Lineage-Specific CYP80 Expansion and Benzylisoquinoline Alkaloid Diversity in Early-Diverging Eudicots.

Menispermaceae species, as early-diverging eudicots, can synthesize valuable benzylisoquinoline alkaloids (BIAs) like bisbenzylisoquinoline alkaloids (bisBIAs) and sinomenines with a wide range of structural diversity. However, the evolutionary mechanisms responsible for their chemo-diversity are not well understood. Here, a chromosome-level genome assembly of Menispermum dauricum is presented and demonstrated the occurrence of two whole genome duplication (WGD) events that are shared by Ranunculales and specific to Menispermum, providing a model for understanding chromosomal evolution in early-diverging eudicots. The biosynthetic pathway for diverse BIAs in M. dauricum is reconstructed by analyzing the transcriptome and metabolome. Additionally, five catalytic enzymes - one norcoclaurine synthase (NCS) and four cytochrome P450 monooxygenases (CYP450s) - from M. dauricum are responsible for the formation of the skeleton, hydroxylated modification, and C-O/C-C phenol coupling of BIAs. Notably, a novel leaf-specific MdCYP80G10 enzyme that catalyzes C2'-C4a phenol coupling of (S)-reticuline into sinoacutine, the enantiomer of morphinan compounds, with predictable stereospecificity is discovered. Moreover, it is found that Menispermum-specific CYP80 gene expansion, as well as tissue-specific expression, has driven BIA diversity in Menispermaceae as compared to other Ranunculales species. This study sheds light on WGD occurrences in early-diverging eudicots and the evolution of diverse BIA biosynthesis.

'Sharpshooter' in Botanic Garden: the tale of a rare plant-insect interaction.

Here we report a unique plant-insect interaction between the leafhopper Aloka depressa (tribe Phlogisini) and the host liana, Diploclisia glaucescens, from a Botanic Garden located at the southern edge of Western Ghats in India. Field observations and SEM micrographs were employed to derive evidences on this rare plant-insect interaction. 20-Hydroxyecdysone (20E), insect moulting hormone, was detected and quantified in the host plant D. glaucescens using HPTLC-densitometry. 20E was isolated and characterized from D. glaucescens using column chromatography, 1H-, 13C-NMR and HR-MS. 20E was also detected in A. depressa excrement using HPTLC-densitometry. The leafhopper A. depressa is functioning as a 'sharpshooter' drawing nutrients from the host liana, D. glaucescens, and flinging the waste fluid as droplets through their tail ends. SEM micrographs of A. depressa revealed its external morphological features, characteristic of a sharpshooter. We quantified 20E (0.44-1.44%, dry wt.) in various parts of D. glaucescens. 20E (1.47%, dry wt.) was also detected in the excrement of A. depressa. This plant (D. glaucescens)-insect (A. depressa) association crucially is not damaging the host liana. Considering the diseases caused by sharpshooting leafhoppers in the Americas, this association and the survival of the host plant (D. glaucescens) is illustrating a unique plant-insect interaction.

Effect of Palrnatine on lipopolysaccharide-induced acute lung injury by inhibiting activation of the Akt/NF|-κB pathway.

Inflammation plays an important role in the development of acute lung injury (ALI). Severe pulmonary inflammation can cause acute respiratory distress syndrome (ARDS) or even death. Expression of proinflammatory interleukin-|1ß (IL-|1ß) and inducible nitric oxide synthase (iNOS) in the process of pulmonary inflammation will further exacerbate the severity of ALI. The purpose of this study was to explore the effect of Palrnatine (Pa) on lipopolysaccharide (LPS)-induced mouse ALI and its underlying mechanism. Pa, a natural product, has a wide range of pharmacological activities with the potential to protect against lung injury. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed to detect the expression and translation of inflammatory genes and proteins in vitro and in vivo. Immunoprecipitation was used to detect the degree of P65 translocation into the nucleus. We also used molecular modeling to further clarify the mechanism of action. The results showed that Pa pretreatment could significantly inhibit the expression and secretion of the inflammatory cytokine IL-1ß, and significantly reduce the protein level of the proinflammatory protease iNOS, in both in vivo and in vitro models induced by LPS. Further mechanism studies showed that Pa could significantly inhibit the activation of the protein kinase B (Akt)/nuclear factor-κB (NF-κB) signaling pathway in the LPS-induced ALI mode and in LPS-induced RAW264.7 cells. Through molecular dynamics simulation, we observed that Pa was bound to the catalytic pocket of Akt and effectively inhibited the biological activity of Akt. These results indicated that Pa significantly relieves LPS-induced ALI by activating the Akt/NF-κB signaling pathway.

