High-level biosynthesis of enantiopure germacrene D in yeast.

Germacrene D, a sesquiterpenoid compound found mainly in plant essential oils at a low level as (+) and/or (-) enantiomeric forms, is an ingredient for the fragrance industry, but a process for the sustainable supply of enantiopure germacrene D is not yet established. Here, we demonstrate metabolic engineering in yeast (Saccharomyces cerevisiae) achieving biosynthesis of enantiopure germacrene D at a high titer. To boost farnesyl pyrophosphate (FPP) flux for high-level germacrene D biosynthesis, a background yeast chassis (CENses5C) was developed by genomic integration of the expression cassettes for eight ergosterol pathway enzymes that sequentially converted acetyl-CoA to FPP and by replacing squalene synthase promoter with a copper-repressible promoter, which restricted FPP flux to the competing pathway. Galactose-induced expression of codon-optimized plant germacrene D synthases led to 13-30 fold higher titers of (+) or (-)-germacrene D in CENses5C than the parent strain CEN.PK2.1C. Furthermore, genomic integration of germacrene D synthases in GAL80, LPP1 and rDNA loci generated CENses8(+D) and CENses8(-D) strains, which produced 41.36 µg/ml and 728.87 µg/ml of (+) and (-)-germacrene D, respectively, without galactose supplementation. Moreover, coupling of mitochondrial citrate pool to the cytosolic acetyl-CoA, by expressing a codon-optimized ATP-citrate lyase of oleaginous yeast, resulted in 137.71 µg/ml and 815.81 µg/ml of (+) or (-)-germacrene D in CENses8(+D)* and CENses8(-D)* strains, which were 67-120 fold higher titers than in CEN.PK2.1C. In fed-batch fermentation, CENses8(+D)* and CENses8(-D)* produced 290.28 µg/ml and 2519.46 µg/ml (+) and (-)-germacrene D, respectively, the highest titers in shake-flask fermentation achieved so far. KEY POINTS: • Engineered S. cerevisiae produced enantiopure (+) and (-)-germacrene D at high titers • Engineered strain produced up to 120-fold higher germacrene D than the parental strain • Highest titers of enantiopure (+) and (-)-germacrene D achieved so far in shake-flask.

Unraveling the terpene synthase family and characterization of BsTPS2 contributing to (S)-( +)-linalool biosynthesis in Boswellia.

Boswellia tree bark exudes oleo-gum resin in response to wounding, which is rich in terpene volatiles. But, the molecular and biochemical basis of wound-induced formation of resin volatiles remains poorly understood. Here, we combined RNA-sequencing (RNA-seq) and metabolite analysis to unravel the terpene synthase (TPS) family contributing to wound-induced biosynthesis of resin volatiles in B. serrata, an economically-important Boswellia species. The analysis of large-scale RNA-seq data of bark and leaf samples representing more than 600 million sequencing reads led to the identification of 32 TPSs, which were classified based on phylogenetic relationship into various TPSs families found in angiosperm species such as TPS-a, b, c, e/f, and g. Moreover, RNA-seq analysis of bark samples collected at 0-24 h post-wounding shortlisted 14 BsTPSs that showed wound-induced transcriptional upregulation in bark, suggesting their important role in wound-induced biosynthesis of resin volatiles. Biochemical characterization of a bark preferentially-expressed and wound-inducible TPS (BsTPS2) in vitro and in planta assays revealed its involvement in resin terpene biosynthesis. Bacterially-expressed recombinant BsTPS2 catalyzed the conversion of GPP and FPP into (S)-( +)-linalool and (E)-(-)-nerolidol, respectively, in vitro assays. However, BsTPS2 expression in Nicotiana benthamiana found that BsTPS2 is a plastidial linalool synthase. In contrast, cytosolic expression of BsTPS2 did not form any product. Overall, the present work unraveled a suite of TPSs that potentially contributed to the biosynthesis of resin volatiles in Boswellia and biochemically characterized BsTPS2, which is involved in wound-induced biosynthesis of (S)-( +)-linalool, a monoterpene resin volatile with a known role in plant defense.

