Characterization of terpenoids and norisoprenoids from base and retail Qingke Baijiu by GC×GC-TOFMS and multivariate statistical analysis

Qingke(highland barley)Baijiu is a special Chinese Baijiu which is mainly produced from Qinghai-Tibet Plateau.Since the pine board is used as the bottom of the fermentation pit,we deduced that the terpenoids and norisoprenoids in pine board might be introduced into Qingke Baijiu.Thus,the terpenoids and norisoprenoids in Qingke Baijiu were investigated by headspace solid phase microextraction(HS-SPME)combined with comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry(GC×GC-TOFMS).The results showed thatγ-terpinene(0.70-530.72μg/L),α-phellandrene(0.34-256.66μg/L),longicyclene(0-38.55μg/L),α-pinene(1.21-35.54μg/L)and limonene(0.93-23.69μg/L)were the top 5 terpenoids/norisoprenoids in Qingke Baijiu.Pulegone was detected and reported for the first time in Baijiu,and the concentrations in Qingke Baijiu were 0.78μg/L(fresh)and 3.90μg/L(7 years old).According to the principal component analysis(PCA)plot,young,aged,and retail Qingke Baijiu could be differentiated clearly.Fold change(FC)and t-tests analysis indicated thatβ-pinene,γ-terpinene,andα-selinene were the most different terpenoids/norisoprenoids between young and aged Qingke Baijiu,and longifolene was the most different terpenoids/norisoprenoids between base and retail Qingke Baijiu.The terpenoids and norisoprenoids,such as(E)-β-ionone,isoborneol andβ-cyclocitral,could be potential markers indicating the ageing process of Qingke Baijiu.

Identification of key genes involved in flavonoid and terpenoid biosynthesis and the pathway of triterpenoid biosynthesis in Passiflora edulis

Passion fruit(Passiflora edulis Sims) is a vine of the Passiflora genus in the Passifloraceae family. The extracted components include flavonoids and terpenoids, which have good anti-anxiety and anti-inflammatory effects in humans.In this study, we analyzed the transcriptomes of four tissues of the ‘Zixiang’ cultivar using RNA-Seq, which provided a dataset for functional gene mining. The de novo assembly of these reads generated 96 883 unigenes, among which 61 022 unigenes were annotated(62.99% yield). In addition to its edible value, another important application of passion fruit is its medicinal value. The flavonoids and terpenoids are mainly derivatives of luteolin, apigenin, cycloartane triterpenoid saponins and other active substances in leaf extracts. A series of candidate unigenes in the transcriptome data that are potentially involved in the flavonoid and terpenoid synthesis pathways were screened using homologybased BLAST and phylogenetic analysis. The results showed that the biosynthesis of triterpenoids in passion fruit comes from the branches of the mevalonate(MVA) and 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate(MEP/DOXP) pathways, which is different from the MVA pathway that is used in other fruit trees. Most of the candidate genes were found to be highly expressed in the leaves and/or flowers. Quantitative real-time PCR(qRT-PCR) verification was carried out and confirmed the reliability of the RNA-Seq data. Further amplification and functional analysis of these putative unigenes will provide additional insight into the biosynthesis of flavonoids and terpenoids in passion fruit.

Remodeling Isoprene Pyrophosphate Metabolism for Promoting Terpenoids Bioproduction

Terpenoids are the largest family of natural products.They are made from the building block isoprene pyrophosphate(IPP),and their bioproduction using engineered cell factories has received a great deal of attention.To date,the insufficient metabolic supply of IPP remains a great challenge for the efficient synthesis of terpenoids.In this work,we discover that the imbalanced metabolic flux distribution between the central metabolism and the IPP supply hinders IPP accumulation in Bacillus subtilis(B.subtilis).Therefore,we remodel the IPP metabolism using a series of genetically encoded two-input-multioutput(TIMO)circuits that are responsive to pyruvate or/and malonyl-CoA,resulting in an IPP pool that is significantly increased by up to four-fold.As a proof-of-concept validation,we design an IPP metabolism remodeling strategy to improve the production of three valuable terpenoids,including menaquinone-7(MK-7,4.1-fold),lycopene(9-fold),andβ-carotene(0.9-fold).In particular,the titer of MK-7 in a 50-L bioreactor reached 1549.6 mg·L^(-1),representing the highest titer reported so far.Thus,we propose a TIMO genetic circuits-assisted IPP metabolism remodeling framework that can be generally used for the synergistic fine-tuning of complicated metabolic modules to achieve the efficient bioproduction of terpenoids.

