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The current study aimed to investigate the drug delivery potential of chitosan‐conjugated Spondias pinnata phytocompounds for anticancer and antibacterial applications. The phytochemical composition of the aqueous extract of S. pinnata plant leaves revealed seven major compounds, including stearic acid, 2H‐Indol‐2‐one, beta amyrin, oleic acid, octadecanoic acid, 7‐hexadecenoic acid, and phytol. Additionally, five minor compounds were identified through GC–MS analysis. SEM analysis of chitosan‐conjugated S. pinnata phytocompounds revealed amorphous particles. This demonstrates the attainment of optimized larger crystallites, which differ in size and shape extensively. The antioxidant potential of both the chitosan‐conjugated S. pinnata phytocompounds and S. pinnata leaf extracts was evaluated via DPPH and ABTS assays, and the results revealed that the chitosan‐conjugated S. pinnata phytocompounds exhibited significant scavenging activity, with IC50 values of 18.20 and 33.15 μg/mL, respectively. Chitosan‐conjugated S. pinnata phytocompounds also demonstrated antibacterial activity against four clinically significant infections, with zones of inhibition ranging from 16 ± 0.07, 19 ± 0.10, 17 ± 0.09, and 19 ± 0.11 mm against Escherichia coli (MTCC 452), Salmonella typhi (MTCC 733), Klebsiella pneumonia (MTCC 39), and Pseudomonas aeruginosa (MTCC 1688), respectively. Furthermore, the cytotoxicity of the chitosan‐conjugated S. pinnata phytocompounds was assessed against A549 lung cancer cells, and the results revealed a significant reduction in cell viability (33.85) at higher concentrations of 150 μg/mL. The IC50 values of S. pinnata leaf extract (149.2 mg/mL) and chitosan‐conjugated S. pinnata (126.4 mg/mL) toward A549 lung cancer cells were recorded. Overall, the results of the present study highlight the therapeutic applications of chitosan‐conjugated S. pinnata phytocompounds, particularly in the context of their anticancer and antibacterial activities. [ABSTRACT FROM AUTHOR]

Objectives: Alpha-amyrin (AA) is a pentacyclic triterpene that exhibits erratic gastrointestinal absorption and poor blood-brain barrier permeability. The study aims to isolate AA from the stem bark of Ficus benghalensis L. (Fb) (Moraceae), purify it, and formulate a nanoemulsion (NE) that may improve its bioavailability, characterization, and intranasal (IN) administration to Swiss albino mice to check its neurobehavioral effects in aluminum-induced neurotoxicity. Materials and Methods: AA was isolated from the stem bark of Fb by Soxhlet extraction, purified by analytical methods, prepared chitosandecorated NE of the same, and characterized. It was then administered through IN route to aluminum-treated Swiss albino mice for 28 days to check its effect on neurobehavioral parameters. Results: IN delivery of chitosan-decorated AA, NE resulted in significant improvement in neurobehavioral parameters. It reduced the fall-off period in the rotarod test and the escape latency in the Morris water maze test, and animals showed improved learning and spatial memory in the elevated plus maze. The transfer latency of animals improved with treatment compared with the aluminum-induced groups, indicative of the neuroprotective role of the drug. Conclusion: IN administration of AA, NE isolated from the stem bark of Fb improved neurobehavioral parameters in aluminum-induced neurotoxicity in Swiss albino mice. [ABSTRACT FROM AUTHOR]

Sterols are known for a plethora of 250 different structures. Between 5 and 10% of them usually occur with varying abundance ratios (~ four orders of magnitude) and total amounts (0.4–1000 mg/100 g oil) in samples. Yet, quantitative data are mostly restricted to the few major sterols which are available as reference standards. Here, we developed a gas chromatography with mass spectrometry method operated in selected ion monitoring mode (GC/MS-SIM) that enabled the quantitation of 30 (silylated) sterols although only ten were available as reference standards. This could be obtained by studying the full-scan mass spectra of these ten sterol standards and 20 additional sterols measured in seven oils. In the next step, sterols were assigned to different groups. Values for quantification were then selected on the premise that response factors were constant within a sterol group. The deviation of the response factors within one sterol group was frequently below ± 10% and otherwise about ± 11–12%. Using mean response factors for all sterols, the novel GC/MS-SIM quantification method was superior to GC/FID which was exemplarily applied to two oils. Between eight and 21 of the 30 studied sterols and pentacyclic triterpenols were detected and quantified in 18 vegetable oils and two vegetable fats. The much higher number of sterols that could be quantified resulted in higher sterol amounts and the method and data may be useful for food authentication. [ABSTRACT FROM AUTHOR]

Background: Aluminium is a pervading metal that poses a serious threat causing severe brain damage and neuro-degeneration. Alpha amyrin a pentacyclic triterpene despite its potency has not been properly utilized to treat neurodegenerative disorders because of its erratic GI absorption and poor BBB permeability. Purpose: To determine the role of alpha amyrin nano-emulsion in neuroinflammation induced by aluminium through an in vivo and in silico approach. Materials and Methods: An in silico approach was opted to determine the possible interactions of the drug with various inflammatory markers and was performed using Autodock. A chitosan-decorated nano-emulsion of alpha amyrin was prepared, characterized, and administered intranasally to Wistar albino rats for 42 days. The role of the treatment on neuroinflammation was measured by checking levels of SOD, catalase, inflammatory markers like IL-6, TNF-α, and acetylcholine esterase, and glutamate levels in the brain. Also, histopathology studies were performed in detail. Results: The results confirm that alpha amyrin interacts well with the inflammatory markers with a good docking score, thereby inhibiting them. The administration of nano-emulsion brings about improvement in the anti-oxidant status of the brain, reduction in neuroinflammatory markers, and acetylcholine esterase levels in rat brains. Histopathology of rat brains shows that alpha amyrin nano-emulsion administration brought about neuro-protection as it increased the number of intact neurons, and lowered neuronal damage, gliosis and pyknosis brought about by aluminium. Conclusion: The results and findings confirm the pro-active role of alpha amyrin nano-emulsion in quashing neuro-degeneration and neuro-inflammation in aluminum-induced neuro-toxicity. [ABSTRACT FROM AUTHOR]

Terminalia catappa L., which is also known as Indian almond, tropical almond, and ketapang, belongs to the family of Combretaceae and it forms layers of canopy, which provides shade to locals. The parts of the plant such as bark, fruit, leaf, rhizomes, and roots have been traditionally used in folk medicines for several treatment purposes, demonstrating its numerous biological activities. The current study evaluated phytochemical constituents in its leaf responsible for its biology activities and toxicity analysis by brine shrimp lethality test for ethanolic leaf extract of T. catappa L. (EKLE) to set a safe limit for future applications in studies. Phytochemical compounds such as squalene, phytol, DL-a-tocopherol, ß-sitosterol, stigmasterol, a-amyrin, and ß-amyrin were identified in EKLE through GC-MS analysis, which is believed to contribute to its biology activities such as antibacterial. This is the first time to report ß-sitosterol in the leaf of T. catappa L., though previous studies have reported in the bark of the tree and other parts of its genus. This is the first time to identify ß-amyrin in this tree. The LC50 value in the brine shrimp assay was above 100 µg/mL, suggesting the extract is biologically safe and non-toxic for humans. However, the application of the extract shall not be more than 11.61 mg/mL. [ABSTRACT FROM AUTHOR]

