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Triterpenes are among the most diverse plant natural products, and their diversity is closely related to various triterpene skeletons catalyzed by different 2,3-oxidosqualene cyclases (OSCs). Celastrol, a friedelane-type triterpene with significant bioactivities, is specifically distributed in higher plants, such as Celastraceae species. Friedelin is an important precursor for the biosynthesis of celastrol, and it is synthesized through the cyclization of 2,3-oxidosqualene, with the highest number of rearrangements being catalyzed by friedelane-type triterpene cyclases. However, the molecular mechanisms underlying the catalysis of friedelin production by friedelane-type triterpene cyclases have not yet been fully elucidated. In this study, transcriptome data of four celastrol-producing plants from Celastraceae were used to identify a total of 21 putative OSCs. Through functional characterization, the friedelane-type triterpene cyclases were separately verified in the four plants. Analysis of the selection pressure showed that purifying selection acted on these OSCs, and the friedelane-type triterpene cyclases may undergo weaker selective restriction during evolution. Molecular docking and site-directed mutagenesis revealed that changes in some amino acids that are unique to friedelane-type triterpene cyclases may lead to variations in catalytic specificity or efficiency, thereby affecting the synthesis of friedelin. Our research explored the functional diversity of triterpene synthases from a multispecies perspective. It also provides some references for further research on the relative mechanisms of friedelin biosynthesis.

The structure and complete nuclear magnetic resonance (NMR) assignments of orbifomitellic acid, a novel lanostane triterpene isolated from the fruiting bodies of a Gabonese Ganoderma orbiforme (Polyporaceae), are reported. Within the vast catalogue of lanostanes documented from Ganoderma spp., orbifomitellic acid is the first disclosing a -COOH group at C-4.

Pistacia chinensis subsp. integerrima (J. L. Stewart ex Brandis) Rech. F. is a valuable medicinal plant used in south Asian communities for the treatment of asthma, diarrhea, diabetes, liver diseases, fever, pain and inflammation. This review critically evaluates the available information on P. integerrima's ethnobotany, ethnopharmacology, phytochemistry, pharmacology and toxicology. Electronic databases such as Google Scholar, PubMed, Springer Link, and so forth, books and theses were used to find relevant information about P. integerrima using keywords such as "Pistacia integerrima," "P. integerrima," "Ethnopharmacology," "Phytochemistry," "Traditional uses". A number of in vitro and in vivo pharmacological activities have been reported; however, the most promising and attractive activity observed was its role in Alzheimer, diabetes, convulsions, cancer, asthma, diabetes, diarrhea and as an immunomodulatory, analgesic and antiinflammatory. In addition, Pistagremic acid exerted anti-Alzheimer's activity based on a hitherto unknown mechanism through interference with the amyloidogenic pathway. Most of the pharmacological activities were linked with traditional uses. A range of compounds have been reported from P. integerrima extracts including triterpenes, volatile oils, flavonoids, fatty acids, phenolic, phytosterols, tannins and oligosaccharides as well as unknown triterpenes and flavonoids. Pistagremic acid, a novel triterpene, was attributed to most of the activities. in vivo toxicological studies in animal suggested a toxic dose of 1,500 mg kg-1 , for its methanolic extract. All reported pharmacological activities were carried out in vitro and a gap in research, that is, preclinical and clinical investigation exists. Its outstanding activity as an antiglycating agent is the most promising and a so far unique activity and needs further evaluation. In-depth research and clinical trials on human subjects in order to investigate P. integerrima pharmacological activity, clinical efficacy and safety are crucial next steps.

Introduction Hederacoside C (HDC) is a bioactive natural triterpenoid saponins constituent originating from traditional Chinese medicines, playing an important role in the treatment of acute respiratory infections and chronic inflammatory bronchitis. Meanwhile, it is recognised by Korea as a botanical drug. Objectives In order to develop an integrated template approach to analysing screening and identification of the metabolites of traditional Chinese medicines. This study will provide available information for further pharmaceutical studies of HDC and other triterpene saponins. Methodology An analysis strategy based on ultrahigh-performance liquid chromatography quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) technique combined with automatic fragment ion search (FISh) was firstly exploited for the characterisation metabolites of HDC in vivo and in vitro. Accurate full mass scan combined with an on-line FISh annotations approach was developed to rapidly identify all the potential metabolites of HDC. Furthermore, FISh accurately located the structure of the target compound in a large number of mass spectral data. Results A total of 34 metabolites were detected and tentatively identified by analysing comprehensive biological samples. The results clearly demonstrated that HDC underwent general metabolic reactions including dealkylation, reduction, oxidation, desaturation, dehydration, cysteine conjugation, GSH conjugation, taurine conjugation, and glycine conjugation to produce 26 phase I and eight phase II metabolites. Conclusion In the present study, UHPLC-Q-Exactive Orbitrap MS technique combined with FISh provided a rapid and efficient platform to characterise metabolites of HDC in vivo and in vitro. The proposed method could develop an integrated template approach to screen and identify the constituents and metabolites of traditional Chinese medicines.