Comparative Chemical Composition and Acetylcholinesterase (AChE) Inhibitory Potential of Cinnamomum camphora and Cinnamomum tamala.

Cinnamomum species have applications in the pharmaceutical and fragrance industry for wide biological and pharmaceutical activities. The present study investigates the chemical composition of the essential oils extracted from two species of Cinnamomum namely C. tamala and C. camphora. Chemical analysis showed E-cinnamyl acetate (56.14 %), E-cinnamaldehyde (20.15 %), and linalool (11.77 %) contributed as the major compounds of the 95.22 % of C. tamala leaves essential oil found rich in phenylpropanoids (76.96 %). C. camphora essential oil accounting for 93.57 % of the total oil composition was rich in 1,8-cineole (55.84 %), sabinene (14.37 %), and α-terpineol (10.49 %) making the oil abundant in oxygenated monoterpenes (70.63 %). Furthermore, the acetylcholinesterase inhibitory activity for both the essential oils was carried out using Ellman's colorimetric method. The acetylcholinesterase inhibitory potential at highest studied concentration of 1 mg/mL was observed to be 46.12±1.52 % for C. tamala and 53.61±2.66 % for C. camphora compared to the standard drug physostigmine (97.53±0.63 %) at 100 ng/ml. These multiple natural aromatic and fragrant characteristics with distinct chemical compositions offered by Cinnamon species provide varied benefits in the development of formulations that could be advantageous for the flavor and fragrance industry.

Role of ACC-deaminase synthesizing Trichoderma harzianum and plant growth-promoting bacteria in reducing salt-stress in Ocimum sanctum.

Salinity is a significant concern in crop production, causing severe losses in agricultural yields. Ocimum sanctum, also known as Holy Basil, is an important ancient medicinal plant used in the Indian traditional system of medicine. The present study explores the use of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing strains of plant-growth-promoting bacteria (PGPB) namely Str-8 (Halomonas desiderata), Sd-6 (Brevibacterium halotolerans), Fd-2 (Achromobacter xylosoxidans), Art-7 (Burkholderia cepacia), and Ldr-2 (Bacillus subtilis), and T. harzianum (Th), possessing multi-functional properties like growth promotion, stress alleviation, and for enhancing O. sanctum yield under salt stress. The results showed that co-inoculation of Th and PGPBs enhanced plant height and fresh herb weight by 3.78-17.65% and 7.86-58.76%, respectively; highest being in Th + Fd-2 and Th + Art-7 compared to positive control plants. The doubly inoculated plants showed increased pigments, phenol, flavonoids, protein, sugar, relative water content, and nutrient uptake (Nitrogen and Phosphorous) as compared to monocultures and untreated positive control plants. In addition, co-inoculation in plants resulted in lower Na+, MDA, H2O2, CAT, APX activities, and also lower ACC accumulation (49.75 to 72.38% compared to non-treated salt- stressed plant) in O. sanctum, which probably played a significant role in minimizing the deleterious effects of salinity. Finally, multifactorial analysis showed that co-inoculation of Th and PGPBs improved O. sanctum growth, its physiological activities, and alleviated salt stress compared to single inoculated and positive control plants. These microbial consortia were evaluated for the first time on O. sanctum under salt stress. Therefore, the microbial consortia application could be employed to boost crop productivity in poor, marginalized and stressed agricultural fields. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01328-2.

Volatile Compounds Governed by Single Recessive Gene Impart Aroma in Sponge Gourd (Luffa cylindrica L. Roem).