Triterpenoid saponins from the leaves and stems of Pericampylus glaucus and their insulin mimetic activities.

During an attempt to discover insulin mimetics, thirteen new triterpenoid saponins (1-13), including three phytolaccagenic acids (1, 2, and 12) and ten serjanic acids (3-11 and 13), as aglycones were isolated from a 70% ethanol extract of leaves and stems from Pericampylus glaucus. The chemical structures of compounds 1-13 were determined through spectroscopic data analysis, including NMR, IR, and HRESIMS. All isolated compounds (1-13) were evaluated using 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose (2-NBDG) as a fluorescent-tagged glucose probe to determine their stimulatory effects on glucose uptake in differentiated 3 T3-L1 adipocyte cells. Consequently, four compounds (4, 7, 11, and 12) exhibited stimulatory effects on glucose uptake.

Tiliacora triandra (Colebr.) Diels Leaf Aqueous Extract Inhibits Hepatic Glucose Production in HepG2 Cells and Type 2 Diabetic Rats.

This study investigated the effects of Tiliacora triandra (Colebr.) Diels aqueous extract (TTE) on hepatic glucose production in hepatocellular carcinoma (HepG2) cells and type 2 diabetic (T2DM) conditions. HepG2 cells were pretreated with TTE and its major constituents found in TTE, epicatechin (EC) and quercetin (QC). The hepatic glucose production was determined. The in vitro data were confirmed in T2DM rats, which were supplemented daily with 1000 mg/kg body weight (BW) TTE, 30 mg/kg BW metformin or TTE combined with metformin for 12 weeks. Results demonstrate that TTE induced copper-zinc superoxide dismutase, glutathione peroxidase and catalase genes, similarly to EC and QC. TTE decreased hepatic glucose production by downregulating phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) and increasing protein kinase B and AMP-activated protein kinase phosphorylation in HepG2 cells. These results correlated with the antihyperglycemic, antitriglyceridemic, anti-insulin resistance, and antioxidant activities of TTE in T2DM rats, similar to the metformin and combination treatments. Consistently, impairment of hepatic gluconeogenesis in T2DM rats was restored after single and combined treatments by reducing PEPCK and G6Pase genes. Collectively, TTE could potentially be developed as a nutraceutical product to prevent glucose overproduction in patients with obesity, insulin resistance, and diabetes who are being treated with antidiabetic drugs.

Tiliacora triandra extract and its major constituent attenuates diabetic kidney and testicular impairment by modulating redox imbalance and pro-inflammatory responses in rats.

BACKGROUND: Literature has demonstrated that diabetes is associated with renal complication and testicular dysfunctions. The current study explored the potential of Tiliacora triandra extract and its major component against diabetic kidney and testicular damages in rats. METHODS: Diabetes was induced by high fat diet/streptozotocin (HFD/STZ) and treated orally with Tiliacora triandra extract (TTE, 100 and 400 mg kg-1 body weight) and its major component, 5,7-dihydroxy-6-oxoheptadecanoic acid (DHA, 25 mg kg-1 body weight) for 30 consecutive days. Testicular activities of testicular enzymes, serum levels of testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), sperm parameters and urinalysis for protein and albumin levels were evaluated. Renal and testicular biomarkers of oxidative stress and pro-inflammation were analysed along with histology. RESULTS: The experimental diabetes induced significant alterations in the levels and activities of indices evaluated compared to non-diabetic normal rats. The 28-day treatment of diabetic rats with TTE and DHA markedly improved activities of testicular enzymes, restored levels of testosterone, LH and FSH and sperm parameters compared to untreated diabetic rats. TTE and DHA abrogated proteinuria and reversed urine albumin level. Testicular and renal oxidative stress and pro-inflammation were attenuated in diabetic rats treated with TTE and DHA. The diabetes-mediated histopathological damage was alleviated in the kidney and testis. CONCLUSION: The protective effect of TTE and DHA against diabetes induced kidney and testicular damages may be related to its antioxidant and anti-inflammatory activities. © 2020 Society of Chemical Industry.