INTRODUCTION: Alismatis Rhizoma (AR) has been widely used to treat various diseases. Its complex chemical composition has caused certain difficulties in the analysis of this traditional Chinese medicine. Therefore, it is necessary to establish a method for the rapid classification and identification of the chemical constituents of AR. OBJECTIVE: This article describes a method for the rapid classification and identification of major triterpenoids in AR. METHODOLOGY: The samples were analysed by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS). The assay was performed on a Waters ACQUITY UPLC BEH C₁₈ column (100 mm × 2.1 mm, 1.7 μm) with 0.1% formic acid in water (A), and acetonitrile (B) as mobile phase by gradient elution at a flow rate of 0.3 mL/min. In the positive ion mode, the fragment information was obtained and compared with the characteristic fragments and neutral losses described in the literature. Then, the rapid classification and identification of the chemical components from AR were achieved. RESULTS: Finally, 25 triterpene compounds of AR were identified. CONCLUSIONS: The method established in this study achieved the rapid classification and identification of chemical components in AR, which promotes the development of research methods to study the constituents of traditional Chinese medicine.

The aim of the present work is to isolate a series of triterpene derivatives with rhamnosyl linking acetyl groups from Glechoma longituba according to the structural characteristics of previously described triterpene saponins. The extract ion chromatography spectrum of the crude extract of G. longituba was detected and analyzed by HPLC-HR-ESI-MS to determine possible components, and these metabolites were traced and separated by combining high-resolution mass spectrometry and predicted liquid chromatography retention time. Three 11α, 12α-epoxypentacyclic oleanolic acid triterpene saponins (glechomanosides H-J) and one ursane triterpene aldehyde saponin with a C-28 aldehyde group were isolated from G. longituba. The structure of these compounds was confirmed by NMR and compared with those of previously characterized compounds. The strategy described in this report enables a rapid, reliable, and complete analysis of glycoside compounds containing different numbers of acetyl groups at different positions on the sugar.

Squalene is a naturally occurring triterpene with wide industrial applications. Due to limited natural resources, production of this valuable lipid in yeast is of high commercial relevance. Typically low levels of squalene in yeast can be significantly increased by specific cultivation conditions or genetic modifications. Under normal conditions, excess squalene is stored in lipid droplets (LD), while in a Saccharomyces cerevisiae mutant unable to form LD it is distributed to cellular membranes. We present here the evidence that squalene accumulation in this LD-less mutant treated with squalene monooxygenase inhibitor terbinafine induces growth defects and loss of viability. We show that plasma membrane malfunction is involved in squalene toxicity. We have found that subinhibitory concentrations of terbinafine increased the sensitivity of LD-less mutant to several membrane-active substances. Furthermore, squalene accumulation in terbinafine-treated LD-less cells disturbed the maintenance of membrane potential and increased plasma membrane permeability to rhodamine 6G. LD-less cells treated with terbinafine showed also high sensitivity to osmotic stress. To confirm the causal relationship between squalene accumulation, loss of viability and impaired plasma membrane functions we treated LD-less cells simultaneously with terbinafine and squalene synthase inhibitor zaragozic acid. Reduction of squalene levels by zaragozic acid improved cell growth and viability and decreased plasma membrane permeability to rhodamine 6G in terbinafine-treated LD-less cells. Our results support the hypothesis that plasma membrane malfunction is involved in the mechanisms of squalene lipotoxicity in yeast cells with defective lipid storage.

Medicago truncatula is an established model plant, not only for legume biology, but also for the study of specialized metabolism, which serves to create defense barriers and responses to biotic and abiotic external threats. Triterpene saponins (TS) and flavonoids are the two major classes of specialized metabolites in M. truncatula. Here, we provide a detailed overview of TS biosynthesis and regulation and link this to the spatial and functional organization of TS in M. truncatula. Depending on specific oxidations of the triterpene aglycone backbone, M. truncatula TS can be divided further into two types, the hemolytic and non‐hemolytic TS. TS biosynthesis is positively regulated by jasmonate phytohormones, which is mediated, at least in part, through specific basic helix‐loop‐helix transcription factors. Furthermore, TS production is subjected to post‐translational regulation by protein‐protein interactions or endoplasmic reticulum‐associated degradation of key enzymes of the TS biosynthesis pathway, which allows surveying appropriate TS balances.

Introduction Most secondary metabolites from plants have a prominent defensive role and repellency against predators and microbial pathogens. These properties largely vary among plant species and offer potential applications as biologically active compounds in medicine as well in agriculture. Objectives We propose a new procedure that combine different spectroscopic techniques and multivariate data analysis to determine the chemical composition and the relative amounts of each metabolites and/or each class of organic compounds. The approach was used for a rapid identification of secondary metabolites from leaf and root of eight Mediterranean plants species. Methodology The polar and the apolar extracts of two leaves and roots of each plant were analysed by proton nuclear magnetic resonance (1 H-NMR) and gas chromatography coupled to mass spectrometry (GC-MS), respectively. Multivariate data analysis was used for a faster interpretation of data. Results The metabolic fingerprint of the Mediterranean plants, Acanthus mollis, Dittrichia viscosa, Festuca drymeja, Fraxinus ornus, Fagus sylvatica, Hedera helix, Quercus ilex, and Typha latifolia, showed a complex chemical composition, being specific for each species and plant tissue. Two alditols, mannitol and quercitol, were found in manna ash (Fraxinus ornus) and holm oak (Q. ilex) polar leaf extracts, respectively. The highest levels of aromatic compounds were found in D. viscosa and T. latifolia. Fatty acids were the predominant class of compounds in all apolar extracts under investigation. Triterpene were almost exclusively found in roots, except for holm oak, where they constitute 58% of total extract. Steroids were widespread in leaf extracts. Conclusion The major advantages of the proposed approach are versatility and rapidity, thus making it suitable for a fast comparison among species and plant tissue types.