Drug development begins with medicinal plants as valuable materials. Euphorbia hirta (EH) and Nauclea latifolia (NL) are important medicinal plants having applications in the management of infectious diseases. This research aimed at evaluating EH and NL leaves for their anthelmintic property and establishment of probable dauer diapause effect induced via interaction with daf-12 nuclear receptor in worms using standard procedures. Powdered plant samples were extracted using cold maceration for 72h. Qualitative, quantitative phytochemicals and antioxidants were determined using standard procedures. Metal concentrations were determined using Atomic Absorption Spectrophotometer. Anthelmintic assay was done by observing paralysis and death times of worms. Molecular docking of compounds isolated from plants was done using PyRx-Virtual Screening Tool workspace. SwissADME of the most promising compound was done with online tool. Molecular Dynamics simulation done using UCSF Chimera and graphic processor unit (GPU) version of the PMEMD.CUDA having AMBER package. Different concentrations (10, 30, and 50 mg/mL) of methanol and hexane leaf extracts of EH and NL displayed appreciable anthelmintic activity against Pheritima pasthuma, Taenia solium and Fasciola gigantica in the following order, P. pasthuma >F. gigantica >T. solium. The antagonist effect of Beta amyrin and Benzene-1,2,4-trimethyl on the key Amino acid residues in the binding site of Daf-12 nuclear receptor confer a pronounced dauer diapause effect in EH and NL. In silico assay showed Betaamyrin having promising activity and appreciable stability in the binding site of the target and low bioavailability. This study justifies the reasons behind EH and NL usage as anthelmintic drugs locally. [ABSTRACT FROM AUTHOR]

Proso millet (Panicum miliaceum) is neglected in human nutrition. Thanks to the composition of the grains, millet is suitable for people with celiac disease and it is also useful in the prevention of cardiovascular diseases. For screening the substances in all plant parts of millet via GC–MS, two varieties, Hanacká Mana and Unicum, were used. Substances from the group saccharides, amino acids, fatty acids, carboxylic acids, phytosterols and others were identified in the roots, leaves, stems, and seeds. The highest level of saccharides was found in the stems (83%); amino acids in the roots (6.9%); fatty acids in the seeds (24.6%); carboxylic acids in the roots (3%), phytosterols in the seeds (10.51%); other substances, such as tetramethyl-2-hexadecenol (1.84%) and tocopherols (2.15%), in the leaves; retinal in the roots (1.30%) and squalene in the seeds (1.29%). Saccharides were the dominant group in all plant parts of proso millet followed by fatty acids. The dominant saccharides in all parts of the millet plant were sucrose, fructose and psicose. On the contrary, turanose, trehalose, glucose and cellobiose belonged to the least represented sugars. Additionally, amyrin, miliacin, campesterol, stigmasterol, β-sitosterol, and others were identified. Varietal variability can be assumed, e.g., in retinal, miliacin or amyrin content. [ABSTRACT FROM AUTHOR]

Abstract Background Amyrin is an important triterpenoid and precursor to a wide range of cosmetic, pharmaceutical and nutraceutical products. In this study, we metabolically engineered the oleaginous yeast, Yarrowia lipolytica to produce α- and β-amyrin on simple sugar and waste cooking oil. Results We first validated the in vivo enzymatic activity of a multi-functional amyrin synthase (CrMAS) from Catharanthus roseus, by expressing its codon-optimized gene in Y. lipolytica and assayed for amyrins. To increase yield, prevailing genes in the mevalonate pathway, namely HMG1, ERG20, ERG9 and ERG1, were overexpressed singly and in combination to direct flux towards amyrin biosynthesis. By means of a semi-rational protein engineering approach, we augmented the catalytic activity of CrMAS and attained ~ 10-folds higher production level on glucose. When applied together, protein engineering with enhanced precursor supplies resulted in more than 20-folds increase in total amyrins. We also investigated the effects of different fermentation conditions in flask cultures, including temperature, volumetric oxygen mass transfer coefficient and carbon source types. The optimized fermentation condition attained titers of at least 100 mg/L α-amyrin and 20 mg/L β-amyrin. Conclusions The design workflow demonstrated herein is simple and remarkably effective in amplifying triterpenoid biosynthesis in the yeast Y. lipolytica.

HCV is a hepatotropic RNA virus recognized for its frequent virulence and fatality worldwide. Despite many vaccine development programs underway, researchers are on a quest for natural bioactive compounds due to their multivalent efficiencies against viral infections, considering which the current research aimed to figure out the target-specificity and therapeutic potentiality of α, β, and δ subunits of amyrin, as novel bioactive components against the HCV influx mechanism. Initially, the novelty of amyrin subunits was conducted from 203 pharmacophores, comparing their in-silico pharmacokinetic and pharmacodynamic profiles. Besides, the best active site of CD81 was determined following the quantum tunneling algorithm. The molecular dynamic simulation was conducted (100 ns) following the molecular docking steps to reveal the parameters- RMSD (Å); Cα; RMSF (Å); MolSA (Å2); Rg (nm); PSA (Å); SASA (Å2), and the MM-GBSA dG binding scores. Besides, molecular strings of CD81, along with the co-expressed genes, were classified, as responsible for encoding CD81-mediated protein clusters during HCV infection, resulting in the potentiality of amyrins as targeted prophylactics in HCV infection. Finally, in vivo profiling of the oxidative stress marker, liver-specific enzymes, and antioxidant markers was conducted in the DMN-induced mice model, where β-amyrin scored the most significant values in all aspects. [ABSTRACT FROM AUTHOR]

Background: Amyrin is an important triterpenoid and precursor to a wide range of cosmetic, pharmaceutical and nutraceutical products. In this study, we metabolically engineered the oleaginous yeast, Yarrowia lipolytica to produce α- and β-amyrin on simple sugar and waste cooking oil. Results: We first validated the in vivo enzymatic activity of a multi-functional amyrin synthase (CrMAS) from Catharanthus roseus, by expressing its codon-optimized gene in Y. lipolytica and assayed for amyrins. To increase yield, prevailing genes in the mevalonate pathway, namely HMG1, ERG20, ERG9 and ERG1, were overexpressed singly and in combination to direct flux towards amyrin biosynthesis. By means of a semi-rational protein engineering approach, we augmented the catalytic activity of CrMAS and attained ~ 10-folds higher production level on glucose. When applied together, protein engineering with enhanced precursor supplies resulted in more than 20-folds increase in total amyrins. We also investigated the effects of different fermentation conditions in flask cultures, including temperature, volumetric oxygen mass transfer coefficient and carbon source types. The optimized fermentation condition attained titers of at least 100 mg/L α-amyrin and 20 mg/L β-amyrin. Conclusions: The design workflow demonstrated herein is simple and remarkably effective in amplifying triterpenoid biosynthesis in the yeast Y. lipolytica. [ABSTRACT FROM AUTHOR]