As a vegetable crop, sponge gourd is widely consumed worldwide due to its health promoting and nutraceutical value. This study describes genetics of an aromatic genotype VRSG-7-17 and deciphers the genetic control and volatile compound composition of sponge gourd. To study the inheritance of this trait, a cross was made between aromatic light-green-fruited VRSG-7-17 and non-aromatic dark-green-fruited VRSG-194 genotypes. The F1s were found to be non-aromatic and have a green fruit colour. Chi-square (χ2) analysis of backcross and F2 population segregating for aroma suggested that the inheritance of aroma in VRSG-7-17 is governed by a single recessive gene in a simple Mendelian fashion. The SPME-GC/MS analysis of the volatile compounds suggested that the compounds responsible for Basmati rice-like aroma were mainly hexanal, 1-octen-3-ol, 3-octanone and limonene. The aroma persists in the cooked VRSG-7-17 fruits, that did not lose fragrance traits at high temperatures. The inheritance of fruit colour was found to be controlled by a single gene with incomplete dominance. The segregation analysis showed that the aroma and fruit colour were not linked, and they segregated independently. The findings will lead to understanding the inheritance of the aromatic compounds in the sponge gourd and may be utilised in the breeding programmes for developing improved aromatic varieties.

A combination of linalool and linalyl acetate synergistically alleviates imiquimod-induced psoriasis-like skin inflammation in BALB/c mice.

Introduction: Psoriasis is a chronic inflammatory skin disorder characterized by keratinocyte hyperproliferation and differentiation with increased immune cell infiltration. The anti-psoriatic effect of lavender oil has been reported. However, its phytoconstituents, linalool (L) and linalyl acetate (LA), showed a distinctive affinity with psoriasis targets. Objectives: This investigation was aimed to determine the combined effect of L and LA in ameliorating psoriasis-like skin inflammation and its safety in long-term topical uses. Methods: The combined effect of L and LA was compared with their individual effects. The anti-psoriatic activity was performed using imiquimod (IMQ)-induced psoriasis in BALB/c mice and evaluated to reduce PASI and CosCam scores and Th-1 and Th-17 cell-specific cytokine levels. The acute and repeated dose dermal toxicities were investigated as per the OECD guidelines. Results: L and LA combination (LLA) in the 1:1 w/w ratio at 2% concentration showed a synergistic effect. The combination showed 76.31% and 71.29% recovery in PASI and CosCam Scores; however, L2% and LA2% showed 64.28% and 47.61% recovery in PASI and 64.75 and 56.76% recovery in CosCam scores, respectively. It showed >90% and >100% recovery in Th-17 and Th-1 cell-specific cytokines, respectively, and restored epidermal hyperplasia and parakeratosis toward normal compared with psoriatic mice. A marked reduction in NF-κB, cck6, and the IL-17 expression was also observed in the LLA-treated group. This combination was safe in a therapeutically effective dose for 28 days as no significant changes were observed in organ and body weights, liver and kidney parameters, and differential leukocyte counts. Conclusion: This study proves the synergy between L and LA in a 1:1 w/w ratio at 2% in the treatment of psoriasis-like skin inflammation and provides strong scientific evidence for its safe topical use.

UGT86C11 is a novel plant UDP-glycosyltransferase involved in labdane diterpene biosynthesis.