Ardisia crenata Sims (Primulaceae) occurs in natural habitats in two varieties, bearing red or white fruits. While roots of the red-berried ardisia are valued as a medicinal product, the pharmacological activity of which is attributed to triterpene saponins, including ardisiacrispin A, data on the white-berried variety are scarce. A TLC-densitometric method was developed and validated to estimate the levels of saponins, calculated as ardisiacrispin A, in different plant parts in both varieties. Their content amounted to 22.17±4.75 and 25.72±1.46 mg/g d.w. in roots, and 2.64±0.74 and 3.43±0.70 mg/g d.w. in fruits of red-berried and white-berried ardisia, respectively. Assessment of cytotoxicity of ardisiacrispin A and A. crenata extracts on a panel of human cancer cell lines revealed a similar effect of root extracts from both varieties, with the highest potency against melanoma WM793 and colon cancer Caco2. Thus, roots of the white-berried variety may be treated as a substitute for red-berried ardisia and serve as an alternative source for the acquisition of plant material rich in bioactive saponins.

Fu-Ke-Zai-Zao pills, the famous traditional Chinese medicine formula, composed of 42 medicinal herbs, have been widely used to treat various gynecological diseases. However, the chemical constituents and metabolic profiling of Fu-Ke-Zai-Zao pills remain largely unknown, which hampers improvement of the quality control and pharmacological elucidation of this formula. In the present study, a sensitive and selective ultra high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry method was developed to separate and identify the comprehensive chemical constituents of Fu-Ke-Zai-Zao pills. According to the results, a total of 83 compounds were identified, including phenylpropionic acids, flavonoids, terpenoids, triterpene saponins, and phthalides, and 81 compounds were first reported in Fu-Ke-Zai-Zao pills. Moreover, the absorbed components and metabolites in rat plasma after intragastric administration of Fu-Ke-Zai-Zao pills were also detected by the same analytical method. A total of 36 compounds were identified, including 21 prototypes and 15 metabolites. The latter were generated through the metabolic pathways of methylation and glucuronidation, and glucuronidated metabolites were the main constituents in the plasma. This is the first systematic study on the chemical constituents and metabolic profiling of Fu-Ke-Zai-Zao pills, and the results provide valuable chemical information for further elucidating pharmacological effects and mechanism of action of Fu-Ke-Zai-Zao pills.

Saponins are naturally occurring secondary metabolites, which can be widely found in a number of plant families. They can be found in different parts of plants usually as multicomponent mixtures. They are composed of the triterpene or steroidal aglycone to which a number of sugars in the form of one, two, or three sugar chains are attached. Depending on the chemical structure, they show different biological activities. The common feature of these compounds is surface activity, so they can be used as emulsifiers, and in the past, they were used as soap substitutes. Some of them have found application in food industry, whereas the others are being considered as antinutritional substances. Owing to the fact that saponins occur in quite complex mixtures, their separation to individual compounds is still a challenge. Also, their determination with chromatographic techniques creates some problems as they are lacking chromophores allowing ultraviolet (UV) detection. Precolumn derivatization, light scattering detection, and liquid chromatography coupled with mass detector are the methods of choice for their determination. Keywords: saponins; glycosides; steroids; triterpenes; analytical methods

Vangueria agrestis is a shrub indigenous to tropical Africa, belonging to family Rubiaceae and is traditionally used as a decoction for treatment of fever, pain, and malaria. This study was undertaken to investigate the chemical constituents based on precursor exact mass and fragment ion information. The chemical profiling and structural characteristics of chemical constituents from methanolic extracts of dried aerial parts and roots of V. agrestis and dietary supplements were analyzed using ultra-high-performance liquid chromatography/ion mobility quadrupole time-of-flight mass spectrometry coupled with UNIFI platform and multivariate analysis in both negative and positive ion modes. A non-targeted ultra-high-performance liquid chromatography-mass spectrometry analysis was carried out to profile the chemical constituents of crude extracts of V. agrestis, and 73 compounds, including reference compounds, were identified. The fragments of flavonoids, monoterpene, and triterpene glycosides revealed the characteristic cleavage of glycosidic linkages, and the fragmentation pattern provided the identity of the sugars. This analytical method provides a quick method for quality assessment of dietary supplements. Finally, a chemometrics approach with multivariate statistical tools was used to visualize the differences between root and aerial parts of plant samples and to find the potential chemical markers that differentiate among these parts of V. agrestis samples and dietary supplements.