Background: The search for bioactive organic products for the treatment of various diseases is a growing concern worldwide, because these bioactive natural products are associated with fewer side effects and are easily accessible. Aim: The present study analysed the phytochemical constituents and cytotoxic effects of the leaf, bark and fruit extracts of Cordia grandicalyx Oberm. Setting: Plant samples were collected from Ga-Mashishimale village in Ba-Phalaborwa Municipality, South Africa. Methods: Antioxidant activity was determined through 1,1-diphenyl-2-picrylhydrazyl radical scavenging method, whilst cytotoxic assay was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide method. Plant extracts were subjected to phytochemical profiling using both qualitative and quantitative methods. The extracts were also subjected to fractionation using column chromatography and thin-layer chromatography, and the sub-fractions with considerable yields were identified using nuclear magnetic resonance (NMR). Results: The aqueous extracts of the bark and leaves had significant (p 0.001) antioxidant activity as compared to negative controls and ascorbic acid. Additionally, appreciable amounts of total phenolic and alkaloid contents were recorded on polar extracts, notably 200 mg/GAE and 140 mg/quercetin equivalents for leaf cold water and leaf hot water extracts, respectively. All extracts were not toxic to cells, whilst the positive control (H2O2) led to almost 100% demise of cells. Two compounds were isolated from the leaf acetone extracts collected from fraction 20 to 30 and fraction 101 to 120 and identified as α˗amyrin and β-amyrin by NMR spectral analysis. Conclusion: The study provided evidence supporting the screening of plants for the discovery of therapeutic compounds. The study also revealed that all the different C. grandicalyx extracts were less toxic to cells and may provide scientific backing for continued use of the plant in mixtures for the treatment of diseases.

Background: This study describes the isolation of chemical constituents from the leaves and bark of Phyllanthus acidus, a medicinal plant widely used for the treatment of several ailments. Materials and Methods: The crude extract of P. acidus was chromatographed on a gravity column dry packed with silica gel and was fractionated by increasing proportions of acetone in CH2Cl2. The purified isolates were subjected to NMR for structure elucidation. Their structures were identified mainly by using 1H-NMR and by comparing current NMR data with those reported in the literature. Results: The results showed that the air-dried leaves extracts of the plant afforded mixture of β-sitosterol and stigmasterol while the bark yielded mixture of phyllanthol, α-amyrin, β-amyrin and lupeol. Conclusion: The air-dried of DCM extract of P. acidus resulted in the isolation of the mixture of phyllanthol, α- amyrin, β-amyrin and lupeol from the bark and mixture of β-sitosterol and stigmasterol from the leaves of the plant. It is important to conduct detailed pharmacological and toxicity investigations as well as clinical studies using different doses or concentrations to verify its traditional uses. [ABSTRACT FROM AUTHOR]

Background: The search for bioactive organic products for the treatment of various diseases is a growing concern worldwide, because these bioactive natural products are associated with fewer side effects and are easily accessible. Aim: The present study analysed the phytochemical constituents and cytotoxic effects of the leaf, bark and fruit extracts of Cordia grandicalyx Oberm. Setting: Plant samples were collected from Ga-Mashishimale village in Ba-Phalaborwa Municipality, South Africa. Methods: Antioxidant activity was determined through 1,1-diphenyl-2-picrylhydrazyl radical scavenging method, whilst cytotoxic assay was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide method. Plant extracts were subjected to phytochemical profiling using both qualitative and quantitative methods. The extracts were also subjected to fractionation using column chromatography and thin-layer chromatography, and the sub-fractions with considerable yields were identified using nuclear magnetic resonance (NMR). Results: The aqueous extracts of the bark and leaves had significant (p < 0.001) antioxidant activity as compared to negative controls and ascorbic acid. Additionally, appreciable amounts of total phenolic and alkaloid contents were recorded on polar extracts, notably 200 mg/GAE and 140 mg/quercetin equivalents for leaf cold water and leaf hot water extracts, respectively. All extracts were not toxic to cells, whilst the positive control (H2O2) led to almost 100% demise of cells. Two compounds were isolated from the leaf acetone extracts collected from fraction 20 to 30 and fraction 101 to 120 and identified as α˗amyrin and β-amyrin by NMR spectral analysis. Conclusion: The study provided evidence supporting the screening of plants for the discovery of therapeutic compounds. The study also revealed that all the different C. grandicalyx extracts were less toxic to cells and may provide scientific backing for continued use of the plant in mixtures for the treatment of diseases. [ABSTRACT FROM AUTHOR]

Cascabela thevetia (Thevetia peruviana) giant milkweed, is known for its pharmacological importance for centuries. The chemical constituents of Thevetia peruviana leaves are amyrin, amyrin acetate, β-sitosterol, urosolic acid, cardenolides, calotropin. The anti-arthritic activity of Thevetia peruviana leaves in comparison with Diclofenac was evaluated through in-vitro studies. In-vitro studies revealed anti-arthritic effects (p< 0.05) of crude extracts of aqueous, methanolic, ethanolic and chloroform of Thevetia peruviana leaves on bovine serum albumin as evident for % inhibition protein denaturation. Different extracts of Thevetia peruviana leaves were found to possess in-vitro antiarthritic activity against BSA, using diclofenac as the reference standard which are Dose-dependent (100µg/ml, 250µg/ml, 500µg/ml). Chloroform extract has shown better activity comparing the other extracts. [ABSTRACT FROM AUTHOR]

The traditional method for the identification of paper from different origins relies on microscopy to observe the fiber morphology, which requires professional experience. In this paper, pyrolysis-gas chromatographymass spectrometry was utilized to analyze five types of traditional Chinese handmade papers, e.g., ramie paper, bamboo paper, bark fiber paper (mulberry paper and kozo paper), and Langdu paper. The results demonstrated that this method can be used to differentiate the four types of handmade papers. A high phenolic compound content was detected in bamboo paper; macromolecular triterpenoids, e.g., ß - amyrin, a - amyrin, and stigmastan-3,5-diene were found in mulberry paper and kozo paper; while those compounds were not found in ramie paper. In particular, a large quantity of phytosterol compounds were found for the first time in Langdu paper. This not only can be used to differentiate Langdu paper from others, but also it helps to explain why Langdu paper can resist insect and mildew activities. Results of the study can provide experimental reference for characterization of traditional handmade Chinese papers via pyrolysis-gas chromatography-mass spectrometry. [ABSTRACT FROM AUTHOR]

This study aimed to compare the anti-inflammatory activity of 10-year-old Dendropanax morbifera LEV (DM) leaf extracts. The leaves were collected during different seasons (May, August, and November), and the extracts were prepared using different methods (hot water, 30% ethanol, or 60% ethanol). Lipopolysaccharide-stimulated RAW264.7 cells were treated with these extracts for 12 h. The anti-inflammatory effects were evaluated by measuring the production of nitrite; prostaglandin E2 (PGE2); and inflammatory cytokines such as interleukin (IL)-6, IL-1β, and tumor necrosis factor-alpha, in addition to the mRNA expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 and activation of the nuclear factor κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathways. The amyrin and polyphenol compositions of the extracts were analyzed using a triple time-of-flight mass spectrometer and high-performance liquid chromatography. The 30% ethanol extract harvested in May and 60% ethanol extracts collected in August and November displayed the highest inhibitions of nitrite, PGE2, and inflammatory cytokines. The 60% ethanol extract harvested in August suppressed activation of the NF-κB and MAPK signaling pathways. The contents of amyrin and polyphenol compounds were highly dependent on the ethanol concentration used during each season. These results suggest that ethanol extracts of DM leaves may have the potential to regulate inflammatory responses. [ABSTRACT FROM AUTHOR]

