of machine learning approaches. Extremely good discrimination (R2 > 0.98, Q2 > 0.97, and accuracy = 1.0) with sensitivity and specificity of 100% was achieved using this metabolic fingerprinting strategy. Our study demonstrated that FT-NIR-based rapid metabolic fingerprinting can be used as a robust analytical method to authenticate several important medicinal herbs.

Precursors and elicitor induced enhancement of cell biomass and phenolic compounds in cell suspensions of Indian basil-Ocimum basilicum (CIM-Saumya).

CIM-Saumya is an improved, methyl chavicol rich variety of Ocimum basilicum (Family-Lamiaceae), developed by Council of Scientific and Industrial Research-Central Institute of Medicinal and Aromatic Plants. This plant possesses analgesic, anti-ulcerogenic, anti-inflammatory, anti-oxidant, cardiac stimulant, Central Nervous System depressant, hepatoprotective and immunomodulator activities due to the presence of various phytoconstituents. Among them rosmarinic acid, caffeic acid and ferulic acid are the three major phenolic compounds responsible for its therapeutic utility. These compounds are produced in very low amounts in the in vivo plants. Therefore, the present study has been conducted for establishment of cell suspensions, optimization of inoculums size, growth kinetics and screening of elicitor and precursors for the accumulation of cell biomass and the production of the three important phenolic compounds in cell suspension of O. basilicum (CIM-Saumya). Leaf derived friable callus was used for establishing the cell suspension in liquid Murashige and Skoog's medium fortified with 1 g/L casein hydrolysate + 2.26 µM 2,4-dichlorophenoxyacetic acid + 0.465 µM kinetin + 2.68 µM naphthalene acetic acid. The growth kinetic analysis pattern of cell suspension revealed the maximum biomass increments (% BI = 486.7) and production of RA 8.086 mg/g dry weight was found in 30th day harvested cells. Whereas, the other two phenolic compounds i.e. ferulic acid (0.0125 mg/g dry weight) and caffeic acid (0.38 mg/g dry weight) was recorded highest on 25th day of growth cycle. In the present study, one biotic elicitor i.e. yeast extract and three precursors [peptone, tryptone and lactalbumin hydrolysate] were tested, among them, lactalbumin hydrolysate (100 mg/L; added at 16th day) treated cells recorded highest estimated phenolic compounds yield (251.5 mg/L; 6.81 fold compared to the control) and biomass increments i.e. % BI = 1207 with 1.85 fold compared to the control. The highest rosmarinic acid content i.e. 25.47 mg/g DW (4.4 fold compared to the control) and 24.42 mg/g dry weight (4.1 folds compared to the control) was noticed in 30th day harvested cells treated with yeast extract (1 g/L on 0 day) and lactalbumin hydrolysate (100 mg/L added on 16th day), respectively. While caffeic acid content (0.91 mg/g dry weight) showed 2.9 folds higher compared to the control in cells treated with peptone 200 mg/L added on 16th day of culture cycle. All the treated cells showed enhanced phenylalanine ammonia-lyase enzyme activity with highest specific activity in lactalbumin hydrolysate followed by tryptone, peptone, and yeast extract. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01316-6.

Towards the development of phytoextract based healthy ageing cognitive booster formulation, explored through Caenorhabditis elegans model.