Alkaloids of Abuta panurensis Eichler: In silico and in vitro study of acetylcholinesterase inhibition, cytotoxic and immunomodulatory activities.

Natural products obtained from species of the genus Abuta (Menispermaceae) are known as ethnobotanicals that are attracting increasing attention due to a wide range of their pharmacological properties. In this study, the alkaloids stepharine and 5-N-methylmaytenine were first isolated from branches of Abuta panurensis Eichler, an endemic species from the Amazonian rainforest. Structure of the compounds was elucidated by a combination of 1D and 2D NMR spectroscopic and MS and HRMS spectrometric techniques. Interaction of the above-mentioned alkaloids with acetylcholinesterase enzyme and interleukins IL-6 and IL-8 was investigated in silico by molecular docking. The molecules under investigation were able to bind effectively with the active sites of the AChE enzyme, IL-6, and IL-8 showing affinity towards the proteins. Along with the theoretical study, acetylcholinesterase enzyme inhibition, cytotoxic, and immunomodulatory activity of the compounds were assessed by in vitro assays. The data obtained in silico corroborate the results of AChE enzyme inhibition, the IC50 values of 61.24µM for stepharine and 19.55µM for 5-N-methylmaytenine were found. The compounds showed cytotoxic activity against two tumor cell lines (K562 and U937) with IC50 values ranging from 11.77 µM to 28.48 µM. The in vitro assays revealed that both alkaloids were non-toxic to Vero and human PBMC cells. As for the immunomodulatory activity, both compounds inhibited the production of IL-6 at similar levels. Stepharine inhibited considerably the production of IL-8 in comparison to 5-N-methylmaytenine, which showed a dose dependent action (inhibitory at the IC50 dose, and stimulatory at the twofold IC50 one). Such a behavior may possibly be explained by different binding modes of the alkaloids to the interleukin structural fragments. Occurrence of the polyamine alkaloid 5-N-methylmaytenine was reported for the first time for the Menispermaceae family, as well as the presence of stepharine in A. panurensis.

GC-HRMS screening of bioactive compounds responsible for antimicrobial and antioxidant activities of blood fruit (Haematocarpus validus Bakh. F. Ex Forman) of North-East India.

The aims of this study are to evaluate phytochemical composition, antimicrobial and antioxidant activities of different solvent-assisted blood fruit pulp extracts. The extracts of blood fruit were prepared with various solvents namely, water, ethyl acetate, methanol and acetone. These extracts were divided into two groups, first group of extract were thermosonicated (treated) and the second group were not exposed to thermosonication (untreated). The sample extracts were then evaluated for their antimicrobial properties against various microorganisms. The results revealed that the antimicrobial activity of thermosonicated ethyl acetate extracts at 12 mg/mL concentration exhibited the highest inhibition zone of 19.4 mm against Staphylococcus aureus. The phenolic content of ethyl acetate extract was found to be the highest as compared to that of other solvent extracts and the values were 1508.08 and 1478.34 mg GAE/100 g for treated and untreated samples, respectively. The treated ethyl acetate extract antioxidative activity by DPPH, ORAC, ABTS, and FRAP assay were 1627.68, 2119.82, 938.11, and 2360.18 µmol TE/g, respectively. The minimum bactericidal concentration (MBC - 1.5 mg/mL) against the selected foodborne pathogens was obtained with thermosonicated ethyl acetate blood fruit extract. In the present study, it was observed that the thermosonicated ethyl acetate extract exhibits maximum antioxidant and antimicrobial activities than any other examined solvent extracts. The major bioactive volatile compounds found in treated ethyl acetate extracts were 2-bromotetradecane, tetracosane, heptadecane, eicosane and palmitic acid. These bioactive compounds can be utilizable for the development of functional, nutraceutical and pharmacological products.