This paper reports a pioneering study of an unknown historical drug formulation preserved in the Spezieria of Santa Maria della Scala in Rome, founded at the end of the seventeenth century by the Discalced Carmelites. Due to limited literature related to pharmaceutical remedies and drugs of the Early Modern Era (between the XV and XVIII centuries) and the complexity in their formulations, the study of these drugs represents a great challenge. The untargeted nature of the selected drug required a multi-analytical approach with complementary techniques to formulate a compositional hypothesis: FT-IR spectroscopy, gas chromatography-associated/mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) were successfully employed to identify different organic compounds. Systematic archaeobotanical research was performed as well, allowing us to acquire data related to the possible genus of plants from which these natural compounds derive and their geographical origin. The unknown drug formulation turned out to be a complex mixture used as an ointment with an anti-inflammatory purpose. It mainly contains a mixture of Venetian turpentine; a Pine resin (colophony) from the Pinaceae family; an exudate of a plant from South America, whose identified components are triterpenic compounds such as alpha- and beta-amyrins, betulin and lupeol; and saturated fatty acids which act as carriers and/or to reduce the viscosity of abovementioned exudates and resins. The study of historical drugs is important not only in order to know the practices handed down by the speziali in the past but also to reconstruct historical recipes, which can inspire new dermatological, cosmetic, hygienic and current healing products. Graphical abstract [ABSTRACT FROM AUTHOR]

Micromeria dalmatica Benth. is a Balkan endemic medicinal and aromatic plant widely used as herb or spice. Methanolic and aqueous extracts of three populations of M. dalmatica were evaluated for free radical scavenging activity against DPPH radicals. All studied extracts showed significant activity. No difference was found in the antiradical properties between methanolic and aqueous extracts. Comparative analysis of metabolites of plant material of the studied populations was done by GC-MS. The results showed that M. dalmatica is rich in essential nutrients as phenolic and organic acids, triterpenes (amyrin), sterols, fatty acids (saturated and unsaturated). The content of these important biologically active substances determines M. dalmatica as a valuable spice and medicinal plant. [ABSTRACT FROM AUTHOR]

Datura metel Linn is used traditionally for the treatment of various diseases including relaxation of smooth muscles, relief of fever, as well as gastrointestinal disorder. This study deals with the bio-guided isolation of an active, amyrin-type triterpenoid, namely 3-oxo-6- β -hydroxy- β -amyrin (daturaolone; 1), from the chloroform fraction of Datura metel L. (Angel's trumpet) fruits and its gastrointestinal motility, antipyretic, and muscle relaxation effects in animal models. The chemical structure of daturaolone (1) was elucidated by NMR spectroscopy and crystallography techniques. The chloroform fraction and daturaolone (1) were assessed for the GIT motility test. Data exhibited in charcoal meal GI transit test show that chloroform fraction and daturaolone (1) significantly reduce GIT motility and increased intestinal transit time, comparable to the standard (atropine), a muscarinic receptor blocking agent. Muscle relaxant potency of the extract and daturaolone (1) was assessed in various animal paradigms. In the inclined plane screening test, it produced a significant (P < 0.05) muscle relaxation potential in a dose-dependent manner after 30, 60, and 90 min. Likewise, the muscle relaxation potential of the extract and daturaolone (1) was strongly complemented by the chimney and traction test, representing a dominant effect after 60 min of sample administration. The chloroform fraction showed good antipyretic activity, and while daturaolone (1) exhibited significant activity at a higher dose, the maximum effect (84.64%) was at 20 mg/kg i.p. In acute toxicity screening test, the chloroform extract (100, 250, 500, and 1,000 mg/kg) and daturaolone (1) (5, 10, 20, and 50 mg/kg) were found safe. In conclusion, the chloroform extract and daturaolone (1) exhibited strong gastrointestinal motility, muscle relaxation, and antipyretic activity in different animal models and intestinally, was found safe at higher tested doses. [ABSTRACT FROM AUTHOR]

Cork is a water-impermeable, suberin-based material harboring lignin, (hemi)cellulose, and extractable small molecules (primarily triterpenoids). Extractables strongly influence the properties of suberin-based materials. Though these previous findings suggest a key role for triterpenoids in cork material quality, directly testing this idea is hindered in part because it is not known which genes control cork triterpenoid biosynthesis. Here, we used gas chromatography and mass spectrometry to determine that the majority (>85%) of non-polar extractables from cork were pentacyclic triterpenoids, primarily betulinic acid, friedelin, and hydroxy-friedelin. In other plants, triterpenoids are generated by oxidosqualene cyclases (OSCs). Accordingly, we mined Quercus suber EST libraries for OSC fragments to use in a RACE PCR-based approach and cloned three full-length OSC transcripts from cork (QsOSC1-3). Heterologous expression in Saccharomyces cerevisiae revealed that QsOSC1-3 respectively encoded enzymes with lupeol synthase, mixed α- and β-amyrin synthase, and mixed β-amyrin and friedelin synthase activities. These activities together account for the backbone structures of the major cork triterpenoids. Finally, we analyzed the sequences of QsOSC1-3 and other plant OSCs to identify residues associated with specific OSC activities, then combined this with analyses of Q. suber transcriptomic and genomic data to evaluate potential redundancies in cork triterpenoid biosynthesis. [ABSTRACT FROM AUTHOR]

Protium heptaphyllum (Aubl.) Marchand (PH) trees are endemic to the tropical region of South America, mostly Brazil. Antibacterial, antinociceptive, anti-inflammatory, anxiolytic, antidepressant and anti-hyperlipidemic/anti-hypercholesterolemic effects were reported for its resinous exudate Protiumheptaphyllum resin (PHR). This work aims to provide a qualitative and quantitative consistent chemical profiling of the major constituents of this resin and two extracts enriched in acid (acidic triterpene concentrated extract, ATCE) and neutral triterpenes (α and β-amyrin concentrated extract, AMCE). GC–MS/GC–FID was used for volatile terpene fraction, a validated GC–MS method was developed for quantification of neutral α and β-amyrin and HPLC–APCI HRMS2 was used for acidic triterpenes analysis. The chemical investigation reported 29 molecules, including 14 volatile terpenes, 6 neutral triterpenes and 11 acid triterpenes. The most abundant compounds were α-amyrin (251.28 g kg−1, 123.98 g kg−1 and 556.82 g kg−1 in PHR, ATCE and AMCE, respectively), β-amyrin (172.66 g kg−1, 95.39 g kg−1 and 385.58 g kg−1 in PHR, ATCE and AMCE, respectively), 3-oxo-tirucalla-7,24-dien-21-oic acid (80.64 g kg−1, 157.10 g kg−1 and 15.31 g kg−1 in PHR, ATCE and AMCE, respectively) and 3α-hydroxy-tirucalla-8,24-dien-21-oic acid (77.71 g kg−1, 130.40 g kg−1 and 11.64 g kg−1 in PHR, ATCE and AMCE, respectively). Results showed specific enrichment of acidic and neutral triterpenoids in the two respective extracts.

