Saccharomyces cerevisiae. However, some nonionic surfactants added to the aqueous buffer system improved the bioreduction process by decreasing the reverse and side reaction rates in addition to effectively increasing the average positive reaction rate. Further, a shorter carbon chain length of hydrophilic or hydrophobic moieties in surfactants resulted in a higher yield of (S)-2-octanol. The alkylphenol ethoxylate surfactants had a less influence than polyoxyethylenesorbitan trialiphatic surfactants on the product e.e. It suggested that the product e.e. resulting from the change of carbon chain length of the hydrophobic moieties varied markedly compared with the change of carbon chain length of the hydrophilic moiety. Emulsifier OP-10 and Tween 20 markedly enhanced the yield and product e.e. at the concentration of 0.4 mmol L−1 with a yield of 73.3 and 93.2%, and the product e.e. of 99.2 and 99.3%, respectively, at the reaction time of 96 h.In an aqueous buffer system, serious reverse and side reactions were found in the asymmetric reduction of 2-octanone with  Saccharomyces cerevisiae contains alcohol dehydrogenases and other redox enzymes -2-octanol  of products due to the reversible and side reactions caused by excessive substrates and/or products.The yeast Supplementation of culture medium with surface-active agents shifts the physiologic properties of yeast and other cells -2-octanol, (S)-2-octanol and (S)-(+)-PEIC (phenylethyl isocyanate) were supplied by Aldrich (USA). Emulsifier OP-6 (MW470), Tween 20 (MW1226.5), Tween 40 (MW1284.6), Tween 60 (MW1311.7) and Tween 80 (MW1309.6) were products of Sinopharm Chemical Reagent Co., Ltd. (China). Emulsifier OP-8 (MW559), Emulsifier OP-10 (MW647), sodium dodecyl sulfate (SDS), Emulsifier OP-12 (MW735) and cetyltriethylammonium bromide (CTAB) were obtained from Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. (China). All other chemicals were of analytical grade. Glucose toolkit was purchased from BHKT Clinical Reagent Co. Ltd. (China).2-Octanone was purchased from Merck-Schuchardt (Germany). (−1): glucose 10.0, (NH4)2SO4 5.0, KH2PO4 1.0, MgSO4·7H2O 0.5, KCl 0.5, ZnSO4 0.01, Fe2(SO4)3 0.01, natural pH.Medium . The harvested cells were used for the following experiments.Conditions: 100 mL medium with 10 g lyophilized cells in a 250 mL-flask was placed in a rotary incubator set at 32 °C and 200 r min2 (99.999%) as the carrier gas, with a pressure front of the column at 100 kpa, the detector temperature 210 °C, the injector temperature 190 °C and the column temperature 170 °C.The concentrations of 2-octanone and 2-octanol were determined by GC  equipped with a flame ionization detector and a non-polar fused silica capillary column AC1-0.25 . The GC conditions included NS)-(+)-PEIC and then left at 45 °C for 2 h. A fused silica capillary column and carrier gas (with column pressure front 65 kpa) were used. The injector and the detector were maintained at 250 and 270 °C, respectively. The retention times of (R)- and (S)-enantiomer were 18.8 min and 19.5 min.The enantiomer resolution was based on the derivation of 2-octanol with optically pure isocyanate. 10 μL sample was mixed with 50 μL toluene and 2 μL (v) was calculated using the concentration of 2-octanol  as a function of time (t)Reaction rate . At a reaction time of 96 h, the v represents the average reaction rate.During the initial 3 h, the c2-ol,t is the concentration of 2-octanol  formed at time (t), and c2-one,0 is the concentration of 2-octanone before the reaction.S)-2-octanolS and AR are the peak areas of (S)-2-octanol and (R)-2-octanol, respectively.Enantiomeric excess of 11SO4−) and cationic CTAB (C16H33N(CH3)3+), are different from the nonionic surfactant Emulsifier OP-6. Emulsifier OP-6 consists of alkylphenol ethoxylates (APEO) and does not dissociate in water. Nonionic surfactants show interesting features such as higher stability, lower surface tension, lower critical micelle concentration, higher aggregation number of micelles and better solubilization. To investigate the effect of surfactants in asymmetric reduction of 2-octanone with S. cerevisiae, we introduced surfactants into the aqueous media and the yield and product e.e. value of the reaction were analyzed. The results are shown in Fig. S)-2-octanol catalyzed by the whole cell S. cerevisiae, led to low yield and product e.e. value. Accordingly, the