Glycosyltransferases constitute a large family of enzymes across all domains of life, but knowledge of their biochemical function remains largely incomplete, particularly in the context of plant specialized metabolism. The labdane diterpenes represent a large class of phytochemicals with many pharmacological benefits, such as anti-inflammatory, hepatoprotective, and anticarcinogenic. The medicinal plant kalmegh (Andrographis paniculata) produces bioactive labdane diterpenes; notably, the C19-hydroxyl diterpene (andrograpanin) is predominantly found as C19-O-glucoside (neoandrographolide), whereas diterpenes having additional hydroxylation(s) at C3 (14-deoxy-11,12-didehydroandrographolide) or C3 and C14 (andrographolide) are primarily detected as aglycones, signifying scaffold-selective C19-O-glucosylation of diterpenes in planta. Here, we analyzed UDP-glycosyltransferase (UGT) activity and diterpene levels across various developmental stages and tissues and found an apparent correlation of UGT activity with the spatiotemporal accumulation of neoandrographolide, the major diterpene C19-O-glucoside. The biochemical analysis of recombinant UGTs preferentially expressed in neoandrographolide-accumulating tissues identified a previously uncharacterized UGT86 member (ApUGT12/UGT86C11) that catalyzes C19-O-glucosylation of diterpenes with strict scaffold selectivity. ApUGT12 localized to the cytoplasm and catalyzed diterpene C19-O-glucosylation in planta. The substrate selectivity demonstrated by the recombinant ApUGT12 expressed in plant and bacterium hosts was comparable to native UGT activity. Recombinant ApUGT12 showed significantly higher catalytic efficiency using andrograpanin compared with 14-deoxy-11,12-didehydroandrographolide and trivial activity using andrographolide. Moreover, ApUGT12 silencing in plants led to a drastic reduction in neoandrographolide content and increased levels of andrograpanin. These data suggest the involvement of ApUGT12 in scaffold-selective C19-O-glucosylation of labdane diterpenes in plants. This knowledge of UGT86 function might help in developing plant chemotypes and synthesis of pharmacologically relevant diterpenes.

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.

Diversity of Essential Oil-Secretory Cells and Oil Composition in Flowers and Buds of Magnolia sirindhorniae and Its Biological Activities.

Magnolia sirindhorniae Noot. & Chalermglin produces fragrant flowers. The volatile oil secretary cells, quantity and quality as well as antioxidant and antimicrobial activities of the oils extracted from buds and flowers, have been investigated. The distribution of essential oil secretory cell in bud and flower revealed that the density and size of the oil cells were significantly higher in flowers compared to buds. In different floral parts, carpel has a higher oil cell density followed by gynophore and tepal. The histochemical analysis revealed the essential oil is synthesized in oil secretory cells. The volatile oil yield was 0.25 % in the buds and 0.50 % in flowers. GC/FID and GC/MS analysis identified 33 compounds contributing 83.2-83.5 % of the total essential oil composition. Linalool is the main constituent contributing 58.9 % and 51.0 % in the buds and flowers oils, respectively. The essential oil extracted from the flowers showed higher antimicrobial efficacy against Klebsiella pneumoniae and Staphylococcus aureus. Similarly, the essential oil isolated from the flowers depicts higher free radical scavenging, and antioxidant activity compared to buds' oil.

A quinoline alkaloid rich Quisqualis indica floral extract enhances the bioactivity.

A volatile alkaloid quinoline-4-carbonitrile (QCN) was isolated from the floral extract of Quisqualis indica. Major compounds were trans-linalool oxide (1.0, 4.5%), methyl benzoate (1.0, 4.0%), 2,2,6-trimethyl-6-vinyl-tetrahydropyran-3-one (7.4, 17.8%), 2,2,6-trimethyl-6-vinyl-tetrahydropyran-3-ol (1.0, 1.2%), (E,E)-α-farnesene (29.1, 16.1%), QCN (5.7, 1.3%) in live and picked flowers, respectively. Flower compositions were altered due to change in enzymatic reaction at the time of picking. Some rearrangements of oxygenated terpenoids occurred in the process of hydrodistillation to obtain essential oil. Chemical synthesis of QCN and its selectively reduced products derived from QCN were prepared through green reaction process. The catalytic modification of QCN has produced quinoline-4-methylamine; the later compound has shown enhanced bio-activities. QCN and floral extract (absolute) have shown potential anti-inflammatory and antioxidant activities. Besides, floral absolute has shown significant anti-inflammatory and antioxidant activities due to improved QCN (19.7%) content to synergize amongst terpenoids and benzenoids as compared to the essential oil with 1.1% of QCN.

Anti-psoriatic effect of Lavandula angustifolia essential oil and its major components linalool and linalyl acetate.