The Wen-Jing decoction, a traditional Chinese medicine formula, has been used as a blood-activating and stasis-eliminating drug to treat gynaecological syndromes, such as dysmenorrhea, amenorrhea, and menstrual disorders. However, its pharmacodynamic material basis and mechanism of action have not been thoroughly elucidated to date. The goal of this study was to characterize and identify multiple constituents and metabolites in Wen-Jing decoction. An ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry method was established and validated in the present study for the first time. A total of 101 compounds, including 11 monoterpene glycosides, 19 flavonoids, 49 triterpene saponins, 5 phthalides, 3 phytoecdysones, and 14 others, were unambiguously or tentatively characterized by comparing their retention times and MS data with reference standards or with data reported in the literature. After oral administration of Wen-Jing decoction, 27 compounds, including nine prototype compounds and 18 metabolites were detected in rat plasma. Thus, the ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry method was found to be efficient for in-depth structural elucidation of chemical compounds in complex matrices of herbal medicines, which will provide useful chemical information for quality control and mechanism-of-action research.

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, β-amyrin or cycloartenol, and a multifunctional LsOSC2 formed α-amyrin as a major product along with β-amyrin. Two CYP716 family P450s (CYP716A265, CYP716A266) catalyzed C-28 oxidation of α-amyrin, β-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.

(3S,4R)-23,28-Dihydroxyolean-12-en-3-yl (2E)-3-(3,4-dihydroxyphenyl)acrylate (1 a), which possesses significant neuritogenic activity, was isolated from the traditional Chinese medicine (TCM) plant, Desmodium sambuense. To confirm the structure and to assess biological activity, we semi-synthesized 1 a from commercially available oleanolic acid. A series of novel 1 a derivatives was then designed and synthesized for a structure-activity relationship (SAR) study. All synthetic derivatives were characterized by analysis of spectral data, and their neuritogenic activities were evaluated in assays with PC12 cells. The SAR results indicate that the number and position of the hydroxy groups on the phenyl ring and the triterpene moiety, as well as the length of the (saturated or unsaturated) alkyl chain that links the phenyl ring with the triterpene critically influence neuritogenic activity. Among all the tested compounds, 1 e [(3S,4R)-23,28-dihydroxyolean-12-en-3-yl (2E)-3-(3,4,5-trihydroxyphenyl)acrylate] was found to be the most potent, inducing significant neurite outgrowth at 1 μm.

Summary Squalene and botryococcene are linear, hydrocarbon triterpenes that have industrial and medicinal values. While natural sources for these compounds exist, there is a pressing need for robust, renewable production platforms. Oilseeds are an excellent target for heterologous production because of their roles as natural storage repositories and their capacity to produce precursors from photosynthetically‐derived carbon. We generated transgenic Arabidopsis thaliana plants using a variety of engineering strategies (subcellular targeting and gene stacking) to assess the potential for oilseeds to produce these two compounds. Constructs used seed‐specific promoters and evaluated expression of a triterpene synthase alone and in conjunction with a farnesyl diphosphate synthase (FPS) plus 1‐deoxyxylulose 5‐phosphate synthase (DXS). Constructs directing biosynthesis to the cytosol to harness isoprenoid precursors from the mevalonic acid (MVA) pathway were compared to those directing biosynthesis to the plastid compartment diverting precursors from the methylerythritol phosphate (MEP) pathway. On average, the highest accumulation for both compounds was achieved by targeting the triterpene synthase, FPS and DXS to the plastid (526.84 μg/g seed for botryococcene and 227.30 μg/g seed for squalene). Interestingly, a higher level accumulation of botryococcene (a non‐native compound) was observed when the biosynthetic enzymes were targeted to the cytosol (>1000 μg/g seed in one line), but not squalene (natively produced in the cytosol). Not only do these results indicate the potential of engineering triterpene accumulation in oilseeds, but they also uncover some the unique regulatory mechanisms controlling triterpene metabolism in different cellular compartments of seeds.

Squalene, a valuable acyclic triterpene, can be used as a chemical commodity for pharmacology, flavor, and biofuel industries. Microbial production of squalene has been of great interest due to its limited availability, and increasing prices extracted from animal and plant tissues. Here we report genetic perturbations that synergistically improve squalene production in Saccharomyces cerevisiae. As reported previously, overexpression of a truncated HMG-CoA reductase 1 (tHMG1) led to the accumulation 20-fold higher squalene than a parental strain. In order to further increase squalene accumulation in the tHMG1 overexpressing yeast, we introduced genetic perturbations-known to increase lipid contents in yeast-to enhance squalene accumulation as lipid body is a potential storage of squalene. Specifically, DGA1 coding for diacylglycerol acyltranferase was overexpressed to enhance lipid biosynthesis, and POX1 and PXA2 coding for acyl-CoA oxidase and a subunit of peroxisomal ABC transporter were deleted to reduce lipid β-oxidation. Simultaneous overexpression of tHMG1 and DGA1 coding for rate-limiting enzymes in the mevalonate and lipid biosynthesis pathways led to over 250-fold higher squalene accumulation than a control strain. However, deletion of POX1 and PXA2 in the tHMG1 overexpressing yeast did not improve squalene accumulation additionally. Fed-batch fermentation of the tHMG1 and DGA1 co-overexpressing yeast strain resulted in the production of squalene at a titer of 445.6 mg/L in a nitrogen-limited minimal medium. This report demonstrates that increasing storage capacity for hydrophobic compounds can enhance squalene production, suggesting that increasing lipid content is an effective strategy to overproduce a hydrophobic molecule in yeast.