presence of Emulsifier OP-6 and CTAB, except SDS, showed a better yield and product e.e. value. The nonionic Emulsifier OP-6 was the best of the surfactants investigated.As typical ionic surfactants, the anionic SDS (CHS)-enantiomer (vS) increased while the rate of (R)-enantiomer (vR) decreased. In detail, the VS increased from 0.072 to 0.97 mmol L−1 h−1 and 0.92 mmol L−1 h−1, as well as the VR decreased from 0.009 to 0.002 mmol L−1 h−1 and 0.003 mmol L−1 h−1, respectively in the media with Emulsifier OP-6 and CTAB. The apparent reverse reaction rate (vrev) approached 0, which indicated no evidence of reverse reaction. In the system containing SDS, the phenomenon was quite different from the system containing Emulsifier OP-6 or CTAB.In the reaction system with surfactants, it was apparent that not only the positive reaction was improved, but also the side and reverse reactions inhibited by Emulsifier OP-6 and CTAB. Based on the synthesis rate of the enantiomers calculated Table , it was 14H21(OC2H4)6OH), Emulsifier OP-8 (C14H21(OC2H4)8OH), Emulsifier OP-10 (C14H21(OC2H4)10OH) and Emulsifier OP-12 (C14H21(OC2H4)12OH), contain an ethoxylate with the unit number ranging from 6 to 12. As seen in the Table The Emulsifier OP series, including Emulsifier OP-6 10COO(OC2H4)20C6H8O(OH)3], Tween 40 , Tween 60  and Tween 80 . These compounds contain the same hydrophilic end but different hydrophobic terminal groups, like C12H23CO, C16H31CO, C18H35CO and C18H33CO , are obtained based on their degree of ethoxylate polymerization, for example, Tween 20  oxidoreductase (EC 1.6.99.3) activities. The results obtained, showed that Triton X-100 inhibited both membrane-bound and solubilized NADH-dependent redox activities  vesicles isolated from the yeast e.e. value. The e.e. value decreased rapidly at a higher level of surfactant concentration. It confirmed that the properties of hydrophilic and hydrophobic groups, as well as the concentration of surfactants, but not the length of side chain of hydrophilic end, were the most important factors contributing to the product e.e. value.However, the carbon chain length of ethoxylate in APEO series surfactants showed a mild influence on the product S. cerevisiae-mediated reduction of 2-ocatanone depending on the length of hydrophobic end. The shorter the carbon chain, the higher the yield. Increased biocompatibility associated with the longer hydrophobic end lowered the cell membrane permeability, and hence resulting in a lower yield.Compared with Emulsifier OP series surfactants, the Tween series surfactants affected the yield in the Glc and the yield, based on the findings of Emulsifier OP series compounds (with fixed hydrophobic end and varied hydrophilic end) and Tween series compounds (with fixed hydrophilic end and varied hydrophobic end). These results illustrated that nonionic surfactants not only changed the cell membrane permeability  as the hydrophobic end, the average e.e. value reached 94.7%, which dramatically dropped to 49.5% when the hydrophobic end was C17H33CO (Tween 80).The carbon chain length at the hydrophobic and hydrophilic ends, and the concentration of surfactants were important factors affecting the product ue Figs. . Howeverend Fig. , which wend Fig. . With C1e.e. value compared with the Emulsifier OP series surfactants. The carbon chain length of hydrophobic end had a significantly greater impact than the length of hydrophilic end in nonionic surfactants. The nonionic surfactants Emulsifier OP-10 and Tween 20 improved the yield and product e.e. value effectively. At a concentration of 0.4 mmol L−1, the reaction yield at 96 h was 73.3 and 93.2%, respectively. Furthermore, the e.e. value was 99.2 and 99.3%, which were significantly higher compared with the reaction without surfactants.The shorter carbon chain in the nonionic surfactants, improved the yield, irrespective of the length of hydrophobic or hydrophilic ends. The Tween series surfactants strongly affected the product