Purpose: To investigate the anxiolytic and antidepressant effects of α- and β-amyrins in a mouse model of mild traumatic brain injury (mTBI), and the underlying mechanism(s) of action. Methods: Male Swiss mice (n = 165), weighing 25 - 40 g (mean weight = 32.5 ± 7.5 g), were used in this study. Existing mTBI model was modified and optimized for mild injury to brain capable of producing neurobehavioral changes. Forced swim test (FST) and tail suspension test (TST) were used to measure the antidepressant effects of α- and β-amyrins, while elevated plus maze (EPM) and light-dark model (LDM) were used for anxiety assessment. The probable mechanism of action of amyrin was also investigated through kinetics of serotonin uptake and activities of monoamine oxidases A (MAO A) and B (MAO B) in mouse hippocampus and cortex. Results: Induction of mTBI produced anxiety- and depression-like behaviors in mice. The 5th hitting righting time was 259 ± 25.11 s while duration of apnea was 27.33 ± 3.84 s. Apnea was significantly reduced on 5th and 7th hits, when compared with 4th and 6th hits (p < 0.05). The immobility time of mice was significantly reduced in FST and TST. A combination of the two forms of amyrin was more effective in reducing duration of immobility, relative to when each was used alone (p < 0.05). Amyrin significantly and dose-dependently increased entries, and time spent in open arms and light zone (p < 0.05). Mice in mTBI group exhibited a high degree of hopelessness, when compared with control group (p < 0.05). However, amyrin at a dose of 50 mg/kg significantly reduced the degree of hopelessness in the mice (p < 0.05). The specific activity of MAO A in hippocampal tissue (265.00 ± 12.07 μmol/min/g protein) was significantly higher than that of cortex (61.85 ± 5.14 μmol/min/g protein). In both tissues, there were no significant differences in the activity of MAO B among the groups (p > 0.05). Amyrin significantly reversed the effects of mTBI on the levels of amino acids in mice hippocampus and cortex (p < 0.05). The results of synaptosomal uptake of serotonin show that fluoxetin exhibited competitive inhibition of serotonin uptake, while amyrin exhibited mixed type inhibition. Conclusion: The results obtained show that α- and β-amyrins exert potent anxiolytic and antidepressant effects via mechanisms involving MAO and GABA in the hippocampus. [ABSTRACT FROM AUTHOR]

Protium heptaphyllum Aubl. belongs to the Burseraceae family. It is commonly called 'almecegueira' and is known to produce an amorphous resin which has constituents such as α‐ and β‐amyrin, taraxastan‐3‐oxo‐20‐ol and sitostenonein. The α‐ and β‐amyrin from P. heptaphyllum have pharmacological activities in several systems, such as central and peripheral nervous system, gastrointestinal tract and immunological system. In this study, our objective was to review pharmacological activities and to gather more information on the mixture of α‐ and β‐amyrin obtained from P. heptaphyllum to guide future preclinical and clinical studies using this compound. This review consisted of searches performed using scientific databases such as PubMed, SciELO, LILACS, SciFinder and Science Direct. Some uses of α‐ and β‐amyrin have been partially confirmed by previous studies demonstrating analgesic, anti‐inflammatory, anticonvulsant, antidepressive, gastroprotective, hepatoprotective, antipancreatitic, anticholytic, antihyperglycemic and hypolipidemic effects. It is noteworthy that there are no α‐ and β‐amirin toxicity tests described in the literature as recommended in the international guidelines, and such tests are one of the research stages to proceed in clinical and preclinical trials if this compound was to be used. [ABSTRACT FROM AUTHOR]

Introduction: Hoya plants are also called wax plants due to the waxy appearance of their leaves and flowers. Most species are cultivated as ornamental plants. In the Philippines, there are at least 109 species of Hoya; 88 of these are endemic to the country. One of the endemic species is Hoya meliflua often confused with H. diversifolia, which can also be found in other countries. This study is part of our research on the chemical constituents of Philippine native Hoyas. Methods: The compounds were isolated by silica gel chromatography and identified by NMR spectroscopy. Results: Chemical investigation of the dichloromethane extract from the leaves of Hoya meliflua afforded squalene and mixtures of β-amyrin (1a), α-amyrin (1b) and lupeol (1c) in about 1:1:0.25 ratio; oleanone (2a), ursenone (2b) and lupenone (2c) in about 1:1:0.3 ratio; β-amyrin cinnamate (3a), α-amyrin cinnamate (3b) and lupenyl cinnamate (3c) in about 0.5:0.3:1 ratio; and β-sitosterol and stigmasterol in about 5:1 ratio. Conclusion: The results of our study indicate that Hoya meliflua shares similar chemical characteristics with other members of the genus Hoya. The triterpenes and sterols obtained from H. meliflua were also identified from other Hoya species. It is interesting to note that although most Hoya plants have no known biological activity and medicinal property, the compounds isolated from H. meliflua possess diverse bioactivities. [ABSTRACT FROM AUTHOR]

Abstract A phytochemical investigation of the stem barks of the Malaysian Croton oblongus Burm.f. (Syn. Croton laevifolius Blume) (Euphorbiaceae) yielded seven previously undescribed ent-neo- clerodane diterpenoids, laevifins A - G and the known crovatin (3). Structures were established by a combination of spectroscopic methods including HRESIMS, NMR spectroscopy and X-ray crystallography. The absolute configuration of crovatin and laevifins A-G was established by comparison of experimental ECD and theoretical TDDFT ECD calculated spectra. This is the first report on the occurrence of the sesquiterpenoid cryptomeridiol in a Croton species. In vitro cytotoxicity assays on laevifins A, B and G showed moderate activities against the MCF-7 cancer cell line (IC 50 102, 115 and 106 μ M, respectively) while β -amyrin and acetyl aleuritolic acid showed good anti-inflammatory activity on the LPS-induced NF-κB translocation inhibition in RAW 264.7 cells assay with IC 50 values of 23.5 and 35.4 μ g/mL, respectively. Graphical abstract Seven previously undescribed ent-neo- clerodane diterpenoids (1 , 2 , 4 - 8) were isolated from Croton oblongus Burm.f. Image 1 Highlights • The stem barks of Malaysian Croton oblongus Burm.f. (Euphorbiaceae) was investigated. • Seven previously undescribed ent-neo- clerodane diterpenoids, laevifins A - G were isolated. • The absolute configurations were determined using Electronic Circular Dichroism. • This is the first occurrence of the sesquiterpenoid cryptomeridiol in Croton. [ABSTRACT FROM AUTHOR]

A reverse phase high performance liquid chromatographic (RP-HPLC) method was developed for identification and estimation of 18-β-glycyrrhetinic acid (GA) in HepG2 cell line. The analysis was carried out using a JASCO HPLC system with a C-18 (3 μm) Supelco reversed phase column (150 x 4.7 mm) using a mobile phase of 80% CH3OH and 20% of CH3CN: tetrahydrofuran: water (10:80:10, v/v/v). The method was linear in the concentration range of 1.5–120 μg /mL (n = 5). The LOD and LOQ were determined based on standard deviation of the y-intercept and the slope of the calibration curve. The LOD and LOQ values were found to be 11.46 μg/mL and 34.72 μg/mL, respectively. The mean percentage recovery by standard addition experiments of GA is 92.4 % ± 5.2%. The intracellular GA concentration value, obtained as mean of five different determinations, was 45.8 ± 7.45 μg/mL. We have developed a HPLC-UV method for quantitative determination of GA inside cells, with advantages in the cost reduction and economy of the analytical process. [ABSTRACT FROM AUTHOR]