Terpenoids as potential chemopreventive and therapeutic agents in liver cancer

Despite significant advances in medicine, liver cancer, predominantly hepatocellular carcinoma remains a major cause of death in the United States as well as the rest of the world. As limited treatment options are currently available to patients with liver cancer, novel preventive control and effective therapeutic approaches are considered to be reasonable and decisive measures to combat this disease. Several naturally occurring dietary and non-dietary phytochemicals have shown enormous potential in the prevention and treatment of several cancers, especially those of the gastrointestinal tract. Terpenoids, the largest group of phytochemicals, traditionally used for medicinal purposes in India and China, are currently being explored as anticancer agents in clinical trials. Terpenoids (also called "isoprenoids") are secondary metabolites occurring in most organisms, particularly plants. More than 40 000 individual terpenoids are known to exist in nature with new compounds being discovered every year. A large number of terpenoids exhibit cytotoxicity against a variety of tumor cells and cancer preventive as well as anticancer efficacy in preclinical animal models. This review critically examines the potential role of naturally occurring terpenoids, from diverse origins, in the chemoprevention and treatment of liver tumors. Both in vitro and in vivo effects of these agents and related cellular and molecular mechanisms are highlighted. Potential challenges and future directions involved in the advancement of these promising natural compounds in the chemoprevention and therapy of human liver cancer are also discussed.

Engineering Terpenoid Indole Alkaloids Biosynthetic Pathway in Catharanthus roseus Hairy Root Cultures by Overexpressing the Geraniol 10-hydroxylase Gene

Catharanthus roseus contains important anti-tumor terpenoid indole alkaloids (TIAs) such as vinblastine and vincristine. Cytochrome P450 enzyme geraniol 10-hydroxylase (G10H) is a putative rate-limiting enzyme involved in the TIAs biosynthetic pathway in C. roseus. In this study the g10h gene driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter was introduced into C. roseus through Agrobacterium-mediated transformation. The integration and overexpression of the target gene (g10h) in hairy root lines were confirmed by polymerase chain reaction and RT-QPCR analysis respectively. Overexpression of g10h in transgenic hairy root lines significantly enhanced the accumulations of monomeric alkaloid ajmalicine and dimeric alkaloids, vincristine and vinblastine. Total TIAs production in hairy roots reached (9.51) mg/g DW, over 3-fold higher than that in the untransformed root lines. This is the first report that engineering of g10h into TIAs-producing plant species results in significant enhancement of TIAs accumulation in cultured hairy roots. This study demonstrates that the putative rate-limiting step catalyzed by G10H is indeed the real rate-limiting step involved in the TIAs biosynthetic pathway in C. roseus, which is one of the key targets for promoting TIAs production by genetic engineering.

Interactions Between Exogenous Bt Insecticidal Protein and Cotton Terpenoid Aldehydes

The contents of terpenoid aldehydes in Bt transgenic cotton and their non-Bt parental varieties were analyzed by the HPLC method. Statistical analysis of variance showed that Bt insecticidal protein Bt-ICP expression has no negative effect on the synthesis of gossypol, total heliocides and total resistant terpenoids. The results of the combined dosage test of Bt-ICP and gossypol in vitro showed that there is no interaction between gossypol and Bt-ICP on the mortality of cotton bollworm larvae Helicoverpa armigera (Hubnner). It is indicated that the actions of Bt-ICP and gossypol on cotton bollworm are additive. Therefore, it is advantageous to combine Bt-ICP with cotton terpenoid aldehydes in controlling cotton bollworm.