Bioassay-guided isolation and identification of anti-ulcer ecdysteroids from the seeds of Sphenocentrum jollyanum Pierre (Menispermaceae).

Sphenocentrum jollyanum seeds (MeOH extract and n butanol fraction) exhibited urease inhibitory activity (IC50 40.0 ± 0.92, 28.6 ± 0.41). The Ethyl acetate (EtOAc) fraction gave significant antacid activity with an increase in the baseline pH value of 1.2 to 1.61 ± 0.00 and 1.53 ± 0.00 at 50 and 100 mg, respectively, compared to the antacid activity of sodium bicarbonate (1.53 ± 0.00, 1.47 ± 0.00). Five known ecdysteroid compounds isolated from S. jollyanum ethyl acetate and n butanol fractions are Pinnatasterone (1), Polypodine B (2), 20-hydroxyecdysone (3), 20, 26-dihydroxyecdysone, (4) and Atrotosterone A (5). The compounds' structures were determined using extensive 1D and 2D NMR experiments, and the molecular mass for each of the compounds was confirmed by FAB-MS. Compounds 1-5 were evaluated for their urease inhibitory and antacid activities. Fractions were active in comparison with the standard drug acetohydroxamic acid, and sodium bicarbonate, respectively. Compounds 2, 3 and 1 showed significant urease inhibitory activity (IC50 7.0 ± 0.56, 13.8 ± 0.49 and 14.1 ± 0.59), respectively. The activity of compounds 4 and 5 were moderate compared to that of acetohydroxamic acid (IC50 value 20.3 ± 0.43). Very few compounds have been isolated from this plant despite the numerous biological activities reported for it. The antacid and urease inhibitory activities of this plant and isolated compounds are described for the first time.

Comparison of the antimalarial activity of a Colombian traditional Uitoto remedy with laboratory preparations.

BACKGROUND & OBJECTIVES: In Colombian Amazonia, Uitoto indigenous people use a preparation of Curarea toxicofera (Menispermaceae) to prevent and treat malaria. To open the way for the production of a standardized herbal remedy, we compared the activity of the traditional preparation with laboratory preparations. METHODS: People were interviewed on their mode of use and preparation of what is considered the best remedy against fevers in this area. The herbal remedy was prepared according to the healer's recommendations. The plant was also submitted to continuous distillation and percolation extraction. The preparations were then tested against Plasmodium falciparum, in vitro. Traditional preparation and extract obtained by percolation were tested on Plasmodium berghei infected mice. Chemical profiles were also explored by thin-layer chromatography. RESULTS: Yields of extraction were around 7% in the preparations (percolation was the most efficient). The phytochemical profile showed a mix of steroids, flavonoids and alkaloids qualitatively similar in all preparations. In vitro, the extracts showed inhibitory concentration 50 <10µg/mL: the traditional preparation was almost three times less active than laboratory preparations. In vivo, percolation was also more active than traditional preparation, inhibiting 78% of the parasite growth at 400mg/kg/day by oral route. INTERPRETATION & CONCLUSION: Pharmacological activities suggest that both the original remedy (prepared according to traditional pharmacopeia) and the extracts obtained by percolation extraction exhibit relevant antiparasitic activity. C. toxicofera should therefore be considered for the elaboration of an improved traditional medicine by implementing toxicological studies and carefully following quality control guidelines for its preparation.

Effect of tiliamosine, a bis, benzylisoquinoline alkaloid isolated from Tiliacora acuminata (Lam.) Hook. f. & Thom on the immature stages of filarial mosquito Culex quinquefasciatus say (Diptera: Culicidae).