ETHNO-PHARMACOLOGICAL RELEVANCE: Lavender oil (LO) is an aromatic/essential oil extracted from Lavandula angustifolia and traditionally used as an aromatherapy massage oil due to its anti-inflammatory and wound healing property and also for providing the relief in other skin conditions such as psoriasis, dermatitis and eczema. However, LO has not been evaluated scientifically for psoriasis like skin inflammation. AIM OF THE STUDY: This study was aimed to investigate the LO and its major components linalool (L) and linalyl acetate (LA) against psoriasis like skin inflammation. MATERIALS AND METHODS: Anti-psoriatic activity was done using Imiquimod (IMQ) induced psoriasis like skin inflammation in BALB/c mice. Assessment of anti-psoriatic effect of LO, L and LA was done on the basis of change in ear thickness, psoriasis area severity index (PASI) scoring at alternative day, CosCam scoring using skin analyzer equipped with SkinSys software, biochemical, immunohistochemical and histological investigations. Level of effectiveness against psoriasis was investigated by percent reduction in PASI scores, CosCam scores and level of Th-1 and Th-17 cell expressing cytokines, as compared to the diseased mice. RESULTS: Topical application of LO 10% showed 73.67% recovery in PASI and 87% in Th-17 cell-specific cytokines towards normal as compared to disease group. L and LA were identified as the major components of LO and favoured ligands for selected psoriasis targets. At 2% topical dose, L and LA showed 64% and 47.61% recovery in PASI scores, respectively. Both, L and LA showed significant recovery in Th-1 specific TNF-α and IL-1ß however, only L showed significant recovery of Th-17 cytokines (IL-17 and IL-22). In contrast to LA (which restored granulosis), L restored epidermal hyperplasia and parakeratosis toward the normal condition. On the other hand, L also reduced the expression of NF-κß, ccr6 and IL-17, while LA reduced the expression of NF-κß only. At 10% topical dose, LO was observed to be slight irritant while at 2% topical dose, L and LA were found non-irritant to the skin. CONCLUSION: This study proves the effectiveness of LO and its major phytoconstituents linalool and linalyl acetate against IMQ induced psoriasis like skin inflammation and provides the scientific evidence for topical use of lavender oil.

Engineering a Carotenoid-Overproducing Strain of Azospirillum brasilense for Heterologous Production of Geraniol and Amorphadiene.

Escherichia coli and Saccharomyces cerevisiae have been used extensively for heterologous production of a variety of secondary metabolites. Neither has an endogenous high-flux isoprenoid pathway, required for the production of terpenoids. Azospirillum brasilense, a nonphotosynthetic GRAS (generally recognized as safe) bacterium, produces carotenoids in the presence of light. The carotenoid production increases multifold upon inactivating a gene encoding an anti-sigma factor (ChrR1). We used this A. brasilense mutant (Car-1) as a host for the heterologous production of two high-value phytochemicals, geraniol and amorphadiene. Cloned genes (crtE1 and crtE2) of A. brasilense encoding native geranylgeranyl pyrophosphate synthases (GGPPS), when overexpressed and purified, did not produce geranyl pyrophosphate (GPP) in vitro Therefore, we cloned codon-optimized copies of the Catharanthus roseus genes encoding GPP synthase (GPPS) and geraniol synthase (GES) to show the endogenous intermediates of the carotenoid biosynthetic pathway in the Car-1 strain were utilized for the heterologous production of geraniol in A. brasilense Similarly, cloning and expression of a codon-optimized copy of the amorphadiene synthase (ads) gene from Artemisia annua also led to the heterologous production of amorphadiene in Car-1. Geraniol or amorphadiene content was estimated using gas chromatography-mass spectrometry (GC-MS) and GC. These results demonstrate that Car-1 is a promising host for metabolic engineering, as the naturally available endogenous pool of the intermediates of the carotenoid biosynthetic pathway of A. brasilense can be effectively utilized for the heterologous production of high-value phytochemicals.IMPORTANCE To date, the major host organisms used for the heterologous production of terpenoids, i.e., E. coli and S. cerevisiae, do not have high-flux isoprenoid pathways and involve tedious metabolic engineering to increase the precursor pool. Since carotenoid-producing bacteria carry endogenous high-flux isoprenoid pathways, we used a carotenoid-producing mutant of A. brasilense as a host to show its suitability for the heterologous production of geraniol and amorphadiene as a proof-of-concept. The advantages of using A. brasilense as a model system include (i) dispensability of carotenoids and (ii) the possibility of overproducing carotenoids through a single mutation to exploit high carbon flux for terpenoid production.