Impact of oxygen supply on production of terpenoids by microorganisms: State of the art

Terpenoids are a class of high value-added natural products with a variety of biological functions.Genetically engineered microorganisms,such as those of Escherichia coli and Saccharomyces cerevisiae,have merits in producing plant or fungus-derived terpenoids,due to their mature genetic manipulation,simple nutrient demand and fast growth.Oxygen,as a key environmental factor,is particularly important to microbial metabolism and growth,and suitable oxygen supply is viewed as a prerequisite for realizing highly efficient production of terpenoids by engineered microorganisms.In this article,the role of oxygen in regulating terpenoid bioproduction is overviewed from the viewpoints of cellular carbon metabolism,energy metabolism and terpenoid anabolism.Strategies on adjusting oxygen availability to microorganisms,including genetic modification of cellular metabolism related with oxygen utilization,are summarized and discussed,to provide helpful information for further improvement of terpenoid biosynthesis by microbes.

The constitutions of terpenoids and hormones of leaves in red-flesh citrus mutants and their wild types

To study the profiles and relationships of terpenoids and phytohormones in leaves of red-flesh citrus,we examined four red-flesh mutants that accumulate lycopene in the fruit and their corresponding wild types.We compared their contents of monoterpenoid and sesquiterpenoid volatiles,triterpenoid limonoid aglycones,and tetraterpenoid carotenoids.The types and concentrations of these terpenoids in the leaves varied among genotypes,and the leaf terpenoid profiles differed between red-fleshmutants and theirwild types.However,lycopenewas not detected in citrus leaves of all eight varieties,including the fourmutants.According to phytohormone accumulation in the leaves,the citrus varieties could be classified into a low-phytohormone group(‘Red Anliu’sweet orange,‘Anliu’sweet orange,and‘Cara Cara’navel orange),a high-phytohormone group(‘Seike’navel orange),and a high-jasmonic acid group(‘Red-flesh Guanxi’pummelo,‘Guanxi’pummelo,‘Chuhong’pummelo,and‘Feicui’pummelo).The contents of terpenoid volatiles were very low in leaves of‘Red-flesh Guanxi’and‘Guanxi’pummelo;therefore,they are ideal materials for verifying the functions of genes related to terpenoid volatiles in overexpression analyses.Sesquiterpenoid volatiles were positively correlated with phytohormones of abscisic acid,jasmonic acid,and salicylic acid.Taken together,lycopene was undetected in the leaves of redflesh citrus mutants with abundant lycopene in their juice sacs,which suggested a tissue-specific accumulation pattern of lycopene in citrus red-flesh mutants.Furthermore,leaf volatile profiles could be suitable to develop fingerprint chromatograms of citrus resources,since leaf volatile profiles were dominated by several compounds that varied among genotypes in their combinations and concentrations.

Study on effect and mechanism of Celastrus Orbiculatus total terpenoids in regulating lipid metabolism

Objective To investigate the effect and mechanism of Celastrus Orbiculatus total terpenoids on lipid metabolism in hyperlipidemia mice.Methods ICR mice were selected as investigated subject.The hyperlipemia mice models were made with feeding high-fat forage and were randomly divided into six groups:the normal group,the model group,the positive control group(treated with simvastatin)and the three groups treated with Celastrus Orbiculatus total terpenoids with low,medium and high dosage,respectively.Each group included eight mice.The control group was fed normal forage,but other groups were fed high fat forage.All groups were allowed to drink water freely.Since the first day when the models were made,intragastric administration had been adopted.The normal group was fed normal forage without intragastric administration;the model group was received physiological saline 20 mL·kg-1·d-1 with intragastric administration;the positive control group received simvastatin 2.6 mg·kg-1·d-1 with intragastric administration;the three treated groups received Celastrus Orbiculatus total terpenoids 60 mg·kg-1·d-1,120 mg·kg-1·d-1,200 mg·kg-1·d-1 with intragastric administration respectively.Each group was weighed once a week.On the basis of establishing hyperlipidemia mice model,blood lipids,lipid metabolic enzyme,antioxidative capacity were investigated after 21 days feeding of high-fat forage.Results Compared with model group,TC and TG in mice treated with Celastrus Orbiculatus total terpenoids all reduced and HDL-C raised obviously(P<0.01).Celastrus orbiculatus total terpenoids was shown to decreased MDA content in both serum and liver,increased serum SOD activity and inhibited the activity of the cholesteryl ester transfer protein(CETP).Conclusions Celastrus Orbiculatus total terpenoids could remarkably modulate the lipid metabolic disorder in hyperlipidemia mice,and has a certain regulating function on lipoprotein,inferring that it could reduce the occur of atherosclerosis.The mechanism of regulating lipid metabolism might be related with decreasing the activity of CETP and increasing antioxidative capacity.