Pholiota adiposa is a kind of edible mushroom which has long been known for its health care applications. To reveal the exact mechanism of its protective functions in humans, in this study we isolated and identified the active compound PB3 of P. adiposa for the first time by a combination of chromatography techniques, including NKA macroporous resin and Sephadex G-15. PB3, with molecular mass of 267.2 Da and molecular formula of C10H13N5O4 discovered by mass spectrum (MS) was identified to be adenosine. Mice were injected intraperitoneally with purified fraction PB3. Seven days after injection, we found a 1.5-fold increase of IL10 at the mRNA level, while a down regulated expression of IL-2, IL-6 and IFN-γ to 49.0%, 56.9% and 73.4%, respectively, was detected in spleen by real-time quantitative PCR. What’s more, SOD expression level was significantly increased by 1.6-fold compared to control. Fraction PB3 displayed anti-inflammatory potency and heightened SOD activity on the transcriptional level, which could be considered of further pharmaceutical or medication value. Pholiota adiposa is one of the edible mushrooms grows wildly in China, which have been the subject of considerable attention for their health-care applications. It is rich in essential amino acids, vitamins, trace elements and proteins, including bio-active enzymes + of PB3 was 268.1040 Da, which corresponded to a molecular mass of 267.2 Da . Sephadex G15 was purchased from Hui De Yi Co., Ltd. . Trizol and RT-PCR kits were purchased from Dingguo Biotechnology Development Center . 2×SYBR Green Realtime PCR Master Mix was from TOYOBO Co., Ltd. . All other chemicals were of the highest quality available.Male BALB/c mice, 5–6 weeks of age and with a body weight of 20 ± 2 g, were purchased from a local registered animal supplier. The mice were housed under normal laboratory conditions  with free access to standard rodent chow and water.P. adiposa cultured and collected in Henan Luoyang on September 2010, was provided by Mr. Jun Hou from Henan University of Science and Technology and then authenticated. The dried fruit bodies of P. adiposa were powdered and extracted with 70% ethanol (100 g : 2 L) under reflux. After filtration to remove debris, the filtrate (crude extract named P) was concentrated on a rotary evaporator.Dried 2/g, and an average pore diameter of 200–220 Å. P was dissolved in distilled water (500 mg/mL) and then chromatographed on a column of macroporous resin (2.5 × 35 cm). The inactive fraction named PA was eluted with distilled water. The resin was then eluted with 30% (v/v) ethanol, and the resulting fraction was named PB. Finally, the resin was regenerated with 70% (v/v) ethanol to get the fraction named PC. PB was chromatographed on a column of Sephadex G15 (1.2 × 60 cm) which was eluted with distilled water. The fractions collected were named PB1, PB2 and PB3. The fraction PB3 was purified with a Lab Alliance high-performance liquid chromatography (HPLC) system, including two Series III pumps, a 500 absorbance detector set at 260 nm, and connected to a Cs420x Hardware integrator . The columns used were a Merck  reverse-phase C18 ODS preparative (250 × 10 mm) and analytical column (250 × 4.6 mm). The eluent was 15% methanol under 30 °C at a flow rate of 3 mL/min for the preparative column and 1 mL/min for the analytical column.The crude extract P was purified by chromatography with macroporous resins NKA, a kind of apolar cross-linked polystyrene, which has a bead size of 0.3–1.0 mm, a surface area of 570–590 mE. coli O55:B5; Sigma, St. Louis, MO, USA) at a dose of 2 mg/kg. All mice were sacrificed 12 h later and total splenic RNA were isolated by using the Trizol kit. Ten mice were divided randomly into two groups. Control mice (group A) were treated with 0.9% (w/w) NaCl by intraperitoneal injection. PB3 mice (group B) were treated with PB3 (50 mg/kg). All mice were injected once a day for seven days. On the eight day, all mice received an intraperitoneal injection of purified LPS  primer, reverse transcription buffer, and 3 units of reverse transcriptase. It was heated at 65 °C for 5 min, then conducted at 42 °C for 30 min, and finally inactivated by heating to 95 °C for 5 min.2O (7 μL) and 2×SYBR Green Realtime PCR Master Mix (10 μL). The following thermocycler program was used for real-time PCR: 1 min preincubation at 95 °C, followed by 40 cycles of incubation at 94 °C for 15 s, 55 °C for 30 s, 72 °C for 45 s. The primers of super oxygen dehydrogenises (SOD), IL-2, IL-6, IL-10, interferon-γ (INF-γ) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are shown in −ΔΔCt method [Real-time PCR reaction mixture contained upstream primer , downstream primer , cDNA (1 μL), ddHt method  was usedP. adiposa. The fraction PB3 which was identified to be adenosine displayed potent anti-inflammatory effects by enhancing the expression of anti-inflammatory cytokines IL-10, as well as suppressing the expression of inflammatory factors IL-6, IL-2 and IFN-γ. Fraction PB3 helped balance the redox system and immune system on the transcriptional level. This suggests that adenosine might not only have cardioprotective effects, but also potential in the treatment of inflammation.In this study, adenosine was isolated for the first time from the mushroom