Transcriptome analysis and functional characterization of oxidosqualene cyclases of the arjuna triterpene saponin pathway.

Arjuna (Terminalia arjuna) tree has been popular in Indian traditional medicine to treat cardiovascular ailments. The tree accumulates bioactive triterpene glycosides (saponins) and aglycones (sapogenins), in a tissue-preferential manner. Oleanane triterpenes/saponins (derived from ß-amyrin) with potential cardioprotective function predominantly accumulate in the bark. However, arjuna triterpene saponin pathway enzymes remain to be identified and biochemically characterized. Here, we employed a combined transcriptomics, metabolomics and biochemical approach to functionally define a suite of oxidosqualene cyclases (OSCs) that catalyzed key reactions towards triterpene scaffold diversification. De novo assembly of 131 millions Illumina NextSeq500 sequencing reads obtained from leaf and stem bark samples led to a total of 156,650 reference transcripts. Four distinct OSCs (TaOSC1-4) with 54-71 % sequence identities were identified and functionally characterized. TaOSC1, TaOSC3 and TaOSC4 were biochemically characterized as ß-amyrin synthase, cycloartenol synthase and lupeol synthase, respectively. However, TaOSC2 was found to be a multifunctional OSC producing both α-amyrin and ß-amyrin, but showed a preference for α-amyrin product. Both TaOSC1 and TaOSC2 produced ß-amyrin, the direct precursor for oleanane triterpene/saponin biosynthesis; but, TaOSC1 transcript expressed preferentially in bark, suggesting a major role of TaOSC1 in the biosynthesis of oleanane triterpenes/saponins in bark.

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.

Application of essential oils as a natural and alternate method for inhibiting and inducing the sprouting of potato tubers.

Use of harmful chemicals and expensive maintenance of cold-storage conditions for controlling sprouting are among the major problems in potato storage. Here, 20 essential oils (EOs) were tested for their sprouting-inhibiting and sprouting-inducing activities. Overall, treatments of lemon grass (LG) and clove (CL) oils could induce sprouting whereas palmarosa (PR) and ajwain (AZ) oils could inhibit sprouting of potato tubers at normal-room-temperature (25 ±â€¯2 °C) storage. Selected-EOs treatments affected sprouting by modulation of accumulation of reducing sugars, ethylene, and expression of genes involved in tuber-sprouting such as ARF, ARP, AIP and ERF. Surprisingly, 7-days AZ-treatments could inhibit sprouting for 30-days which was mediated via damaging apical meristem. However, LG- and CL-treated tubers could produce enhanced potato yield as well. Present work clearly demonstrates that selected-EOs can be used as a promising eco-friendly approach for inducing/inhibiting sprouting of potato tubers during potato storage and those enhancing sprouting can be used for enhancing productivity.

Oxidosqualene cyclase and CYP716 enzymes contribute to triterpene structural diversity in the medicinal tree banaba.