A Novel Terpenoid from Lappula anocarpa

A novel terpenoid, named lappulanocarpine A1 was isolated from the alcoholic extract of the whole plant of Lappula anocarpa. Its structure was characterized by ID-, 2D-NMR and HR-ESIMS.

Anticancer Activities and Mechanism of Action of Nagilactones, a Group of Terpenoid Lactones Isolated from Podocarpus Species

Nagilactones are tetracyclic natural products isolated from various Podocarpus species.These lactone-based compounds display a range of pharmacological effects,including antifungal,anti-atherosclerosis,anti-inflammatory and anticancer activities reviewed here.The most active derivatives,such as nagilactones C,E and F,exhibit potent anticancer activities against different cancer cell lines and tumor models.A comprehensive analysis of their mechanism of action indicates that their anticancer activity mainly derives from three complementary action:(i)a drug-induced inhibition of cell proliferation coupled with a cell cycle perturbation and induction of apoptosis,(ii)a blockade of the epithelial to mesenchymal cell transi-tion contributing to an inhibition of cancer cell migration and invasion and(iii)a capacity to modulate the PD-L1 immune checkpoint.Different molecular effectors have been implicated in the antitumor activity,chiefly the AP-1 pathway blocked upon activation of the JNK/c-Jun axis.Nag-C is a potent inhibitor of protein synthesis binding to eukaryotic ribosomes and inhibition of different protein kinases,such as RIOK2 and JAK2,has been postulated with Nag-E.The literature survey on nagilactones highlights the therapeutic potential of these little-known terpenoids.The mechanistic analysis also provides useful information for structurally related compounds(podolactones,oidiolactones,inumakilactones)isolated from Podo-carpus plants.

Potential of Terpenoid Bioactive Compound Isolated from Papua Ant Nest as an Alternative Ovarian Cancer Treatment

Objectives: The purpose of this study was to determine anticancer activity of terpenoid bioactive compound isolated from Papua ant nest on ovarian cancer cells in vitro. Methods: This was a laboratory experimental study which aims to determine the potential of the terpenoid bioactive compound isolated from Papua ant nest to inhibit the growth and induce apoptotic process on ovarian cancer cells (SKO-3) in vitro. Result: Terpenoid had capability to inhibit the growth of ovarian cancer cell line (SKO-3) in vitro, with IC50 of 481 ug/ml at 48 hours and 463 ug/ml at 48 hours, respectively. At a concentration of 600 ug/ml, terpenoid was able to induce apoptotic process on ovarian cancer cell lines in vitro with the apoptotic index of 30% at 24 hours, 35% at 48 hours and 37% at 72 hours, respectively. Conclusion: Terpenoid bioactive compound isolated from Papua ant nest had the ability to inhibit the growth and was able to induce apoptotic process on ovarian cancer cell lines (SKO-3) in vitro.

Evaluating Protective Terpenoid Aldehyde Compounds in Cotton (Gossypium hirsutum L.) Roots

Cotton (Gossypium hirsutum L.) has epidermal glands containing terpenoid aldehyde (TA) compounds that protect the plant from pests and diseases. One TA, gossypol, has two forms (+) and (-) that are present in varying amounts. This study evaluated the variation in roots for TA compounds and how environment affected the levels of these compounds. Similar to seed, gossypol was the predominant TA in roots. No heliocides were detected with only trace amounts of other TAs, such as hemigossypolone or hemigossypol, detected in a few lines. Among the glanded lines, there were significant differences in gossypol content. Percent plus gossypol was consistently 4% - 10% higher in roots than seed. One line, “Mac7”, had 12 - 14 ug/mg gossypol in roots and 18 ug/mg in seed as well as >90% (+) gossypol in both roots and seed. Unlike other tissues in glandless cotton, the roots of glandless lines consistently produced detectable amounts of gossypol with 77% to 82% in the (+) form. Multi-year field tests showed that although gossypol levels in the roots were more affected by insect pressure or other environmental conditions than seed, there was selectable variation in upland cotton for root gossypol content. Unlike other parts of a glandless plant, the roots retained a functioning biochemical pathway for gossypol production.