A series of organic core–shell-shaped micro/nanoparticles from twisted enantiomeric macrocycles could be obtained in Schiff base reaction with high yield at room temperature. MH), which was obtained from an achiral precursor by Schiff base reaction. Further studies suggest that a series of unexpected and stable core–shell-based organic micro/nanospheres can be directly precipitated from a simple reaction solution with high yield. A single-crystal X-ray diffraction analysis of MH revealed that the unusual C–H···π interaction triggered self-assembly of the enantiomeric forms in the solid state plays an important role in the formation of the core–shell-shaped organic particles.Functional self-assemblies derived from noncovalent interactions such as lipid vesicles and DNA chiral double helices are a typical feature of natural life activity. Because of this phenomenon, a self-assembly approach for various functional organic particles is a desirable objective in supramolecular chemistry. Here, we report the discovery of enantiomeric conformers from a twisted macrocyclic host ( In fact, numerous recent studies have suggested that artificial molecular chirality plays a significant role in supramolecular self-assembly processes, especially those that give rise to helical 3D superstructures.20Schiff base and related compounds, fascinating organic molecules, are often incorporated into macrocycles,MH) derived from an achiral precursor by Schiff base reaction. Further studies suggest that unexpected and stable core-in-hollow-shell-based organic particles can be directly precipitated from the reaction solution with high yield. Most interestingly, the size of the core–shell-based microspheres can be further smartly tuned by the addition of different volumes of water to the reaction solution. A single-crystal X-ray diffraction analysis of MH revealed that the self-assembly processes of the enantiomeric forms in the solid state play a key role in the formation of such core–shell-based nanoparticle structures.Here, we report the discovery of enantiomeric forms from a twisted macrocyclic host -1,3-dicarboximide (1) with 5,5′-methylene-bis-salicylaldehyde (2) in the presence of a Brønsted acid catalyst in methanol was first evaluated. In the case of the use of acid catalysts such as HCl, CH3COOH, CF3COOH, and HNO3, no precipitation was observed in the reaction, and multiple components that were produced were monitored by 1H NMR spectrometry. Concentrated H2SO4 was used as the catalyst to produce a large amount of light yellow precipitate from the reaction solution . Single-crystal X-ray diffraction provided unequivocal proof for the proposed structure and revealed that the macrocycle adopted a twisted three-dimensional (3D) helical structure in the solid state pyridine-2,6-dicarboxamide units, the dihedral angles were 155° and 149° between the central pyridine ring and the two benzene rings located on the two “arms”  and left-handed helical (M) macrocyclic molecule was linked alternately to form a twisted 1D zigzag chain through intermolecular CH···π interactions between the bridge methylene (sp3-carbon C20 and C54) protons and the phenyl rings. The distances for CH···center of π interactions were 2.74 and 2.84 Å respectively. When viewed along the crystallographic a axis, the frameworks had a single (P) helix (red) and a single (M) helix (blue). The two helical chains packed parallel face to face gave a racemic mixture of two enantiomeric helices , phenol ring carbon (C22) protons in the MH molecules, and solvent molecules (CH3COOC2H5)  . When vilecules, , and theP and M enantiomorphic twisted macrocyclic molecules from simple achiral starting materials via a one-step reaction.Generally, most of the supramolecular nanostructures have been constructed from macrocycles by their amphiphiles in solutions.Scanning electron microscopy (SEM) revealed that the precipitates were composed of microspheres with remarkable size uniformity, and that the contact edge between adjacent balls was clearly visible . The aveNotably, differential scanning calorimetry and thermogravimetry indicated that the pure organic core–shell microspheres displayed high thermal stability Fig. S10. In an e1 and 2, a light yellow precipitate almost immediately appeared as the reaction was stirred at room temperature upon addition of catalytic equivalents of sulfuric acid to the methanol solution. Therefore, the simple and facile fabrication conditions of the particles allowed realization of the construction of core–shell assemblies from the enantiomeric macrocyclic molecules. Direct collection of the precipitates from the solution may be done by controlling the reaction time.As mentioned previously, the core–shell particles were obtained directly from the precipitation from the Schiff base reaction. Actually, the precipitates formed fast in the organic reaction. As shown in a video ESI, which c1H NMR spectra of the precipitates which were produced at different reaction times displayed similar proton peaks with MH  A 2is stage . (3) Theis stage . (4) Somarticles . Consequ1H NMR spectra suggested that such different size appended spherical particles were fabricated from the assemblies of the MH molecules , which are complementary to each other and the orientation of which is stabilized by intermolecular C–H···π interactions. We propose that this plays a crucial role in the formation of the core–shell-based microsphere structure. The present work demonstrated a new phenomenon of organic particle growth, as well as providing a new viewpoint and application of enantiomeric structures. Further studies on the synthesis and properties of such twisted chiral macrocycles are currently being carried out.In summary, we have established a novel and facile method for the fabrication of pure homogeneous and highly thermostable organic nanoparticles with core–shell architecture. The synthesis was done N,N'-(6-Amino-2-pyridyl)-1,3-dicarboximide (1)2)1  and 2  were dissolved in methanol solution (30–40.0 mL) in a round-bottomed flask for 10 min, and conc. H2SO4 (20.0–30.0 μL) was added to the solution. The resulting mixture was stirred (800–1000 rpm) at room temperature (15–30 °C) for 3 h. Then, the reaction mixture was filtered to give the crude solid product, and the residue was washed with methanol three times to afford a light yellow solid compound MH . SEM and TEM images confirmed that the yellow solid is the core–shell based microparticles (∼1.22 μm). Mp > 250 °C. 1H NMR  δ (ppm) 12.77 , 10.36  scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>C–NH), 8.51 , 8.38 , 8.22 , 8.04  scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>CH), 7.39 , 7.33 , 7.14 , 6.97 , 6.87 , 6.13 , 3.58 . 13C NMR  δ 161.68, 159.27, 149.54, 139.19, 138.18, 133.68, 132.22, 131.41, 130.88, 128.65, 126.38, 125.54, 124.78, 118.96, 117.63, 115.92, 39.33, 29.33. ESI-MS (m/z): calcd for [M + H]+ [C68H51N10O8]+m/z = 1135.38; found m/z = 1135.3877.MH (50.0 mg) in a solution mixture . Crystal data for MH: (C68H50N10O8)·2CH3COOC2H5·CH2Cl2, Mr = 1394.30, triclinic, space group P1[combining macron], a = 13.861(5) Å, b = 15.962(6) Å, c = 17.258(6) Å, α = 69.871(6)°, β = 75.157(6)°, γ = 81.221(7)°, V = 3457(2) Å3, Z = 2, Dc = 1.340 g cm–3, R1 = 0.0749 (I > 2σ(I)), wR2 = 0.1296 , GoF = 0.804. CCDC ; 1856480.Single crystals were obtained from a crystal grown by evaporation of 1  and 2  were dissolved in 20.0 mL solution mixture  in a round-bottomed flask for 10 min, and conc. H2SO4 (10.0–15.0 μL) was added to the solution. The resulting mixture was stirred (800–1000 rpm) at room temperature (15–30 °C) for 3 h. Then, the reaction mixture was filtered to give the crude solid product, and the residue was washed with methanol three times to afford a light yellow solid compound MH . SEM and TEM images confirmed that the yellow solid is the core–shell based microparticles (∼990 nm and ∼635 nm). No change was observed for the size and the shape of the microspheres when more water was added to the reaction solution.The authors declare no competing financial interests.Supplementary informationClick here for additional data file.Supplementary movieClick here for additional data file.Crystal structure dataClick here for additional data file.