Ursolic acid and oleanolic acid are pentacyclic triterpenoid compounds with a variety of biological activities. A mixture of ursolic acid and oleanolic acid has higher antitumor activity than the individual acids. We have developed a simultaneous biosynthesis pathway for different proportions of ursolic acid and oleanolic acid in Saccharomyces cerevisiae. The ScLCZ08 strain produced 175.15 mg/L of the ursolic acid precursor α‐amyrin, the highest amount reported. Ursolic and oleanolic acid titers and proportions were optimized using Medicago truncatula amyrin C‐28 oxidase and Arabidopsis thaliana cytochrome P450 reductase. Using glucose and ethanol fed‐batch fermentation strategies, the final ursolic acid and oleanolic acid titers were 123.27 and 155.58 mg/L, respectively, demonstrating 4.77‐fold and 4.95‐fold higher production than the parent strain. The ScLCZ11 strain displayed the highest ursolic acid production obtained via microbial fermentation in fed‐batch culture to date. © 2018 American Institute of Chemical Engineers AIChE J, 64: 3794–3802, 2018 [ABSTRACT FROM AUTHOR]

This study aimed to compare the anti-inflammatory activity of 10-year-old Dendropanax morbifera LEV (DM) leaf extracts. The leaves were collected during different seasons (May, August, and November), and the extracts were prepared using different methods (hot water, 30% ethanol, or 60% ethanol). Lipopolysaccharide-stimulated RAW264.7 cells were treated with these extracts for 12 h. The anti-inflammatory effects were evaluated by measuring the production of nitrite; prostaglandin E2 (PGE2); and inflammatory cytokines such as interleukin (IL)-6, IL-1β, and tumor necrosis factor-alpha, in addition to the mRNA expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 and activation of the nuclear factor κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathways. The amyrin and polyphenol compositions of the extracts were analyzed using a triple time-of-flight mass spectrometer and high-performance liquid chromatography. The 30% ethanol extract harvested in May and 60% ethanol extracts collected in August and November displayed the highest inhibitions of nitrite, PGE2, and inflammatory cytokines. The 60% ethanol extract harvested in August suppressed activation of the NF-κB and MAPK signaling pathways. The contents of amyrin and polyphenol compounds were highly dependent on the ethanol concentration used during each season. These results suggest that ethanol extracts of DM leaves may have the potential to regulate inflammatory responses.

α-Amylase inhibitors play a critical role in the control of diabetes and many of medicinal plants have been found to act as α-amylase inhibitors. Swertia genus, belonging to the family Gentianaceae, comprises different species most of which have been used in traditional medicine of several cultures as antidiabetic, anti-pyretic, analgesic, liver and gastrointestinal tonic. Swertia longifolia Boiss. is the only species of Swertia growing in Iran. In the present investigation, phytochemical study of S. longifolia was performed and α-amylase inhibitory effects of the plant fractions and purified compounds were determined. Aerial parts of the plant were extracted with hexane, chloroform, methanol and water, respectively. The components of the hexane and chloroform fractions were isolated by different chromatographic methods and their structures were determined by 1 H NMR and 13 C NMR data. α-Amylase inhibitory activity was determined by a colorimetric assay using 3,5-dinitro salysilic acid. During phytochemical examination, α-amyrin, β-amyrin and β-sitosterol were purified from the hexane fraction,while ursolic acid, daucosterol and swertiamarin were isolated from chloroform fraction. The results of the biochemical assay revealed α-amylase inhibitory activity of hexane, chloroform, methanol and water fractions, of which the chloroform and methanol fractions were more potent (IC 50 16.8 and 18.1 mg/ml, respectively). Among examined compounds, daucosterol was found to be the most potent α-amylase inhibitor (57.5% in concentration 10 mg/ml). With regard to α-amylase inhibitory effects of the plant extracts, purified constituents, and antidiabetic application of the species of Swertia genus in traditional medicine of different countries, S. longifolia seems more appropriate species for further mechanistic antidiabetic evaluations.

Ilex asprella, a plant widely used as a folk herbal drug in southern China, produces and stores a large amount of triterpenoid saponins, most of which are of the α-amyrin type. In this study, two oxidosqualene cyclase (OSC) cDNAs, IaAS1 and IaAS2, were cloned from the I. asprella root. Functional characterisation was performed by heterologous expression in the yeast Saccharomyces cerevisiae. Analysis of the resulting products by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) showed that both genes encode a mixed amyrin synthase, producing α-amyrin and β-amyrin at different ratios. IaAS1, which mainly produces α-amyrin, is the second triterpene synthase so far identified in which the level of α-amyrin produced is ≥80% of total amyrin production. By contrast, IaAS2 mainly synthesises β-amyrin, with a yield of 95%. Gene expression patterns of these two amyrin synthases in roots and leaves of I. asprella were found to be consistent with the content patterns of total saponins. Finally, phylogenetic analysis and multiple sequence alignment of the two amyrin synthases against several known OSCs from other plants were conducted to further elucidate their evolutionary relationship.

Purpose: To evaluate the antioxidant property of the fruit of Telfairia occidentalis and isolate the components responsible for the antioxidant activity. Methods: The fruit pericarp was macerated with methanol and the extract obtained successively partitioned with n-hexane, dichloromethane and ethyl acetate. The in vitro antioxidant activity of the extract and fractions was evaluated using 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging, reducing power, nitric oxide scavenging, total antioxidant and hydrogen peroxide scavenging assays. The n-hexane fraction, which had the highest DPPH scavenging and total antioxidant activities, was subjected to column and thin layer chromatography to isolate the components. The isolated compounds were identified by ultraviolet-visible (UV), nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy and mass spectrometry. Results: Among the fractions tested, n-hexane had the best total antioxidant activity of 99.44% at 20 mg/ml (p < 0.05) compared to ascorbic acid at 99.71% of 20 mg/ml. This fraction also had the highest DPPH radical scavenging activity of all the fractions (p < 0.05) at all test concentrations. For nitric oxide scavenging activity, the whole extract, and the chloroform and aqueous fractions exhibited activity ranging from 92.29 to 97.66% compared to 98.93% for ascorbic acid. The hydrogen peroxide scavenging activity of the extract and fractions ranged from 92.60 to 96.23% compared with of the standard, ascorbic acid (101.68%). The major components of the n-hexane fractions were a- amyrin and β-amyrin. Conclusion: The fruit pericarp of Telfairia occidentalis possesses good DPPH radical scavenging activity. This is the first time the antioxidant activity of the fruit (pericarp) and the presence of a- and β- amyrins in Telfairia occidentalis have been reported. [ABSTRACT FROM AUTHOR]

Rationale: Riverine particulate organic matter is generally considered to be refractory with respect to further decomposition in the ocean. In order to check the validity of this paradigm, there is a real need for tracers sufficiently stable and specific to monitor the degradation of terrestrial higher plant material in the environment. 3β‐Hydroxy‐urs‐12‐en‐11‐one and 3β‐hydroxy‐olean‐12‐en‐11‐one (autoxidation products of α‐ and β‐amyrin) were previously proposed for such use. Methods: Electron ionization mass spectrometry (EIMS) fragmentation pathways of 3β‐hydroxy‐urs‐12‐en‐11‐one and 3β‐hydroxyolean‐12‐en‐11‐one trimethylsilyl (TMS) derivatives were investigated. These pathways were deduced by: (i) low‐energy collision‐induced dissociation (CID)‐gas chromatography/tandem mass spectrometry (GC/MS/MS), (ii) accurate mass measurement and (iii) deuterium labelling. Quantification of these compounds in total lipid extracts of natural samples was then carried out in multiple reaction monitoring (MRM) mode. Results: CID‐MS/MS analyses, accurate mass measurement and deuterium‐labelling experiments allowed us to elucidate EIMS fragmentations of 3β‐hydroxy‐urs‐12‐en‐11‐one and 3β‐hydroxyolean‐12‐en‐11‐one TMS derivatives. Some specific fragmentation pathways, useful in addition to chromatographic retention times for further characterization, could be selected. As an application of some of the described fragmentations, TMS derivatives of these oxidation products were characterized and quantified in MRM mode in different natural samples. Conclusions: EIMS fragmentations of 3β‐hydroxy‐urs‐12‐en‐11‐one and 3β‐hydroxyolean‐12‐en‐11‐one TMS derivatives exhibit specific fragment ions, which appear to be very useful for the quantification of these oxidation products in natural samples (riverine particulate matter, wet and dry deposited atmospheric particles). [ABSTRACT FROM AUTHOR]