The geographic impact on the quality of Alismatis Rhizoma (derived from the tuber of Alisma orientale), a reputable diuretic traditional Chinese medicine, has seldom been evaluated. Here a metabolomics-driven approach targeting the bioactive protostane triterpenes was developed, by incorporating UHPLC with quadrupole time-of-flight mass spectrometry-based untargeted metabolite profiling and multiple reaction monitoring quantitative assay, to probe the triterpene differences between Alismatis Rhizoma samples collected from Sichuan, Fujian, and Jiangxi Provinces. Following the metabolomics workflows, the samples from Sichuan and Jiangxi displayed distinct differences in their triterpene profiles, whereas those from Fujian showed remarkable intra-class variation. Twenty-three triterpenes were identified to contribute most to the differentiated clustering. A sensitive, precise, repeatable, and accurate quantitative assay method was established on a hybrid triple quadrupole-linear ion trap mass spectrometer to quantify the contents of eight triterpene compounds. Taking into account the metabolomics and quantitation results, alisol B 23-acetate and alisol A are significantly different in Alismatis Rhizoma from Sichuan and Jiangxi Provinces, and they may have the potential for geographic discrimination. These results illustrate how geographic difference impacts the triterpene chemistry of Alismatis Rhizoma. Metabolomics-driven chemical comparison is suitable for the quality evaluation of traditional Chinese medicine.

In this study, a capillary zone electrophoresis (CZE) method was first developed to identify three microconstituents of isomeric pentacyclic triterpene acids (PTAs including oleanolic acid (OA), ursolic acid (UA) and betulinic acid (BA)) in Forsythiae Fructus (FF). The baseline separation of PTAs by CZE were eventually achieved in a background electrolyte (BGE) containing 50.0 mmol/L borax and 0.5 mmol/L β-cyclodextrin (β-CD) at pH 9.5 within 13.0 min. Herein, it was not only the compositions of BGE were detail investigated for rapid and good separation, but also the binding ratio and the equilibrium constants (K) for OA, UA and BA with β-CD was estimated by double reciprocal equation to well understand the separation mechanism. The proposed method allowed the LODs of PTAs were averaged at 1.50 μg/mL with UV detection (at 200 nm). The interday RSD of migration time and peak area were around 2.0 and 4.7% (n = 5), respectively. Thus, the content of PTAs in 19 FF real samples distinguished from maturation stages and geographical areas in China was quantified with the proposed method. Depending on the amount of each PTA in FF, it was demonstrated these microconstituents might benefit to identify their harvested time even qualities.

Summary Squalene is a linear intermediate to nearly all classes of triterpenes and sterols and is itself highly valued for its use in wide range of industrial applications. Another unique linear triterpene is botryococcene and its methylated derivatives generated by the alga Botryococcus braunii race B, which are progenitors to fossil fuel deposits. Production of these linear triterpenes was previously engineered into transgenic tobacco by introducing the key steps of triterpene metabolism into the particular subcellular compartments. In this study, the agronomic characteristics (height, biomass accumulation, leaf area), the photosynthetic capacity (photosynthesis rate, conductance, internal CO 2 levels) and triterpene content of select lines grown under field conditions were evaluated for three consecutive growing seasons. We observed that transgenic lines targeting enzymes to the chloroplasts accumulated 50–150 times more squalene than the lines targeting the enzymes to the cytoplasm, without compromising growth or photosynthesis. We also found that the transgenic lines directing botryococcene metabolism to the chloroplast accumulated 10‐ to 33‐fold greater levels than the lines where the same enzymes were targeted to in the cytoplasm. However, growth of these high botryococcene accumulators was highly compromised, yet their photosynthesis rates remained unaffected. In addition, in the transgenic lines targeting a triterpene methyltransferase (TMT) to the chloroplasts of high squalene accumulators, 55%–65% of total squalene was methylated, whereas in the lines expressing a TMT in the cytoplasm, only 6%–13% of squalene was methylated. The growth of these methylated triterpene‐accumulating lines was more compromised than that of nonmethylated squalene lines.

Triterpene saponins are main active constituents of Panax notoginseng. Metabolites profiling of 12 triterpene saponins was analyzed by high-performance liquid chromatography-mass spectrometry in leaf, petiole, and root extracts of P. notoginseng. Most of the 20(S)-protopanaxatriol (PPT) type saponins, except ginsenoside Re, were mainly distributed in roots, while 20(S)-protopanaxadiol (PPD) type saponins were detected among various tissues. The total content of PPD-type saponins decreased in the order of leaf, petiole, and root. The expression patterns of four key genes (PnFPS, PnSQS, PnDS, and PnSE) in the triterpene saponin biosynthetic pathway were measured by real-time quantitative PCR (RT-qPCR). All the four investigated genes showed high expression levels in leaf. A gene-to-metabolite network was constructed through canonical correlation analysis. The results indicated that the expression levels of PnFPS, PnSQS, PnDS, and PnSE had high correlation with PPD-type saponins ginsenoside Rb2 , Rb3 , and Rc, while PnSQS was also highly correlated with Rb1 . Combining metabolic profiling, RT-qPCR, and gene-to-metabolite network, we inferred that the leaf of P. notoginseng was the main biosynthesis site of PPD-type saponins Rb2 , Rb3 , and Rc. The contribution to the biosynthesis of ginsenosides Rb2 , Rb3 , and Rc was in the order of PnSE > PnDS > PnSQS > PnFPS. PnSE and PnDS should be the preferred targets to regulate the production of PPD-type saponins Rb2 , Rb3 , and Rc in P. notoginseng by plant metabolic engineering.

