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.

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.

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.

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.

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.

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.

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.

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.

Involvement of an ent-copalyl diphosphate synthase in tissue-specific accumulation of specialized diterpenes in Andrographis paniculata.

Ent-labdane-related diterpene (ent-LRD) specialized (i.e. secondary) metabolites of the medicinal plant kalmegh (Andrographis paniculata) have long been known for several pharmacological activities. However, our understanding of the ent-LRD biosynthetic pathway has remained largely incomplete. Since ent-LRDs accumulate in leaves, we carried out a comparative transcriptional analysis using leaf and root tissues, and identified 389 differentially expressed transcripts, including 223 transcripts that were preferentially expressed in leaf tissue. Analysis of the transcripts revealed various specialized metabolic pathways, including transcripts of the ent-LRD biosynthetic pathway. Two class II diterpene synthases (ApCPS1 and ApCPS2) along with one (ApCPS1') and two (ApCPS2' and ApCPS2″) transcriptional variants that were the outcomes of alternative splicing of the precursor mRNA and alternative transcriptional termination, respectively, were identified. ApCPS1 and ApCPS2 encode for 832- and 817-amino acids proteins, respectively, and are phylogenetically related to the dicotyledons ent-copalyl diphosphate synthases (ent-CPSs). The spatio-temporal patterns of ent-LRD metabolites accumulation and gene expression suggested a likely role for ApCPS1 in general (i.e. primary) metabolism, perhaps by providing precursor for the biosynthesis of phytohormone gibberellin (GA). However, ApCPS2 is potentially involved in tissue-specific accumulation of ent-LRD specialized metabolites. Bacterially expressed recombinant ApCPS2 catalyzed the conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP), the general precursor of diterpenes to ent-copalyl diphosphate (ent-CPP), the precursor of ent-LRDs. Taken together, these results advance our understanding of the tissue-specific accumulation of specialized ent-LRDs of medicinal importance.

A novel approach for development and characterization of effective mosquito repellent cream formulation containing citronella oil.

Citronella essential oil (CEO) has been reported as an excellent mosquito repellent; however, mild irritancy and rapid volatility limit its topical application. It was aimed to develop a nonirritant, stable, and consistent cream of CEO with improved residence time on skin using an industrial approach. Phase inversion temperature technique was employed to prepare the cream. It was optimized and characterized based on sensorial evaluation, emulsification, and consistency in terms of softness, greasiness, stickiness, and pH. The optimum batch (B5) was evaluated for viscosity (90249.67±139.95 cP), texture profile with respect to firmness (38.67±0.88 g), spreadability (70.33±0.88 mJ), and extrudability (639.67±8.09±0.1 mJ) using texture analyzer along with two most popular marketed products selected as reference standard. Subsequently, B5 was found to be stable for more than 90 days and showed enhanced duration of mosquito repellency as compared to CEO. HS-GC ensured the intactness of CEO in B5. Investigated primary irritation index (PII 0.45) positioned B5 into the category of irritation barely perceptible. The pronounced texture profile and stability of B5 with extended residence time and less PII revealed its potential application in industry and offered a promising alternative to the marketed products of synthetic origin.

Methyl jasmonate-elicited transcriptional responses and pentacyclic triterpene biosynthesis in sweet basil.