The neuroprotective potential of Bombax ceiba L. (Malvaceae) leaves extract (BCLE) was evaluated herein for the first time. Memory impairment was induced in rats by scopolamine (SC) administration for seven days (1.5 mg/kg/day, i.p.). BCLE (200 mg/kg) or donepezil (1mg/kg) was administered orally a week before and then concomitantly with SC for another week. BCLE improved the rats' behaviors estimated by Morris water maze and passive avoidance tests, where the rats showed prolongation in time spent in platform quadrant and step-through latency, respectively, compared to administration of SC alone. BCLE also ameliorated the antioxidant parameters (Catalase with malondialdehyde (MDA), and the SC-induced elevation of acetylcholinesterase (AChE) in rats' brain tissues. Histopathological studies supported the above results. BCLE phytochemical study resulted in isolation and structural elucidation of ß-sitosterol linoleate (first to be isolated from the genus) and ß-amyrin. Twenty-seven compounds (39.35%) were identified in BCLE by gas chromatography/mass spectrometry (GC/MS). The major components were triterpenoids (69.82%), composing mainly of ß-amyrin (45.28%), lupeol (15.03%) and olean-12-en-3-one (6.27%), in addition to fatty acid esters (13.37%) and steroids (7.75%), which were rich in methyl palmitate (9.87%) and stigmasterol acetate (4.91%), respectively. These compounds may be responsible for ameliorating the SC-induced oxidative stress and cholinergic dysfunction. [ABSTRACT FROM AUTHOR]

Background Inflammation is a primary response to infection that can pathologically lead to various diseases including neurodegenerative diseases. The purpose of this study was to evaluate the effect of β-Amyrin, a naturally occurring pentacyclic triterpenoid compound, on inflammation induced by lipopolysaccharide (LPS) and interferone-γ (IFN-γ) in rat microglial cells. Materials and methods Cytotoxicity of β-Amyrin (3–100) μM on microglial cells was evaluated using the MTT assay. Also, the protective effect of various β-Amyrin (2–16 μM) concentrations with LPS/IFN-γ-induced mice microglial cells was studied. The concentrations of TNF-α (Tumor Necrosis Factor-α), IL-1β (Interleukin-1β), IL-6 (Interleukin-6) and PGE-2 (Prostaglandin E2) were evaluated using ELISA. Gene expressions of TNF-α, IL-1β, IL-6, COX-2 (Cyclooxygenase-2), iNOS and arginase-1 were also evaluated using the Real-Time PCR method. Nitrite oxide and urea were measured using biochemical methods. Results The studied concentrations ​​of β-Amyrin had no significant effects on the viability of microglial cells. Interestingly, β-Amyrin concentration dependently and significantly increased the reduced cell proliferation concerning to LPS/IFN-γ exposure (p < 0.001). The concentrations and expression levels of pro-inflammatory factors including TNF-α, IL-1β, IL-6, PGE-2, COX-2 were significantly reduced after β-Amyrin treatment in LPS/IFN-γ-induced microglial cells (p < 0.05–0.001). β-Amyrin also decreased the levels of nitric oxide, increased urea and down regulated the expression of nitric oxide synthesis while arginase-1 expression was enhanced (p < 0.001). The ratio of NO/urea and iNOS/Arg1 were also markedly increased in comparison to the LPS/IFN-g group (p < 0.001). Conclusion β-Amyrin reduces inflammation in microglial cells and can be used as a potential anti-inflammatory agent in central nervous system neurodegenerative diseases such as Alzheimer and multiple sclerosis, by affecting the inflammatory cytokine and differentiation of microglia as resident CNS macrophages. [ABSTRACT FROM AUTHOR]

Background: Mixtures of ursolic acid (1) and oleanolic acid (2) (1:1 and 1:2), oleanolic acid (2), squalene (3), chlorophyll a (4), wrightiadione (5) and α-amyrin acetate (6) were isolated from the dichloromethane (CH2Cl2) extracts of the leaves and twigs of Wrightia pubescens (R.Br.). Objectives: To test for the cytotoxicity potentials of 1-6. Materials and Methods: The antiproliferative activities of 1-6 against three human cancer cell lines, breast (MCF-7) and colon (HT-29 and HCT-116) and a normal cell line, human dermal fibroblast neonatal (HDFn), were evaluated using the PrestoBlueR cell viability assay. Results: Compounds 4, 1 and 2 (1:2), 2, 1 and 2 (1:1), and 5 exhibited the most cytotoxic effects against HT-29 with half maximal inhibitory concentration (IC50) values of 0.68, 0.74, 0.89, 1.70 and 4.07 μg/mL, respectively. Comparing 2 with its 1:1 mixture with 1 (IC50 = 1.70 and 7.18 μg/mL for HT-29 and HCT-116, respectively) and 1:2 mixture with 1 (0.74 and 3.46 μg/mL for HT-29 and HCT-116, respectively), 2 also showed strong cytotoxic potential against HT-29 and HCT-116 (0.89 and 2.33 μg/mL, respectively). Unlike the mixtures which exhibited low effects on MCF-7 (IC50 = 20.75 and 30.06 μg/mL for 1:1 and 1:2, respectively), 2 showed moderate activity against MCF-7 (10.99 μg/mL). Compound 6 showed the highest cytotoxicity against HCT-116 (IC50 = 4.07 μg/mL). Conclusion: Mixtures of 1 and 2 (1:1 and 1:2), 2, 3, 4, 5, and 6 from the CH2Cl2 extracts of the leaves and twigs of W. pubescens (R.Br.) exhibited varying cytotoxic activities. All the compounds except 6 exhibited the strongest cytotoxic effects against HT-29. On the other hand, 6 was most cytotoxic against HCT-116. Overall, the toxicities of 1-6 were highest against HT-29, followed by HCT-116 and MCF-7. All the compounds showed varying activities against HDFn (IC50 <30 μg/mL). [ABSTRACT FROM AUTHOR]

Anemone flaccida Fr. Shmidt (Ranunculaceae), known as ‘Di Wu’ in China, is a perennial herb which has long been used to treat arthritis. The rhizome of A. flaccida contains pharmacologically active components i.e. oleanane-type triterpenoid saponins. Oleanolic acid is natural triterpenoid in plants with diverse biological activities. The biosynthesis of oleanolic acid involves cyclization of 2,3-oxidosqualene to the oleanane-type triterpenoid skeleton, followed by a series of oxidation reactions catalyzed by cytochrome P450 monooxygenase (CYP450). Previously, we identified four possible cytochrome P450 genes belonging to CYP716A subfamily from the transcriptome of A. flaccida . In this study, we identified one of those genes “CYP716A254” encoding a cytochrome P450 monooxygenase from A. flaccida that catalyzes the conversion of the β-amyrin into oleanolic acid. The heterologous expression of CYP716A254 in yeast resulted in oxidation of β-amyrin at the C-18 position to oleanolic acid production. These results provide an important basis for further studies of oleanane-type triterpenoid saponins synthesis in A. flaccida. [ABSTRACT FROM AUTHOR]