The positive effect of herbal supplements on aging and age-related disorders has led to the evolution of natural curatives for remedial neurodegenerative diseases in humans. The advancement in aging is exceedingly linked to oxidative stress. Enhanced oxidative stress interrupts health of humans in various ways, necessitating to find stress alleviating herbal resources. Currently, minimal scientifically validated health and cognitive booster resources are available. Therefore, we explored the impact of plant extracts in different combinations on oxidative stress, life span and cognition using the multicellular transgenic humanized C. elegans, and further validated the same in Mus musculus, besides testing their safety and toxicity. In our investigations, the final product-the HACBF (healthy ageing cognitive booster formulation) thus developed was found to reduce major aging biomarkers like lipofuscin, protein carbonyl, lipid levels and enhanced activity of antioxidant enzymes. Further confirmation was done using transgenic worms and RT-PCR. The cognitive boosting activities analyzed in C. elegans and M. musculus model system were found to be at par with donepezil and L-dopa, the two drugs which are commonly used to treat Parkinson's and Alzheimer's diseases. In the transgenic C. elegans model system, the HACBF exhibited reduced aggregation of misfolded disease proteins α-synuclein and increased the health of nicotinic acetylcholine receptor, levels of Acetylcholine and Dopamine contents respectively, the major neurotransmitters responsible for memory, language, learning behavior and movement. Molecular studies clearly indicate that HACBF upregulated major genes responsible for healthy aging and cognitive booster activities in C. elegans and as well as in M. musculus. As such, the present herbal product thus developed may be quite useful for healthy aging and cognitive boosting activities, and more so during this covid-19 pandemic. Supplementary Information: The online version contains supplementary material available at 10.1007/s13237-022-00407-1.

Altered Developmental and Metabolic Gene Expression in Basil Interspecific Hybrids.

To understand the altered developmental changes and associated gene expression in inter-genomic combinations, a study was planned in two diverse yet closely related species of Ocimum, targeting their hybrid F1 and amphidiploids. The existing developmental variations between F1 and amphidiploids was analyzed through phenotypical and anatomical assessments. The absence of 8330 transcripts of F1 in amphidiploids and the exclusive presence of two transcripts related to WNK lysine-deficient protein kinase and geranylgeranyl transferase type-2 subunit beta 1-like proteins in amphidiploids provided a set of genes to compare the suppressed and activated functions between F1 and amphidiploids. The estimation of eugenol and methyleugenol, flavonoid, lignin and chlorophyll content was correlated with the average FPKM and differential gene expression values and further validated through qRT-PCR. Differentially expressed genes of stomatal patterning and development explained the higher density of stomata in F1 and the larger size of stomata in amphidiploids. Gene expression study of several transcription factors putatively involved in the growth and developmental processes of plants clearly amalgamates the transcriptome data linking the phenotypic differences in F1 and amphidiploids. This investigation describes the influence of interspecific hybridization on genes and transcription factors leading to developmental changes and alleviation of intergenomic instability in amphidiploids.

Genetic improvement of pyrethrum (Tanacetum cinerariifolium Sch. Bip.) through gamma radiation and selection of high yield stable mutants through seven post-radiation generations.

PURPOSE: To increase the size of the flowers for easy plucking, flower yield, pyrethrins content (%), and elite mutant selection in pyrethrum. MATERIALS AND METHODS: To increase pyrethrum production and acclimatize in north Indian plain condition, a genetic improvement program was undertaken to widen the range of variations for size and yield of flowers and pyrethrins content (%) in pyrethrum crop. Pyrethrum seeds of the variety Avadh were irradiated with gamma rays at 20 to 300 Gy doses in Gamma chamber 5000 (cobalt-60 research irradiator). RESULTS: Observations gathered visually in M1 based on vigor, synchronization of flowering, and flower's size. Out of 90 M2 families, 20 mutants were raised in M3 along with the check-in preliminary evaluation trial. The four promising mutants, 1 (20 Gy-3), 7 (40 Gy-5), 10 (40 Gy-8), 14 (60 Gy19-10) was grown for four years in a bench-scale trial (randomized block design, replicated thrice) to test the yield performance and selection of high yielding elite mutant (s). It has been found that pyrethrum is sensitive to gamma rays irradiation and produced a high range of qualitative and quantitative variations. After massive screening over four years, two promising mutants for high dry flower yield and pyrethrins content, namely 7 (40 Gy-5), and 10 (40GY-8) were isolated. CONCLUSIONS: The mutagenesis changed traits mean in positive or negative directions. Pyrethrum plant is highly sensitive to gamma irradiation and produced a high range of variability in the qualitative and quantitative traits. The mutagenesis changed the mean of traits in both positive and negative directions. Due to mutagenic efficacy, two mutants 7 (40 Gy-5), and 10 (40GY-8) were expressed high performance for pyrethrin percent i.e., 87.23 and 59.78% improvement over the check variety 'Avadh', with synchronous flowering. These two mutants are in the pipeline for release as a variety for cultivation in the North Indian plains.