Sweet basil (Ocimum basilicum) is well known for its diverse pharmacological properties and has been widely used in traditional medicine for the treatment of various ailments. Although a variety of secondary metabolites with potent biological activities are identified, our understanding of the biosynthetic pathways that produce them has remained largely incomplete. We studied transcriptional changes in sweet basil after methyl jasmonate (MeJA) treatment, which is considered an elicitor of secondary metabolites, and identified 388 candidate MeJA-responsive unique transcripts. Transcript analysis suggests that in addition to controlling its own biosynthesis and stress responses, MeJA up-regulates transcripts of the various secondary metabolic pathways, including terpenoids and phenylpropanoids/flavonoids. Furthermore, combined transcript and metabolite analysis revealed MeJA-induced biosynthesis of the medicinally important ursane-type and oleanane-type pentacyclic triterpenes. Two MeJA-responsive oxidosqualene cyclases (ObAS1 and ObAS2) that encode for 761- and 765-amino acid proteins, respectively, were identified and characterized. Functional expressions of ObAS1 and ObAS2 in Saccharomyces cerevisiae led to the production of ß-amyrin and α-amyrin, the direct precursors of oleanane-type and ursane-type pentacyclic triterpenes, respectively. ObAS1 was identified as a ß-amyrin synthase, whereas ObAS2 was a mixed amyrin synthase that produced both α-amyrin and ß-amyrin but had a product preference for α-amyrin. Moreover, transcript and metabolite analysis shed light on the spatiotemporal regulation of pentacyclic triterpene biosynthesis in sweet basil. Taken together, these results will be helpful in elucidating the secondary metabolic pathways of sweet basil and developing metabolic engineering strategies for enhanced production of pentacyclic triterpenes.

Monitoring the emission of volatile organic compounds from flowers of Jasminum sambac using solid-phase micro-extraction fibers and gas chromatography with mass spectrometry detection.

Solid-phase micro-extraction (SPME) was studied as a solvent free alternative method for the extraction and characterization of volatile compounds in intact and plucked flowers of Jasminum sambac at different day time intervals using gas chromatography (GC-FID) and gas chromatography-quadrupole mass spectrometry. The analytes identified included alcohols, esters, phenolic compounds, and terpenoids. The main constituents identified in the flower aroma using different fibers were cis-3-hexenyl acetate, (E)-beta-ocimene, linalool, benzyl acetate, and (E,E)-alpha-farnesene. The benzyl acetate proportion decreased from morning to afternoon and then increased in evening collections. PDMS fiber showed a high proportion of (E,E)-alpha-farnesene in jasmine floral aroma. Among other constituents identified, cis-3-hexenyl acetate, linalool, and benzyl acetate were major aroma contributors in plucked and living flowers extracts using PDMS/DVB, Carboxen/PDMS, and DVB/Carboxen/PDMS fibers. PDMS/DVB recorded the highest emission for benzyl acetate while the (E)-beta-ocimene proportion was highest in DVB/Carboxen/PDMS when compared with the rest. The highest linalool content, with increasing proportion from morning to noon, was found using mixed coating fibers. Almost negligible volatile adsorption was recorded for the polyacrylate fiber for intact flower aroma, whereas it was most effective for benzyl acetate, followed by indole under plucked conditions. Moreover, the highest amounts extracted, evaluated from the sum of peak areas, were achieved using Carboxen/PDMS, and DVB/Carboxen/PDMS. Introduction of a rapid, and solvent free SPME method for the analysis of multicomponent volatiles can be successfully employed to monitor the extraction and characterization of flower aroma constituents.

Terpenoid compositions, and antioxidant and antimicrobial properties of the rhizome essential oils of different Hedychium species.

A phytochemical study of the rhizome essential oils of four different Hedychium species was performed by means of GC and GC/MS analyses. H. ellipticum mainly contained 1,8-cineole, sabinene, and terpin-4-ol, while H. aurantiacum possessed terpin-4-ol, para-cymene, and bornyl acetate as the major entities. Similarly, trans-meta-mentha-2,8-diene and linalool were noticed in H. coronarium. Three different collections (I-III) of H. spicatum showed amazing differences in the relative contents of their essential oils, 1,8-cineole and 10-epi-gamma-eudesmol being identified as markers for samples I and II, terpin-4-ol and sabinene being the major compounds in sample III. The rhizome essential oils of the above species were studied for their antioxidant activities by different methods, including their effect on the chelating properties of Fe(2+), DPPH radical-scavenging activity, and reducing power. Antimicrobial screenings of the oils by the paper-disc method were performed against Staphylococcus aureus, Shigella flexneri, Pasteurella multocida, Escherichia coli, and Salmonella enterica enterica, and the respective minimum-inhibitory-concentration (MIC) values were determined. The rhizome essential oils from all Hedychium species exhibited moderate-to-good Fe(2+) chelating activity. H. spicatum from collection site III showed a completely different DPPH radical-scavenging profile than the samples from the other collection sites.