Pentacyclic triterpenes (PCTs) represent a major class of bioactive metabolites in banaba (Lagerstroemia speciosa) leaves; however, biosynthetic enzymes and their involvement in the temporal accumulation of PCTs remain to be studied. We use an integrated approach involving transcriptomics, metabolomics and gene function analysis to identify oxidosqualene cyclases (OSCs) and cytochrome P450 monooxygenases (P450s) that catalyzed sequential cyclization and oxidative reactions towards PCT scaffold diversification. Four monofunctional OSCs (LsOSC1,3-5) converted the triterpene precursor 2,3-oxidosqualene to either lupeol, ß-amyrin or cycloartenol, and a multifunctional LsOSC2 formed α-amyrin as a major product along with ß-amyrin. Two CYP716 family P450s (CYP716A265, CYP716A266) catalyzed C-28 oxidation of α-amyrin, ß-amyrin and lupeol to form ursolic acid, oleanolic acid and betulinic acid, respectively. However, CYP716C55 catalyzed C-2α hydroxylation of ursolic acid and oleanolic acid to produce corosolic acid and maslinic acid, respectively. Besides, combined transcript and metabolite analysis suggested major roles for the LsOSC2, CYP716A265 and CYP716C55 in determining leaf ursane and oleanane profiles. Combinatorial expression of OSCs and CYP716s in Saccharomyces cerevisiae and Nicotiana benthamiana led to PCT pathway reconstruction, signifying the utility of banaba enzymes for bioactive PCT production in alternate plant/microbial hosts that are more easily tractable than the tree species.

RNAi of Sterol Methyl Transferase1 Reveals its Direct Role in Diverting Intermediates Towards Withanolide/Phytosterol Biosynthesis in Withania somnifera.

The medicinal properties of Ashwagandha (Withania somnifera) are accredited to a group of compounds called withanolides. 24-Methylene cholesterol is the intermediate for sterol biosynthesis and a proposed precursor of withanolide biogenesis. However, conversion of 24-methylene cholesterol to withaferin A and other withanolides has not yet been biochemically dissected. Hence, in an effort to fill this gap, an important gene, encoding S-adenosyl l-methionine-dependent sterol-C24-methyltransferase type 1 (SMT1), involved in the first committed step of sterol biosynthesis, from W. somnifera was targeted in the present study. Though SMT1 has been characterized in model plants such as Nicotiana tabacum and Arabidopsis thaliana, its functional role in phytosterol and withanolide biosynthesis was demonstrated for the first time in W. somnifera. Since SMT1 acts at many steps preceding the withanolide precursor, the impact of this gene in channeling of metabolites for withanolide biosynthesis and its regulatory nature was illustrated by suppressing the gene in W. somnifera via the RNA interference (RNAi) approach. Interestingly, down-regulation of SMT1 in W. somnifera led to reduced levels of campesterol, sitosterol and stigmasterol, with an increase of cholesterol content in the transgenic RNAi lines. In contrast, SMT1 overexpression in transgenic N. tabacum enhanced the level of all phytosterols except cholesterol, which was not affected. The results established that SMT1 plays a crucial role in W. somnifera withanolide biosynthesis predominantly through the campesterol and stigmasterol routes.

The essential oil from Citrus limetta Risso peels alleviates skin inflammation: In-vitro and in-vivo study.

ETHNOPHARMACOLOGICAL RELEVANCE: Citrus fruit peels are traditionally used in folk medicine for the treatment of skin disorders but it lacks proper pharmacological intervention. Citrus limetta Risso (Rutaceae) is an important commercial fruit crops used by juice processing industries in all continents. Ethnopharmacological validation of an essential oil isolated from its peels may play a key role in converting the fruit waste materials into therapeutic value added products. AIM OF THE STUDY: To evaluate the chemical and pharmacological (in-vitro and in-vivo) profile of essential oil isolated from Citrus limetta peels (Clp-EO) against skin inflammation for its ethnopharmacological validation. MATERIALS AND METHODS: Hydro-distilled essential oil extracted from Citrus limetta peels (Clp-EO) was subjected to gas chromatography (GC) analysis for identification of essential oil constituents and its anti-inflammatory evaluation through in vitro and in vivo models. RESULTS: Chemical fingerprint of Clp-EO revealed the presence of monoterpene hydrocarbon and limonene is the major component. Pre-treatment of Clp-EO to the macrophages was able to inhibit the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß) in LPS-induced inflammation as well as the production of reactive oxygen species (ROS) in H2O2-induced oxidative stress. In in-vivo study, topical application of Clp-EO was also able to reduce the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear thickness, ear weight, lipid peroxidation, pro-inflammatory cytokines production and ameliorate the histological damage in the ear tissue. In-vitro and in-vivo toxicity study indicate that it is safe for topical application on skin. CONCLUSION: These findings suggested the preventive potential of Clp-EO for the treatment of inflammation linked skin diseases.