HIV/AIDS patients who fail to respond to first-line treatment protocols are switched to second-line ART. Identifying factors that influence effective second-line treatment can improve utilization of limited medical resources. We investigated the efficacy of long-term second-line anti-retroviral therapy (ART) after first-line virologic failure as well as the impact of non-nucleotide reverse transcriptase inhibitor (NNRTI), nucleotide reverse transcriptase inhibitor (NRTI), and protease inhibitor (PI) resistance mutations and medication adherence on ineffective viral suppression.+ T cell count, viral load, and drug resistance genotypes were quantified.A total of 120 patients were evaluated at 6, 12, 18, 24, and 48 months after initiation of second-line ART; a paper questionnaire was administered via a face-to-face interview and venous blood samples were collected. CD4+ T cell counts increased from 170 cells/μL (IQR 100–272) at baseline to 359 cells/μL (IQR 236–501) after 48 months of second-line treatment. Viral load (log10) decreased from 4.58 copies/mL (IQR 3.96–5.17) to 1.00 copies/mL (IQR 1.00–3.15). After switching to second-line ART, nine patients newly acquired the NRTI drug-resistant mutation, M184 V/I. No major PI resistance mutations were detected. Logistical regression analysis indicated that medication adherence < 90% in the previous month was associated with ineffective viral suppression; baseline high/low/moderate level resistance to 3TC/TDF was protective towards effective viral suppression.CD4Long-term second line ART was effective in the Henan region of China. Drug resistance mutations to NRTIs were detected in patients receiving second-line ART, suggesting that drug resistance surveillance should be continued to prevent the spread of resistant strains. Patient medication adherence supervision and management should be strengthened to improve the efficacy of antiviral treatment. In 2003, the “Four Free and One Care” antiretroviral therapy treatment (ART) policy was formally implemented in China . This po+ T cells in HIV-infected patients within 12 months of the switch to second-line treatment; HIV viral loads were lower than at the time of first-line failure in these patients as well [+ T cell counts were 531 after 36 months of second-line ART in patients from northeast China [+ T cell counts within 24 months of switching to second-line therapy [Although second-line treatment regimens include two NRTIs, 3TC and TDF, multidrug resistance and cross-resistance mutations acquired during first-line ART often results in only partial effectiveness of the NRTIs during second-line treatment. Some small sample size studies conducted in China have reported an increase in CD4 as well –11. Dingst China ; thus, l therapy –17. The  therapy . HoweverDrug selection pressure caused by the therapy regimen changes can lead to the accumulation of drug resistance mutations during long-term treatment. An increase in NRTI drug resistance was reported in patients after switching to the second line regimen . ProteasHere, we describe a prospective study of a cohort of patients with AIDS within 4 years of initiation of second-line treatment in Weishi County of Henan Province in China. We examined both the effectiveness of second-line treatment as well as associated factors which can impact treatment; importantly, we also analyzed whether cumulative NRTI resistance mutations acquired during first-line therapy failure affected the efficacy of second-line therapy. We also analyzed changes in PI, NRTI, and NNRTI resistance mutations after long-term second-line therapy.This research was approved by the Institutional Review Board of the first Affiliated Hospital of China Medical University. All participants signed informed consent statements.Patients were recruited from Henan between August 2009 and February 2010. Inclusion criteria were those listed in the Chinese National Free AIDS Antiretroviral Therapy Manual issued in 2009  and in t+ T cell count, HIV viral load, and “in-house” HIV genotype drug resistance test. During second-line therapy, “ineffective viral suppression” is defined as follows: plasma HIV-1 RNA level > 1000 copies/mL after 12 months of treatment; viral rebound which is defined as plasma HIV-1 RNA level > 1000 copies/mL after initial virologic suppression during second-line ART.After 6, 12, 18, 24, and 48 months of second-line therapy, we administered a paper questionnaire through a face-to-face interview and collected blood samples to assess patients’ CD4+ T lymphocyte count: a FACSCalibur flow cytometer  was used to enumerate CD4+, CD8+, and CD3+ T lymphocytes. Peripheral venous blood was collected and treated with EDTA-3 K anticoagulant; T lymphocyte counts were obtained within 24 h.CD47 