In Silico Identification of miRNA and Targets from Chrysopogon zizanioides (L.) Roberty with Functional Validation from Leaf and Root Tissues.

microRNAs are small non-coding RNA molecule that plays an important role in metabolism. Chrysopogon zizanioides (L.) Roberty is an important aromatic plant used in perfumery industries, soil, water conservation, and agricultural practices. In this study, the transcriptomic sequence of vetiver leaf and root was subjected to miRNA identification by the computational methods. miRNA identification was carried out using a homology-based method by C-mii software with several other online tools. A total of 80 miRNA were identified from both leaf and root sequences. Target identification was done by identified miRNA sets. A total of 25 and 31 miRNA families were identified in both leaf and root, respectively, with ten common families involve in different ontological function. miR169 and miR5021 regulate most of the target in leaf and root. In vetiver, many primary and secondary metabolism elements are regulated by miRNA as photo-system, transcription factor, terpenoid metabolism, etc. Here is the first in silico study revealing the specific miRNAs and their target genes for corresponding root and leaf tissues respectively in C. zizanioides.

Induced polygenic variations through γ -rays irradiation and selection of novel genotype in chamomile (Chamomilla recutita [L.] Rauschert).

Purpose: To develop elite mutants in chamomile (Chamomilla recutita [L.] Rauschert) for increasing the quantity and quality of essential oil rich in acetylenic compound (2Z,8Z)-matricaria acid methyl ester by applying γ -rays irradiation. Molecular and chemical analysis was performed for ithe dentification/differentiation of mutant genotype. Materials and methods: Chamomile (Chamomilla recutita [L.] Rauschert) variety Vallary seeds were irradiated by applying γ -rays irradiation at 100, 200, 300, 400, 500, 600, 700, 800, 900 and1000 Gy doses at a dose rate of 55 Gy/min and mutants were isolated and analyzed for the quantity and quality of essential oil. The oil was found to be rich in acetylenic compound (2Z,8Z)-matricaria acid methyl ester and the results obtained were validated using gas chromatography (GC) coupled with either Flame Ionization detection (GC-FID) or mass spectrometer (GC-MS), and nuclear magnetic resonance (NMR). Results: The selected mutant SELM-1 (Selection Mutant-1) showed the production potential of 7.00-7.50 q ha-1 dry flowers and 6.00-6.50 kg ha-1 essential oil yield. Essential oil of mutant SELM-1 contained in [(2Z,8Z)-matricaria acid methyl ester] (76-80%) useful in cosmetic, perfumery, and pharmaceutical industries. Conclusion:γ -rays irradiation method is a very efficient mutation breeding method for chamomile crop. GC-FID or GC-MS and NMR methods are found to be the most powerful methods for screening of essential oil chemical compounds isolated from the mutants. The novel mutant (SELM-1) is very promising in terms of high flower and essential oil yield rich in acetylenic compound (2Z,8Z)-matricaria acid methyl ester (76-80%), hence, it was released as variety in Council of Scientific & Industrial Research (CSIR)-Central Institute of Medicinal and Aromatic Plant (CIMAP), Lucknow U.P. (India) named as CIM-Ujjwala for commercial cultivation.

Biochemical characterization and spatio-temporal analysis of the putative L-DOPA pathway in Mucuna pruriens.