Study of the mechanism underlying inhibitory effect of terpenoids from Daphne plants on liver cancer cells using a network pharmacology-based approach

Daphne plants,belonging to Thymelaeaceae,have been used as traditional Chinese medicine for thousands of years.Previous studies suggested that they exerted anti-tumor activities and reported a number of compounds with anti-tumor activities,isolated from the genus Daphne.However,the targets of these compounds have not been determined yet.In this study,network pharmacology was used to analyze 128 terpenoid compounds from the genus Daphne and 49 target proteins of liver cancer.The results suggested that the target with the most number of directed edges was 5yz0.The compound which docked with 5yz0 best is AR.DT.25(acutilobin D),and the compounds with total docking score greater than 10 were almost all Daphnane diterpenoids.The network pharmacology results predicted that the 5yz0 protein was one of the potential targets of the terpenoids from the genus Daphne and then suggested several new targets and compounds for further liver cancer research.

In Silico Analysis and Feeding Assays of Some Genes in the Early Steps of Terpenoid Biosynthetic Pathway in Camellia Sinensis

Terpenoids play important roles in productivity and product quality in tea(Camellia sinensis),not only because volatile terpenoids contribute significantly to the aroma of teas,but terpenoid-derived light harvesting pigments and phytohormones such as strigolactones implement photosynthesis and branching in plants.All terpenoids are derived from isopentenyl diphosphate(IPP),dimethylallyl diphosphate(DMAPP) produced by methyl-erythritol-phosphate(MEP) and mevalonate(MVA) pathways,the early steps of isoprenoid biosynthesis.For the purpose to determine key genes in the MEP and MVA pathways in C.sinensis,five genes with complete coding sequence and three with partial sequence were obtained based on previously attained transcriptome data and polymerase chain reaction approach in this study.In silico analyses suggested that the five genes with full coding sequence individually encoded acetyl-CoA C-acetyltransferase(CsAACT),3-hydroxy-3-methylglutaryl-CoA synthase(CsHMGS),4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase(CsCMK),1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase(CsHDS),and farnesyl pyrophosphate synthase(CsFPS).Additionally,studies on metabolite-mediated gene expression were performed using tea cell suspensions and leaf-disc cultures.Our data showed that the addition of acetyl-CoA increased the transcript level of CsAACT and CsHMGS by 1.5- and 3-fold,respectively.The addition of deoxy-D-xylulose 5-phosphate(DXP) induced the expression of CsCMK and CsHDS by 2.3- and 1.5-fold,respectively.The CsFPS expression was approximately 2-fold higher when treated with DXP,IPP or DMAPP than the untreated control,but this induction was not observed with the acetyl-CoA treatment.Our studies indicated that expression of CsAACT,CsHMGS,CsCMK,CsHDS,and CsFPS were metabolite-mediated in a different extent.

Recent Advances in Microbial Production of Terpenoids from Biomass-derived Feedstocks

Terpenoids are a diverse class of natural products widely used as pharmaceuticals,perfumes,flavors,and biofuels.Traditionally,terpenoids are obtained from natural sources,such as plants,but their production is limited by the insufficiency of resources and low yields of extraction.Microbial production of terpenoids has emerged as a promising alternative due to that it is sustainable and easy to scale up.This review aims to summarize recent advances in microbial production of terpenoids from inexpensive biomass-derived feedstocks.Metabolic pathways and key enzymes involved in terpenoid biosynthesis are introduced.Microorganisms that can utilize low-cost lignocellulosic feedstocks for terpenoid production are highlighted.The challenges and prospects faced by microbial terpenoid production are proposed.We believe that continuous progress in the fields of biomass transformation and synthetic biology will ultimately achieve industrial production of microbial terpenoids.