Ferula gummosa Boiss. is an industrial and pharmaceutical plant that has been highly recognized for its valuable oleo-gum-resin, namely galbanum. Despite the fabulous value of galbanum, very little information on the genetic and biochemical mechanisms of its production existed. In the present study, the oleo-gum-resin and four organs (root, flower, stem, and leaf) of F. gummosa were assessed in terms of metabolic compositions and the expression of genes involved in their biosynthetic pathways. Results showed that the most accumulation of resin and essential oils were occurred in the roots (13.99 mg/g) and flowers (6.01 mg/g), respectively. While the most dominant compound of the resin was β-amyrin from triterpenes, the most abundant compounds of the essential oils were α-pinene and β-pinene from monoterpenes and α-eudesmol and germacrene-D from sesquiterpenes. Transcriptome analysis was performed by RNA sequencing (RNA-seq) for the plant roots and flowers. Differential gene expression analysis showed that 1172 unigenes were differential between two organs that 934 (79.6%) of them were up-regulated in the flowers and 238 (20.4%) unigenes were up-regulated in the roots (FDR ≤0.001). The most important up-regulated unigenes in the roots were involved in the biosynthesis of the major components of galbanum, including myrcene, germacrene-D, α-terpineol, and β-amyrin. The results obtained by RNA-Seq were confirmed by qPCR. These analyses showed that different organs of F. gummosa are involved in the production of oleo-gum-resin, but the roots are more active than other organs in terms of the biosynthesis of triterpenes and some mono- and sesquiterpenes. This study provides rich molecular and biochemical resources for further studies on molecular genetics and functional genomics of oleo-gum-resin production in F. gummosa. [ABSTRACT FROM AUTHOR]

Amyrins are the immediate precursors of many pharmaceutically important pentacyclic triterpenoids. Although various amyrin synthases have been identified, little is known about the relationship between protein structures and the constituent and content of the products. IaAS1 and IaAS2 identified from Ilex asprella in our previous work belong to multifunctional oxidosqualene cyclases and can produce α-amyrin and β-amyrin at different ratios. More than 80% of total production of IaAS1 is α-amyrin; while IaAS2 mainly produces β-amyrin with a yield of 95%. Here, we present a molecular modeling approach to explore the underlying mechanism for selective synthesis. The structures of IaAS1 and IaAS2 were constructed by homology modeling, and were evaluated by Ramachandran Plot and Verify 3D program. The enzyme-product conformations generated by molecular docking indicated that ASP484 residue plays an important role in the catalytic process; and TRP611 residue of IaAS2 had interaction with β-amyrin through π−σ interaction. MM/GBSA binding free energy calculations and free energy decomposition after 50 ns molecular dynamics simulations were performed. The binding affinity between the main product and corresponding enzyme was higher than that of the by-product. Conserved amino acid residues such as TRP257; TYR259; PHE47; TRP534; TRP612; and TYR728 for IaAS1 (TRP257; TYR259; PHE473; TRP533; TRP611; and TYR727 for IaAS2) had strong interactions with both products. GLN450 and LYS372 had negative contribution to binding affinity between α-amyrin or β-amyrin and IaAS1. LYS372 and ARG261 had strong repulsive effects for the binding of α-amyrin with IaAS2. The importance of Lys372 and TRP612 of IaAS1, and Lys372 and TRP611 of IaAS2, for synthesizing amyrins were confirmed by site-directed mutagenesis. The different patterns of residue−product interactions is the cause for the difference in the yields of two products.

α, β amyrin (ABAM) is a natural mixture of pentacyclic triterpenes that has a wide range of biological activities. ABAM is isolated from the species of the Burseraceae family, in which the species Protium is commonly found in the Amazon region of Brazil. The aim of this work was to develop inclusion complexes (ICs) of ABAM and β-cyclodextrin (βCD) and hydroxypropyl-β-cyclodextrin (HPβCD) by physical mixing (PM) and kneading (KN) methods. Interactions between ABAM and the CD’s as well as the formation of ICs were confirmed by physicochemical characterization in the solid state by Fourier transform infrared (FTIR), scanning electron microscopy (SEM), X-ray diffraction (XRD), thermogravimetry (TG) and differential scanning calorimetry (DSC). Physicochemical characterization indicated the formation of ICs with both βCD and HPβCD. Such ICs were able to induce changes in the physicochemical properties of ABAM. In addition, the formation of ICs with cyclodextrins showed to be an effective and promising alternative to enhance the anti-inflammatory activity and safety of ABAM.

Cycloartenol is biosynthetically the first sterol skeleton, which is metabolized to phytosterols such as β-sitosterol and stigmasterol. β-Amyrin is the most commonly occurring aglycone skeleton for oleanane-type saponins such as glycyrrhizin and saikosaponins. It has been regarded that these cyclic triterpenes are unable to be produced in Escherichia coli, while no reports are available on their production with E. coli. Here, we describe a method to synthesize triterpene skeletons from higher plants, including cycloartenol and β-amyrin. We introduced into E. coli the biosynthetic pathway genes from farnesyl diphosphate (FPP) to cycloartenol or β-amyrin, which contained Arabidopsis ( Arabidopsis thaliana)-derived squalene synthase ( AtSQS) and squalene epoxidase ( AtSQE) genes in addition to the Arabidopsis cycloartenol synthase ( AtCAS1) gene, or the β-amyrin synthase ( EtAS) gene of the petroleum plant Euphorbia tirucalli, along with the isopentenyl diphosphate isomerase ( HpIDI) gene from a green algae Haematococcus pluvialis. The order of genes, HpIDI, AtSQS, AtSQE, driven by transcriptional read-through from a tac promoter to an rrnB terminator, was crucial for their functional expression in E. coli to produce cycloartenol or β-amyrin. The co-expression of a bacterial NADPH-regenerating gene ( zwf or gdh) as well as bacterial redox partner protein genes ( camA and camB, or NsRED and NsFER) was found to increase the amounts of these triterpenes several fold. The present study could open up opportunities not only to carry out functional analysis of a higher-plant-derived oxidosqualene cyclase ( OSC) gene in E. coli but also to produce functional triterpenes that originate from medicinal or herbal plants. [ABSTRACT FROM AUTHOR]

Autoxidation products of specific lipid components of angiosperms (betulin, α- and β-amyrin) and gymnosperms (dehydroabietic acid) and their parent compound in surface sediments from different regions of the Canadian Arctic were quantified. The high proportions of these autoxidation products in all the sediments (mean 60.1 ± 14.0%, 91.8 ± 1.1, 98.7 ± 2.2 and 96.6 ± 5.4% for dehydroabietic acid, betulin and α- and β-amyrin, respectively) confirmed, unambiguously, the important role played by autoxidation in the degradation of vascular plant material in the region and its enhancement in seawater. The enhancement of these processes in Arctic waters could be the result of intense photooxidation during the senescence of the organisms within the terrestrial environment. [ABSTRACT FROM AUTHOR]