MAIN CONCLUSION: Transcriptome analysis and biochemical characterization of the putative l-3,4-dihydroxyphenylalanine (l-DOPA) pathway in Mucuna pruriens (L.) DC have been performed. Spatio-temporal quantification of the putative l-DOPA biosynthetic pathway genes and its correlation with respective metabolites was established. l-tyrosine, l-DOPA, and dopamine from all plant parts were quantified. The de novo transcriptome analysis was performed using leaves of the selected M. pruriens mutant T-IV-9 during maturity. The putative L-DOPA pathway and its regulatory genes were retrieved from transcriptome data and the L-DOPA pathway was biochemically characterized. The spatial and temporal gene expression for the L-DOPA pathway was identified with respect to the chemical constituents. L-tyrosine, L-DOPA, and dopamine contents were highest in leaves during maturity (about 170-day-old plants). The polyphenol oxidase (PPO) was highly expressed in tender stems (230-fold higher as compared to seeds) as well as a high L-DOPA content. The PPO gene was highly expressed in leaves (3367.93 in FPKM) with a 79-fold increase compared to control plants during maturity. L-DOPA was found in every part with varied levels. The highest L-DOPA content was found in mature dried seed (3.18-5.8%), whereas the lowest amount was recorded in mature and dried leaves. The reproductive parts of the plant had a higher amount of L-DOPA content (0.9-5.8%) compared to the vegetative parts (0.2-0.91%). Various amino acid transporters and permeases were expressed in M. pruriens. The transcripts of dopa decarboxylase (DDC) were found in almost all parts of the plant, but its higher content was limited to the leaf.

Inducing mutations through γ-irradiation in seeds of Mucuna pruriens for developing high L-DOPA-yielding genotypes.

PURPOSE: To develop elite genotypes in Mucuna pruriens (L.) DC with high L-DOPA (L-3, 4 dihydroxyphenylalanine) yields, with non-itching characteristics and better adaptability by applying γ-irradiation. Molecular and chemical analysis was performed for screening based on specific characteristics desired for developing suitable genotypes. MATERIALS AND METHODS: Developed, mutant populations were analyzed for L-DOPA % in seeds through TLC (thin layer chromatography), and the results obtained were validated with the HPLC (High performance liquid chromatography). The DNA (Deoxyribonucleic acid) was isolated from the leaf at the initial stage and used for DNA polymorphism. RNA (Ribonucleic acid) was isolated from the leaf during maturity and used for expression analysis. RESULTS: The selected mutant T-I-7 showed 5.7% L-DOPA content compared to 3.18% of parent CIM-Ajar. The total polymorphism obtained was 57% with the molecular marker analysis. The gene expression analysis showed higher fold change expression of the dopadecarboxylase gene (DDC) in control compared to selected mutants (T-I-7, T-II-23, T-IV-9, T-VI-1). CONCLUSION: DNA polymorphism was used for the screening of mutants for efficient screening at an early stage. TLC was found suitable for the large-scale comparative chemical analysis of L-DOPA. The expression profile of DDC clearly demonstrated the higher yields of L-DOPA in selected mutants developed by γ-irradiation in the seeds of the control.

Unravelling the genome of Holy basil: an "incomparable" "elixir of life" of traditional Indian medicine.

BACKGROUND: Ocimum sanctum L. (O. tenuiflorum) family-Lamiaceae is an important component of Indian tradition of medicine as well as culture around the world, and hence is known as "Holy basil" in India. This plant is mentioned in the ancient texts of Ayurveda as an "elixir of life" (life saving) herb and worshipped for over 3000 years due to its healing properties. Although used in various ailments, validation of molecules for differential activities is yet to be fully analyzed, as about 80 % of the patents on this plant are on extracts or the plant parts, and mainly focussed on essential oil components. With a view to understand the full metabolic potential of this plant whole nuclear and chloroplast genomes were sequenced for the first time combining the sequence data from 4 libraries and three NGS platforms. RESULTS: The saturated draft assembly of the genome was about 386 Mb, along with the plastid genome of 142,245 bp, turning out to be the smallest in Lamiaceae. In addition to SSR markers, 136 proteins were identified as homologous to five important plant genomes. Pathway analysis indicated an abundance of phenylpropanoids in O. sanctum. Phylogenetic analysis for chloroplast proteome placed Salvia miltiorrhiza as the nearest neighbor. Comparison of the chemical compounds and genes availability in O. sanctum and S. miltiorrhiza indicated the potential for the discovery of new active molecules. CONCLUSION: The genome sequence and annotation of O. sanctum provides new insights into the function of genes and the medicinal nature of the metabolites synthesized in this plant. This information is highly beneficial for mining biosynthetic pathways for important metabolites in related species.