Clavukoelloids A—M,Thirteen Rare N-Containing Nardosinane Type Sesquiterpenoids from the Soft Coral Clavularia koellikeri

Thirteen new nitrogen-containing nardosinane-type sesquiterpenoids,clavukoelloids A-M(1-13)with unique acrylamide fragments vs.the reported general nardosinane-type sesquiterpenoids containing lactone,were isolated from the soft coral Clavularia koellikeri collected from the South China Sea.The structures and absolute configurations of new compounds were unambiguously determined by analysis of spectroscopic data,CP3,DP4+,ECD spectra,and X-ray diffraction.Compound 6 showed anti-inflammatory activities and hepatoprotective activities in transgenic fluorescent zebrafish.

Alternative Construction of the Core of Lindenane Sesquiterpenoid

Comprehensive Summary Alternative synthetic routes toward the tricyclic core of lindenane sesquiterpenoid are presented herein.The route A features an asymmetric organocatalysis enabling desymmetric silylation of diol to establish the desired tertiary alcohol,while the route B consists of an acetate ring opening of chiral epoxy moiety.The common intermediate(16)in both routes can give rise to the core of lindenane sesquiterpenoid via an intramolecular cyclopropanation.

Five new terpenoids from Viburnum odoratissimum var.sessiliflorum

Five new terpenoids,including two vibsane-type diterpenoids(1,2)and three iridoid allosides(3-5),together with eight known ones,were isolated from the leaves and twigs of Viburnum odoratissimum var.sessiliflorum.Their planar structures and relative configurations were determined by spectroscopic methods,especially 2D NMR techniques.The sugar moieties of the iridoids were confirmed asβ-D-allose by GC analysis after acid hydrolysis and acetylation.The absolute configurations of neovibsanin Q(1)and dehydrovibsanol B(2)were determined by quantum chemical calculation of their theoretical electronic circular dichroism(ECD)spectra and Rh2(OCOCF_(3))_(4)-induced ECD analysis.The anti-inflammatory activities of compounds 1,3,4,and 5 were evaluated using an LPS-induced RA W264.7 cell model.Compounds 3 suppressed the release of NO in a dose-dependent manner,with an IC_(50) value of 55.64μmol·L^(-1).The cytotoxicities of compounds 1-5 on HCT-116 cells were assessed and the results showed that compounds 2 and 3 exhibited moderate inhibitory activities with IC_(50) values of 13.8 and 12.3μmol·L^(-1),respectively.

Meroterpenoids Possessing Diverse Rearranged Skeletons with Anti-inflammatory Activity from the Mangrove-Derived Fungus Penicillium sp. HLLG-122

Fifteen new meroterpenoids, littoreanoids A—O (1—15), including three rearranged skeleton meroterpenoids (1—3), were isolated from the mangrove-derived fungus Penicillium sp. HLLG-122. Compound 1 was a novel berkeleyacetal-derived meroterpenoid featuring an unusual spirocyclic 2-oxaspiro[5.5]undeca-4,7-dien-3-one moiety. Compound 2 possessed an unusual 6/6/6/6/6 pentacyclic system with a novel 1-hydroxy-7,7-dimethyl-2-oxabicyclo[2.2.2]octan-5-yl acetate moiety. Compound 3 was an unusual 6/7/6/5/6/5/4 polycyclic systems containing a β-lactone ring. The structures and absolute configurations of new compounds were elucidated by HRESIMS, NMR spectroscopy, single crystal X-ray diffraction analysis, and electronic circular dichroism calculations. The plausible biosynthetic pathways of 1—3 were also proposed. Compounds 6 and 11 exhibited anti-inflammatory effects with IC_(50) values of 30.41 and 19.02 μmol/L, respectively. The bioactive compound 11 was selected for the investigation of preliminary mechanism using molecular docking and Western blotting experiments. Compound 11 could suppress the levels of TNF-α and IL-6, and down-regulate the protein expression of iNOS and COX-2 in RAW 264.7 cells.