The present study was aimed to check the mosquitocidal activity of tiliamosine isolated from Tiliacora acuminata (Lam.) Hook. f. & Thom against immature stages of Culex quinquefasciatus. Eggs and larvae of Cx. quinquefasciatus were exposed to different concentrations of tiliamosine - 0.5, 1.0, 1.5 and 2.0 ppm - prepared using DMSO. The compound tiliamosine showed good larvicidal activity with LC50 and LC90 values of 1.13 and 2.85 ppm respectively, against third-instar larvae of Cx. quinquefasciatus at 24 h. In control, the larvae exhibited normal movement. Tiliamosine exhibited 91% ovicidal activity at 2.0 ppm concentration after 120 h post-treatment. Lowest concentration of tiliamosine (0.5 ppm) showed 19% egg mortality. Histopathology study of the compound-treated larvae showed serious damage on the larval midgut cells. The treated larvae showed restless movement which was different from that of the control larvae. The larvae exhibited malformation in development. The compound tiliamosine was harmless to non-target organisms P. reticulata and Dragon fly nymph at tested concentrations. The compound was highly active and inhibited AChE in a concentration-dependent manner. Computational analysis of the tiliamosine had strong interaction with AChE1 of Cx. quinquefasciatus. This report clearly suggests that the isolated compound can be used as an insecticide to control mosquito population and thus prevent the spread of vector-borne diseases.

Two new phytoecdysteroids from Sphenocentrum jollyanum Pierre root.

The crude methanol extract of Sphenocentrum jollyanum root exhibited 98% and 80% antimicrobial activity against Aspergillus fumigatus Pinh and Vancomycin resistant enterococcus (VRE) at a concentration of 200 µg/mL, with IC50 11.45 and 12.95 µg/mL, respectively. The ethyl acetate fraction of methanol extract showed in-vitro antimicrobial activity against A. fumigatus Pinh at 83% with IC50 of <8 µg/mL. The phytochemical investigation of ethyl acetate fraction yielded six compounds, which were identified by their NMR, IR and MS spectral analyses as two new phytoecdysteroidal glycosides Sphenocentroside A (1), and Sphenocentroside B (2), and four known phytoecdysteroids: polypodoaurein (3), polypodine B (4), ecdysterone (5), and 20, 26-dihydroxyecdysone (6).

Secondary metabolites from Triclisia gilletii (De Wild) Staner (Menispermaceae) with antimycobacterial activity against Mycobacterium tuberculosis.

Triclisinone (2), a new ochnaflavone derivative, was isolated from the aerial parts of Triclisia gilletii, along with known drypemolundein B (1) and eight other known compounds. The chemical shifts of drypemolundein B (1) have been partially revised based on reinterpretation of NMR spectroscopic data. The eight other secondary metabolites are composed of: (+)-nonacosan-10-ol (3); stigmasterol (4), 3-O-ß-D-glucopyranosylsitosterol (5), 3-O-ß-D-glucopyranosylstigmasterol (6); oleanic acid (7); myricetin (8), quercetin (9) and 3-methoxyquercetin (10). Their structures were elucidated using IR, MS, NMR 1D and 2D, 1H and 13C and comparison with literature data. Furthermore, compounds 1, 2, 5, 6, 8, 9 and the crude extract were tested against Mycobacterium tuberculosis. Compounds 1, 2, 8 and 9 displayed moderate to very good activity against resistant strain (codified AC 45) of M. tuberculosis with minimum inhibitory concentrations MICs ranging from 3.90 to 62.5 µg/mL.

Inhibitory effects of Tiliacora triandra (Colebr.) Diels on cholesterol absorption.

Background Natural supplements and herbal medicines have been attracted to use for managing elevated cholesterol levels. Tiliacora triandra (Colebr.) Diels (TT) or Yanang (in Thai) is commonly used as an ingredient in various types of Thai foods. In this study, we investigated the effect of methanolic TT leaf extract on cholesterol absorption by measuring the uptake and the efflux of cholesterol and cholesterol micellar solubility. In addition, we tested the effect of TT leaf extract on pancreatic lipase activity. Methods The uptake and efflux of cholesterol was determined by quantification of radioactivity in differentiated Caco-2 cells after treatment with radioactive cholesterol. Cholesterol mixed micelles were prepared for cholesterol uptake, efflux and solubility studies. The pancreatic lipase activity was determined using 4-methylumbelliferyl oleate as a substrate. Results Our finding showed that TT extract decreased the uptake of cholesterol by approximately 48% but did not affect the efflux of cholesterol. TT inhibited pancreatic lipase activity with the IC50 at 273.5 µg/mL and also decreased cholesterol micellar solubility. Conclusions These findings suggest that TT leaf extract seems to be a potential candidate as cholesterol-lowering agents.