Onoceroids are a group of triterpenes biosynthesized from squalene or dioxidosqualene by cyclization from both termini. We previously identified a bifunctional triterpene/sesquarterpene cyclase (TC) that constructs a tetracyclic scaffold from tetraprenyl-β-curcumene (C35 ) but a bicyclic scaffold from squalene (C30 ) in the first reaction. TC also accepts the bicyclic intermediate as a substrate and generates tetracyclic and pentacyclic onoceroids in the second reaction. In this study, we analyzed the catalytic mechanism of an onoceroid synthase by using mutated enzymes. TCY167A produced an unnatural tricyclic triterpenol, but TCY167L , TCY167F , and TCY167W formed small quantities of tricyclic compounds, which suggested that the bulk size at Y167 contributed to termination of the cyclization of squalene at the bicyclic step. Our findings provide insight into the unique catalytic mechanism of TC, which triggers different cyclization modes depending on the substrate. These findings may facilitate the large-scale production of an onoceroid for which natural sources are limited.

Danshen (Salvia miltiorrhiza) is a well-known medicinal herb in the oriental medicine. The current study on bioactive triterpenoid in the root of S. miltiorrhiza led to the isolation of a new highly hydroxylated ursane-type triterpene, urs-12-ene-2α,3β,7β,16α-tetraol (1) and five known ones including 2β-hydroxypomolic acid (2), maslinic acid (3), asiatic acid (4), ursolic acid (5), and oleanolic acid (6). Their structures were elucidated on the basis of extensive spectroscopic analyses and comparison with literature data. The antiproliferative testing against HL-60 cells revealed that the new compound 1 and ursolic acid (5) showed weak and moderate activities with IC50 values of 42.2 and 11.7 μM. In addition, compounds 1-3 showed inhibitory effect on ghrelin activity. Copyright © 2017 John Wiley & Sons, Ltd.

Background: Olive oil extraction generates a large amount of residue consisting mainly of the pomace and leaves when using a two-phase centrifugation system. The aim of this study was to assess the content of phenolic and triterpene compounds in the by-products produced in Spanish olive oil mills.; Results: Olive pomace had concentrations of phenolic and triterpene substances lower than 2 and 3 g kg-1 , respectively. The leaves contained a high concentration of these substances, although those collected from ground-picked olives had lost most of their phenolic compounds. Moreover, the sediment from the bottom of the olive oil storage tanks did not have a significant amount of these substances. By contrast, a new by-product called olive pomace skin has been revealed as a very rich source of triterpenic acids, the content of which can reach up to 120 g kg-1 in this waste product, maslinic acid comprising around 70% of total triterpenics.; Conclusion: Among the by-products generated during extraction of olive oil, olive pomace skin has been discovered to be a very rich source of triterpenic acids, which can reach up to 120 g kg-1 of the waste. These results will contribute to the valorization of olive oil by-products. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

A series of triterpene dimers bearing different scaffold were designed and synthesized via CuAAC reaction. Their anti-HCV entry activities were evaluated by HCVpp and VSVpp entry assays. It was found that echinocystic acid (EA) and its dimer were still necessary for maintaining anti-HCV entry activity, and replacement of EA by other triterpenes might significantly decrease its anti-viral activities. Using a linker bearing a piperazine group, compound 14 dramatically increased its potency with IC50 at 2.87 nmol/L. In addition, the undesired hemolytic effect of all these compounds was removed.

Asiaticoside (1), namely, 2α,3β,23-trihydroxy-urs-12-en-28-oic-O-α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside, the major active biomarker of the traditional medicinal plant Centella asiatica, has been synthesized for the first time. The strategy features a late-stage gold(I)-catalyzed glycosidic condensation of a triterpenoid acid and a trisaccharide ortho-hexynylbenzoate. The overall preparation required a total 31 steps with the longest linear sequence occurring over 14 steps in a 6 % overall yield and starting from ursolic acid (3β-hydroxy-urs-12-en-28-oic acid).

The crude methanolic extract obtained from Verbascum nigrum aerial parts (VNE) and its six fractions (VNF1-VNF6) were initially screened regarding their effects on the viability of immortalized HaCaT keratinocytes and A431 epidermoid carcinoma cells (MTT assay, 24 h). None of the tested samples affected the viability of HaCaT cells in a concentration range of 25-150 μg/mL. VNE and VNF4 exhibited significant cytotoxic effects in A431 cells, with IC50 values of 81.92 and 12.27 μg/mL, respectively; the selectivity index was higher than 10 for VNF4. The untargeted LC/HR-MS/MS metabolite profiling led to the tentative annotation of a total number of 23 compounds. Of these, VNE comprised mainly iridoid glycosides (harpagoside, laterioside, acylated aucubin derivatives), whereas VNF4 showed a high abundance of triterpene saponin glycosides (ilwensisaponins A and C, songarosaponins A and B), constituents known for their selective cytotoxic potential.