copies/mL.Viral load: Plasma obtained from patients’ venous blood samples was stored at − 80 °C until testing. HIV viral load was determined using a COBAS AmpliPrep/COBAS TaqMan HIV-1 test . The detection range of the COBAS Assay for viral load quantification is 20–10HIV-1 viral RNA was extracted from 200 μl plasma with a QIAamp Viral RNA Mini Kit . One-step Reverse transcription-polymerase chain reaction (RT-PCR) was carried out using a TaKaRa One-step RNA PCR Kit . The HIV-1 pol gene was amplified using first round primers MAW26  and RT21 ; amplification was achieved using 1 cycle of 50°C for 30 min, 1 cycle of 94 °C for 5 min, and 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min 30 s, with a final extension of 72 °C for 10 min in the first round; and second round primers PRO-1  and RT4R ;amplification was achieved using 1 cycle of 94 °C for 5 min and 30 cycles of 94 °C for 30 s, 63 °C for 30 s, and 72 °C for 2 min 30 s, with a final extension of 72°Cfor 10 min [pol gene; negative control: specimens that are HIV-negative; blank control: amplification without template. The negative and positive controls were extracted, amplified, detected, and analyzed simultaneously with the research sample. Sequences were aligned using Contig software and edited using Bioedit software. The resulting sequences were submitted to the Stanford University HIV drug resistance database (http://hivdb.stanford.edu) for interpretation of putative drug resistance results.Drug-resistant mutation analysis: Mutations in the HIV polymerase gene, Pol, were screened for drug resistance mutations. r 10 min , 26. PosP < 0.05 for two-sided tests was defined as statistically significant.SPSS software (version 17.0) was used to analyze quantitative data. Categorical data was described by rate or ratio and analyzed by either Chi-square test or Fisher’s exact test. Continuous data was described by the mean and standard deviation if data met the hypothesis of normal distribution, otherwise median and inter-quartile ranges (IQRs) were used. Univariate and multivariate logistic regression were performed to identify possible associated factors that may have contributed to viral suppression. + T cell count  were 4.58 (3.96–5.17) copies/ml and 170 (100–272) cells/μl, respectively. After first-line failure, drug resistance rates to 3TC and TDF were 32.5% (38/117) and 48.7% (57/117), respectively  years, 60.8% (73/120) were male, 39.2% were female (47/120). 95.8% (115/120) of patients were infected through paid blood donation(blood transfusion). All HIV Pol sequences were subtype B interpreted by the Stanford University HIV drug resistance database. All 120 patients used first-line therapy regimens (2NRTIs + 1NNRTIs); first-line treatment duration was 5.0 (3.7–5.5)  years. At initiation of second-line therapy, patient baseline viral load (lg10)  and mean CD4Staff members regularly followed up with patients. After switching to second-line treatment, 102, 104, 82, 90, and 85 patients were retained for follow-up at 6, 12, 18, 24, and 48 months, respectively. Of the 16 patients who were lost to follow-up at 6 months, two continued second-line therapy according to drug receipt records from the CDC; however, we were unable to collect blood samples and therapy records from these two patients as they were away from Weishi county, Henan province at 6 months. We were able to retrieve blood samples and therapy information from these patients at 12 month. The number of patients who were lost to follow-up increased to 30 at 18 months; Another eight patients continued second-line therapy according to drug receipt records from the CDC; however, we were unable to collect blood samples and therapy records from these patients as they were away from Weishi county at the 18 month timepoint. We were able to obtain their blood samples and therapy information at the 24 month timepoint. Finally, 35 patients were lost to follow-up at 48 months.+ T cells increased from a baseline of 170 cells/μL (IQR 100–272) at initiation of second-line therapy to 359 cells/μL (IQR 236–501) after 48 months of treatment, which was statistically significant (P < 0.05) .During long-term second-line therapy, patients’ CD4 IQR 100–72 at ini10) decreased from an average baseline level of 4.58 copies/mL (IQR 3.96–5.17) to 1.00 copies/mL (IQR 1.00–3.15) after 48 months of second-line therapy. HIV viral load decreased significantly (P < 0.05) after several different durations of second-line treatment (Table P < 0.001). The proportion of patients with viral loads < 1000 copies/mL at 6, 12, 18, 24, and 48 months was 61.8% (63/102), 71.1% (74/104), 69.5% (57/82), 70.0% (63/90), and 75.3% (64/85), respectively. The proportion and frequency of patients with < 50 HIV-RNA copies/mL at 6, 12, 18, 