Hepatoprotective effect of bisbenzylisoquinoline alkaloid tiliamosine from Tiliacora racemosa in high-fat diet/diethylnitrosamine-induced non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is one of the aggressive forms of non-alcoholic fatty liver disease (NAFLD) and is a potential risk factor of HCC. This study reports the curative effect of tiliamosine on NASH. Tiliamosine was isolated from Tiliacora racemosa Colebr. (Menispermaceae) and its structure was confirmed by studying the physical and spectroscopic data. The effects of tiliamsoine on lipid accumulation and lipotoxicity were evaluated using palmitate-oleate induced steatosis in HepG2 cells. The in vivo efficacy of tiliamosine was evaluated using HFD fed, DEN induced non-alcoholic steatohepatitis Wistar rats. In HepG2 cells, tiliamosine did not affect the cell viability up to 100 µM concentration and showed GI25 value of 264.28 µM. The treatment with tiliamsoine significantly lowered the ORO concentration by 44.17% and triglyceride accumulation by 69.32% at 50 µM concentration (P < 0.005). It also reduced the leakage of LDH and transaminases in PO-BSA induced HepG2 cells. The treatment with tiliamsoine significantly decreased the plasma levels of transaminases, phosphatase and LDH (P < 0.05) in HFD-DEN induced steatohepatitis. The histology and the immunohistochemistry of the hepatic sections were in accordance with the biochemical findings. Preliminary molecular analysis indicated that the hepatic FXR expression was upregulated and TNFα expression was downregulated by the treatment with tiliamsoine. This study provided preliminary evidence on the use of tiliamosine for the treatment of NASH.

Anti-ulcerogenic effect of the methanol extract of Chasmanthera dependens (Hochst) stem on male Wistar rats.

Background Oxidative stress and free radical-mediated processes have been implicated in the pathogenesis of indomethacin-induced gastric ulcer. This study investigated the ability of the methanol extract of Chasmanthera dependens to protect the gastric mucosal from oxidative damage induced by oral administration of indomethacin in rats. Methods The C. dependens stems were chopped into pieces, air-dried, and pulverized into powder. One kilogram of the powder was macerated in 1 L of methanol for 72 h. The mixture was filtered and evaporated using rotatory evaporator to obtain the extract of C. dependens. Adult male rats were divided into eight groups of six animals per group and were pretreated orally with the methanol extract of C. dependens (200, 400, and 800 mg/kg) or cimetidine (CIM), a standard drug (50 mg/kg), for 7 days. Gastric ulcer was induced orally with indomethacin. Ulcerogenic parameters, oxidative stress indices, and histopathological examination of the stomach were assessed to monitor the gastroprotective potential of C. dependens stem. Results Indomethacin caused severe gastric mucosa damage and significant reduction in the gastric mucosa antioxidant system with concomitant increase in the level of lipid peroxidation. Pretreatment with the methanol extract of C. dependens or CIM significantly reduced the formation of ulcer at the different doses administered. Similarly, pretreatments with the extract or CIM improved the antioxidant system, decreased acid output, lipid peroxidation, and improved the architecture of the gastric mucosa in ulcerated rats. Conclusions The results show the gastroprotective effect of the methanolic extract of C. dependens, which may be attributed to its antioxidant properties.

Synthesis of (aminoalkyl)cycleanine analogues: cytotoxicity, cellular uptake, and apoptosis induction in ovarian cancer cells.

Our previous studies demonstrated that cycleanine, a macrocyclic bisbenzylisoquinoline (BBIQ) alkaloid, showed potent anti-ovarian cancer activity via apoptosis induction. Here, we synthesized two novel (aminoalkyl)cycleanine analogues (2 and 3) through a simple and efficient two-step reaction starting from cycleanine isolated from Triclisia subcordata Oliv. These analogues showed greater potency than the unmodified cycleanine in three human ovarian cancer cell lines. Both 2 and 3 induced apoptosis in ovarian cancer cells by activations of caspases 3/7, cleavage of PARP, increase in subG1 cell cycle phase and in the percentage of apoptotic cells. Further confocal fluorescence microscopy analysis confirmed the cellular uptake of alkaloids in ovarian cancer cells by using the unique (alkynyl)cycleanine (3) via click chemistry reaction. Our results suggest that cycleanine could be a hit compound for the future development in attacking ovarian cancer.