Two CYP716A subfamily cytochrome P450 monooxygenases of sweet basil play similar but nonredundant roles in ursane- and oleanane-type pentacyclic triterpene biosynthesis.

The medicinal plant sweet basil (Ocimum basilicum) accumulates bioactive ursane- and oleanane-type pentacyclic triterpenes (PCTs), ursolic acid and oleanolic acid, respectively, in a spatio-temporal manner; however, the biosynthetic enzymes and their contributions towards PCT biosynthesis remain to be elucidated. Two CYP716A subfamily cytochrome P450 monooxygenases (CYP716A252 and CYP716A253) are identified from a methyl jasmonate-responsive expression sequence tag collection and functionally characterized, employing yeast (Saccharomyces cerevisiae) expression platform and adapting virus-induced gene silencing (VIGS) in sweet basil. CYP716A252 and CYP716A253 catalyzed sequential three-step oxidation at the C-28 position of α-amyrin and ß-amyrin to produce ursolic acid and oleanolic acid, respectively. Although CYP716A253 was more efficient than CYP716A252 for amyrin C-28 oxidation in yeast, VIGS revealed essential roles for both of these CYP716As in constitutive biosynthesis of ursolic acid and oleanolic acid in sweet basil leaves. However, CYP716A253 played a major role in elicitor-induced biosynthesis of ursolic acid and oleanolic acid. Overall, the results suggest similar as well as distinct roles of CYP716A252 and CYP716A253 for the spatio-temporal biosynthesis of PCTs. CYP716A252 and CYP716A253 might be useful for the alternative and sustainable production of PCTs in microbial host, besides increasing plant metabolite content through genetic modification.

Chemically characterized Mentha cardiaca L. essential oil as plant based preservative in view of efficacy against biodeteriorating fungi of dry fruits, aflatoxin secretion, lipid peroxidation and safety profile assessment.

The study reports Mentha cardiaca essential oil (EO) as plant based preservative against fungal and aflatoxin contamination of stored dry fruits. Mycoflora analysis of the dry fruits revealed Aspergillus favus LHP-PV-1 as the most aflatoxigenic isolate with highest Aflatoxin B1 content. M. cardiaca EO showed broad fungitoxic spectrum inhibiting the tested moulds contaminating dry fruits. It's minimum inhibitory concentration (MIC), minimum aflatoxin inhibitory concentration (MAIC) and minimum fungicidal concentration (MFC) against A. favus LHP-PV-1 were recorded to be 1.25, 1.0 and 2.25 µL/mL respectively. The EO caused decrease in ergosterol content and enhanced leakage of Ca2+, K+ and Mg2+ ions from treated fungal cells, depicting fungal plasma membrane as the site of antifungal action. The EO showed promising DPPH free radical scavenging activity (IC50 value:15.89 µL/mL) and favourable safety profile with LD50 value (7133.70 mg/kg body wt.) when estimated through acute oral toxicity on mice. Carvone (61.62%) was recorded as the major component of the oil during chemical characterisation through GC-MS. Based on strong antifungal, antiaflatoxigenic and antioxidant potential, the chemically characterised M. cardiaca EO may be recommended as safe plant based preservative and shelf life enhancer of food items. This is the first report on antifungal and antiaflatoxigenic activity of M. cardiaca EO.