Centella asiatica is a well‐known herbal medicine used for antioxidant and antibacterial purposes. Main compounds in the group of triterpene saponins, namely, asiaticoside, asiatic acid, madecassoside, and madecassic acid are considered as the active compounds. These active ingredients were extracted from C. asiatica dried powder by the maceration method. The extracted solution was then used as a starting solution for the gas anti‐solvent (GAS) precipitation. The effects of % ethanol in a mixed solvent of ethanol and methanol (0–100%), carbon dioxide flow rate (3–9 ml/min), and temperature (25–45°C) on the amount of precipitates were investigated using a Box–Behnken design of experiments. It was found that % ethanol in the mixed solvent was significant at 5% significance level on madecassoside, asiaticoside, and total triterpene saponins. Higher amounts of total triterpene saponins could be precipitated when using low % ethanol and low temperature. At the optimal conditions, the amount of madecassoside, asiaticoside, madecassic acid, and asiatic acid were found to be 5,733 ± 537, 2,948 ± 249, 1,678 ± 163, and 436 ± 17 μg/5 ml of extracted solution, respectively. In the antioxidant studies, the highest Trolox equivalent found in the GAS products using 2,2‐diphenyl‐1‐picrylhydrazyl and 2,2‐azion‐bis‐3‐ethylbenzothiazo line‐6‐sulphonic acid assays was 5,622 ± 336 and 10,729 ± 820 μg/5 ml of extracted solution. PRACTICAL APPLICATIONS: The novelty of this work covers the feasibility of applying gas anti‐solvent (GAS) technique for precipitation of bioactive compounds from extracted Centella asiatica and a statistical optimization of the operating parameters on the highest amount of precipitates. This work is important for extraction and purification applications focused on obtaining high purity of bioactive compounds from crude extracts. The use of dense CO₂ as an anti‐solvent not only requires mild operating conditions but also provides a solvent‐free product. Therefore, the application of the GAS process can then be extended to precipitate active ingredients from other herbal extracts.

Saponin PH, akemisaponins E, saponin PJ1 and scheffoleoside A, the main bioactive triterpene saponins of Chinese traditional medicine Akebia trifoliata, contribute to its diuretic pharmacological activity. Because of interactions of the multiple ingredients in vivo, pharmacokinetic studies of multiple triterpenes after administration of A. trifoliata extract are essential to clarify their pharmacological effects. The purpose of this study was to develop an efficient and sensitive UHPLC-MS/MS method for simultaneous determination of these four triterpene saponins in rat plasma. The biosamples were prepared by liquid-liquid extraction with n-butanol. The chromatographic separation was performed on a Phenomenex Luna® C18 (150 × 2 mm, 3 μm) with a mobile phase consisting of acetonitrile and water at a flow rate of 0.5 mL/min. The MS/MS system was operated in a negative multiple reaction monitoring mode, and the precursor-product ion transitions were optimized as m/z 941.6 → 471.1 for saponin PH, 941.7 → 471.2 for akemisaponins E, 1089.7 → 601.1 for saponin PJ1 , 957.6 → 487.4 for scheffoleoside A and 799.5 → 637.3 for ginsenoside Rg1 (Rg1 , internal standard). Method validation parameters (calibration curve linearity, lower limit of detection, recovery, matrix effect, intra- and inter-day precision) were within the acceptable ranges. This is the first reported on the UHPLC-MS/MS detection of saponin PH, akemisaponins E, saponin PJ1 and scheffoleoside A, and applied to a preclinical pharmacokinetic study after oral administration of A. trifoliata extract in rats. This study provides a basis for clinical application and further development of A. trifoliata extract.

Summary Rice (Oryza sativa L.) is one of the most important food crops in the world. In red rice, black, purple, brown, and brown red rice, major bioactive components are gallic, protocatechuic, hydroxybenzoic, p-coumaric,ferulic, sinapic acid, cyanidin-3-O-glucoside, peonidin-3-O-glucoside,flavan-3-ol (+) catechin and (−) epicatechin,flavanols (flavan- 3-ols), isoflavones, γ-oryzanol contents, compositions of steryl, triterpene alcohol ferulates proportions, and tocopherols, etc. This review provides information of pigmented rice in the form of bioactive compounds, concentration, activities and its health's benefit. These bioactive compounds are having immense potential for many health benefit effects and can act as antitumour, anti-atherosclerosis, anti-diabetic, anti-allergicagents, alleviating gallstones, anticancer activity, anti-inflammatoryeffects, and others.

Summary Natural rubber (NR) is an important raw material for a large number of industrial products. The primary source of NR is the rubber tree Hevea brasiliensis, but increased worldwide demand means that alternative sustainable sources are urgently required. The Russian dandelion (Taraxacum koksaghyz Rodin) is such an alternative because large amounts of NR are produced in its root system. However, rubber biosynthesis must be improved in order to develop T. koksaghyz into a commercially feasible crop. In addition to NR, T. koksaghyz also produces large amounts of the reserve carbohydrate inulin, which is stored in parenchymal root cell vacuoles near the phloem, adjacent to apoplastically-separated laticifers. In contrast to NR, which accumulates throughout the year even during dormancy, inulin is synthesized during the summer and is degraded from the autumn onwards when root tissues undergo a sink-to-source transition. We carried out a comprehensive analysis of inulin and NR metabolism in T. koksaghyz and its close relative T. brevicorniculatum, and functionally characterized the key enzyme fructan 1-exohydrolase (1-FEH), which catalyzes the degradation of inulin to fructose and sucrose. The constitutive overexpression of Tk1-FEH almost doubled the rubber content in the roots of two dandelion species without any trade-offs in terms of plant fitness. To our knowledge, this is the first study showing that energy supplied by the reserve carbohydrate inulin can be used to promote the synthesis of NR in dandelions, providing a basis for the breeding of rubber-enriched varieties for industrial rubber production. This article is protected by copyright. All rights reserved.