Thermodynamics and Kinetics of Single-Chain Monellin Folding with Structural Insights into Specific Collapse in the Denatured State Ensemble.

Proteins, which behave as random coils in high denaturant concentrations undergo collapse transition similar to polymers on denaturant dilution. We study collapse in the denatured ensemble of single-chain monellin (MNEI) using a coarse-grained protein model and molecular dynamics simulations. The model is validated by quantitatively comparing the computed guanidinium chloride and pH-dependent thermodynamic properties of MNEI folding with the experiments. The computed properties such as the fraction of the protein in the folded state and radius of gyration (Rg) as function of [GuHCl] are in good agreement with the experiments. The folded state of MNEI is destabilized with an increase in pH due to the deprotonation of the residues Glu24 and Cys42. On decreasing [GuHCl], the protein in the unfolded ensemble showed specific compaction. The Rg of the protein decreased steadily with [GuHCl] dilution due to increase in the number of native contacts in all the secondary structural elements present in the protein. MNEI folding kinetics is complex with multiple folding pathways and transiently stable intermediates are populated in these pathways. In strong stabilizing conditions, the protein in the unfolded ensemble showed transition to a more compact unfolded state where Rg decreased by ≈17% due to the formation of specific native contacts in the protein. The intermediate populated in the dominant MNEI folding pathway satisfies the structural features of the dry molten globule inferred from experiments.

Analysis of six active components in Radix tinosporae by nonaqueous capillary electrophoresis with mass spectrometry.

Nonaqueous capillary electrophoresis with mass spectrometry has advantages for the analysis of active components in herbs. Here, a rapid nonaqueous capillary electrophoresis with mass spectrometry method was developed to separate, identify, and quantify palmatin, columbin, cepharanthine, menisperine, magnoflorine, and 20-hydroxyecdysone in Radix tinosporae. Electrospray ionization MS1-3 spectra of the six components were collected and possible cleavage pathways of main fragment ions were elucidated. The conditions that could affect separation, such as the composition of running buffer and applied voltage, were studied, and the conditions that could affect the mass spectrometry detection, such as the composition and flow rate of sheath liquid, the pressure of nitrogen gas, and the temperature and flow rate of the dry gas, were also optimized. Under the optimized conditions, the correlation coefficient was >0.99. The relative standard deviations of migration time and peak areas were <10%. The recoveries were calculated to be 99.31-107.80% in real samples. It has been demonstrated that the proposed method has good potential to be applied to determine the six bioactive components in Radix tinosporae.

Acanthamoebicidal activity of periglaucine A and betulinic acid from Pericampylus glaucus (Lam.) Merr. in vitro.

Acanthamoeba species are pathogenic protozoa which account for amoebic keratitis, conjunctivitis and granulomatous amoebic encephalitis. These amoebae form cysts which resist drugs and more effective acanthamoebicidal agents are needed. Medicinal plants could be useful in improving the current treatment strategies for Acanthamoeba infections. In the present study, we examined the amoebicidal effects of Pericampylus glaucus (Lam.) Merr., a medicinal plant used for the treatment of conjunctivitis in Malaysia. Pathogenic Acanthamoeba triangularis were isolated from environmental water samples and treated with different concentrations of fractions obtained from Pericampylus glaucus (Lam.) Merr. as well as main constituents for 24-72 h. Chlorhexidine was used as a reference drug. Ethanol fraction of stem showed significant (p < 0.05) inhibition of trophozoites survival. Betulinic acid and periglaucine A from this plant at 100 µg/mL inhibited more than 70% survival of both cysts and trophozoites. The calculated therapeutic index for betulinic acid and periglaucine A was 170 and 1.5 for trophozoites stage and 3.75 and 8.5 for cysts stage. The observed amoebicidal efficacies indicate the beneficial aspects of this plant in the treatment of Acanthamoeba infection. Periglaucine A could also be of value for the treatment of Acanthamoeba infection.