De novo sequencing and comparative analysis of holy and sweet basil transcriptomes.

BACKGROUND: Ocimum L. of family Lamiaceae is a well known genus for its ethnobotanical, medicinal and aromatic properties, which are attributed to innumerable phenylpropanoid and terpenoid compounds produced by the plant. To enrich genomic resources for understanding various pathways, de novo transcriptome sequencing of two important species, O. sanctum and O. basilicum, was carried out by Illumina paired-end sequencing. RESULTS: The sequence assembly resulted in 69117 and 130043 transcripts with an average length of 1646 ± 1210.1 bp and 1363 ± 1139.3 bp for O. sanctum and O. basilicum, respectively. Out of the total transcripts, 59648 (86.30%) and 105470 (81.10%) from O. sanctum and O. basilicum, and respectively were annotated by uniprot blastx against Arabidopsis, rice and lamiaceae. KEGG analysis identified 501 and 952 transcripts from O. sanctum and O. basilicum, respectively, related to secondary metabolism with higher percentage of transcripts for biosynthesis of terpenoids in O. sanctum and phenylpropanoids in O. basilicum. Higher digital gene expression in O. basilicum was validated through qPCR and correlated to higher essential oil content and chromosome number (O. sanctum, 2n = 16; and O. basilicum, 2n = 48). Several CYP450 (26) and TF (40) families were identified having probable roles in primary and secondary metabolism. Also SSR and SNP markers were identified in the transcriptomes of both species with many SSRs linked to phenylpropanoid and terpenoid pathway genes. CONCLUSION: This is the first report of a comparative transcriptome analysis of Ocimum species and can be utilized to characterize genes related to secondary metabolism, their regulation, and breeding special chemotypes with unique essential oil composition in Ocimum.

Regulation of vincamine biosynthesis and associated growth promoting effects through abiotic elicitation, cyclooxygenase inhibition, and precursor feeding of bioreactor grown Vinca minor hairy roots.

Hydroxylase/acetyltransferase elicitors and cyclooxygenase inhibitor along with various precursors from primary shikimate and secoiridoid pools have been fortified to vincamine less hairy root clone of Vinca minor to determine the regulatory factors associated with vincamine biosynthesis. Growth kinetic studies revealed that acetyltransferase elicitor acetic anhydride and terpenoid precursor loganin significantly reduce the growth either supplemented alone or in combination (GI = 140.6 ± 18.5 to 246.7 ± 24.3), while shikimate and tryptophan trigger biomass accumulation (GI = 440.2 ± 31.5 to 540.5 ± 40.3). Loganin also downregulates total alkaloid biosynthesis. Maximum flux towards vincamine production (0.017 ± 0.001 % dry wt.) was obtained when 20-day-old hairy roots were fortified with secologanin (10 mg/l) along with tryptophan (100 mg/l), naproxen (8.4 mg/l), hydrogen peroxide (20 µg/l), and acetic anhydride (32.4 mg/l). This was supported by RT PCR (qPCR) analysis where 2- and 3-fold increase in tryptophan decarboxylase (TDC; RQ = 2.0 ± 0.09) and strictosidine synthase (STR; RQ = 3.3 ± 0.36) activity, respectively, was recorded. The analysis of variance (ANOVA) for growth kinetics, total alkaloid content, and gene expression studies favored highly significant data (P < 0.05-0.01). Above treated hairy roots were also up-scaled in a 5-l stirred-tank bioreactor where a 40-day cycle yielded 8-fold increase in fresh root mass.