Microwave-assisted extraction is compared with a more classical technique, Soxhlet extraction, to determine the content of triterpene acids in olive skins. The samples used in their original unmilled state and milled were extracted with ethyl acetate or methanol as solvents. The optimized operating conditions (e.g., amount and type of solvent, and time and temperature of extractions) to attain the better extraction yields have been established. For the identification and quantitation of the target compounds, an ultra high performance liquid chromatography with tandem mass spectrometry method was employed. The best results were achieved using the microwave-assisted extraction technique, which was much faster than the Soxhlet extraction method, and showed higher efficiency in the extraction of the triterpenic acids (oleanolic and maslinic).

Pentalinon andrieuxii (Mull.Arg.) B.F.Hansen & Wunderlin (Apocynaceae) is a vine native to the Yucatan peninsula, where it is widely used in Mayan traditional medicine to treat, among other ailments, the wounds caused by cutaneous leishmaniasis. Among the secondary metabolites isolated from P. andrieuxii are the triterpene betulinic acid and the chemically unusual tri-norsesquiterpene urechitol A; however, to date, there is no existing knowledge about the accumulation dynamics of the ubiquitous betulinic acid or the novel urechitol A in the plant. In this article, we report on the accumulation of both secondary metabolites in wild individuals of P. andrieuxii; our results show that while the content of betulinic acid in plant leaves bears no apparent relation to plant ontogeny, the content of urechitol A in root tissue is clearly related to plant development.

Malonyl-triterpene saponins (MTSs) attract scientific attentions because of their structural diversities and valuable bioactivities. However, its thermal instability brings a huge amount of challenges for isolation and purification of this class of compounds. To our best knowledge, there has been no report on isolation and analysis of MTSs from genus Caulophyllum. In this study, a strategy combining data acquisition using an energy-resolved technique and the narrow widow extracted ion chromatograms as data mining method was developed for discovery and identification of MTSs in Caulophyllum robustum hair roots by ultra high liquid chromatography coupled to electrospray ionization Fourier transform mass spectrometry. The method was performed at an independent MS full scan using our bottom-up energies by in-source collision induced dissociations with 0, 25, 50 and 100 eV in both positive and negative modes. Precursor ion as well as fragment ion information was simultaneously collected from four energy-resolved MS spectra in a single run of 18 min. The fragmentation pathways of intact deprotonated, protonated and sodium ions of MTSs were proposed for the structural elucidation of Caulophyllum MTSs. A flowchart involving a stepwise procedure based on key fragments from ESI- /ESI+ -FT-MS(1, 1) to MS(1, 4) spectra was constructed for the identification of structural elements in the MTSs. As a result, a total of 23 MTSs were discovered and tentatively identified, which had not been reported from Caulophyllum species before. All of these were potentially new compounds. This study provides an excellent example for discovery and identification of MTSs in herb medicines. Copyright © 2016 John Wiley & Sons, Ltd.

Licorice is among the most popular herbal medicines and frequently used in traditional medicine, food products, and cosmetics. In China, only Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. and Glycyrrhiza glabra L. are officially used and are usually processed with honey prior to use. To maintain the quality of commercially available herbal products, a simple, rapid, and reliable ultra high performance liquid chromatography with triple quadrupole mass spectrometry was developed to investigate the major active constituents of commercially available licorice products. Nineteen components were accurately determined, including eight triterpenoid saponins, one triterpene, and ten flavonoids. Subsequently, multivariate statistical analysis methods were employed to further explore and interpret the experimental data. The results indicated that liquiritin apioside may be considered as a candidate index for the quality control of licorice as well as 18β-glycyrrhizic acid and liquiritin. In addition, both 18β-glycyrrhizic acid and licorice-saponin G2 can be used for discrimination between crude and honey-processed licorice. Furthermore, using 18β-glycyrrhizic acid and liquiritin as markers, this work revealed that the quality of licorice products may have declined in recent years. This highlights the need for additional effort focused on good agricultural practice during the processing of licorice. In summary, this study provides a valuable reference for the quality assessment of licorice.

SCOPE Maslinic acid has been described to exert a chemopreventive activity in colon cancer. Hereby, we determined maslinic acid and its metabolites in the rat intestine previous oral administration as a first step in elucidating whether this triterpene might be used as a nutraceutical. METHODS AND RESULTS Maslinic acid was orally administered at 1, 2, and 5 mg/kg to male Sprague-Dawley for 2 days. At 24 h after the last administration, the content of the duodenum and jejunum, ileum, cecum, and colon was collected and extracted with methanol 80% prior to LC-APCI-MS analysis. The developed method was validated providing suitable sensitivity (LOQ of 5 nM), good recovery (97.8 ± 3.6%), linear correlation, and appropriate precision (