24 and 48 months was 43.1% (44/102), 43.4% (45/104), 42.7% (35/82), 50% (45/90), and 67.1% (57/85), respectively.Viral load (lognt Table . The prosecond-line treatment were HIV subtype B as interpreted by the Stanford University HIV drug resistance database. Rates of drug resistance to NRTIs were 58.1% (68/117), 43.3% (13/30), 31.8% (7/22), 28.0% (7/25), 18.1% (2/11), 31.6% (6/19) at baseline, 6, 12, 18, 24, and 48 months, respectively. Rates of drug resistance rates to NNRTIs were 71.8% (84/117), 56.7% (17/30), 45.5% (10/22), 24.0% (6/25), 36.3% (4/11), and 36.8% (7/19) at baseline, 6, 12, 18, 24, and 48 months, respectively. No major resistance to PIs was detected in our study; minor drug resistance to the PIs, A71V/T and L10I, were detected during second-line therapy.In-house genotype drug resistance tests were performed on samples from patients whose viral loads were > 1000 copies/ml. All sequences obtained during After switching to second line ART, newly acquired NRTIs resistance mutations at position 184, which are associated with resistance to 3TC, were detected in nine HIV/AIDS patients. The M184 V/I mutation was detected in five patients, one patient, two patients, and one patient at the 6, 12, 18, and 48 month timepoints, respectively. Mutation K65R, associated with resistance to TDF, was found in four patients at baseline; however, after switching to second-line ART, K65R mutations were not detected. TAM  and D67N mutations, associated with resistance to TDF, were detected during second-line ART. NRTI drug-resistance mutations were not found in some patients from the baseline point to 48 months after beginning second-line treatment  medication adherence (the past month) below 90% (vs. 90–100%) ; 2) compliance unknown (vs. 90–100%) , these two factors were protective toward ineffective viral suppression. 3) baseline high level resistance to 3TC/TDF (vs. sensitive) ; and 4) baseline low/moderate level resistance to 3TC/TDF (vs sensitive)   of patients achieved viral suppression within 24 months in our study, which was similar to results reported from South Africa in which CD4+ T counts increased by an average of 177cells/L and 75% (74/99) of patients achieved a viral load < 1000 copies/mL within 24 months of beginning second line therapy [+ T counts increased to 189 cells/L and 75.3% (64/85) of patients achieved a viral load < 1000 copies/mL. These results indicate that long-term treatment was effective in our cohort.In our study, patients were recruited from Henan province in China; Henan was one of the earliest areas to begin free first-line ART for HIV-infected citizens and was the first to initiate second-line antiretroviral therapy as described in the National Free Antiretroviral Treatment Program (NFATP). During the 4 years of follow-up, patient CD4 therapy . We alsoDrug-resistance mutations were detected when the viral load reached > 1000 copies/ml during second-line ART follow-up; yet, our results show that newly acquired NRTI resistance mutations were few. The M184 V/I mutation was detected in nine patients after switching to second-line therapy, which can attributed to the use of 3TC in the program. Boyd et al. found that one patient newly acquired M184 V resistance mutations after 96 weeks second-line ART . TDF wasMajor PI resistance mutations were not detected using the “in-house” genotype drug resistance test. However, we obtained HIV Pol sequences using monoclonal methods; the results of a rootless radial tree generated via phylogenetic analysis at the two time points (baseline and after 48 months of second-line treatment) showed significant evidence of viral evolution, suggesting that resistance surveillance should be continued.After switching to the second-line regimen, 31 patients exhibited increased viral load; of these, 28 patients exhibited viral loads > 1000 copies/ml after at least 12 months of second-line therapy. We performed a logistical regression analysis of the data; our results indicated that medication adherence (in the past month) below 90%, compliance or when medication adherence was unknown was associated with ineffective viral suppression. These results are consistent with the work reported by Murphy et al. . In a stIn our cohort, baseline resistance to 3TC/TDF resulted from ineffectiveness of the second-line ART; which was similar to the research reported regarding second line treatment in Africa , 31. PatLong-term second-line antiviral therapy in the Henan region was effective. During the four years of second-line therapy, drug mutations conferring resistance to NRTIs were observed among patients receiving second-line ART, although major drug resistance mutations were not detected in the PI region. Drug resistance monitoring should be continued to prevent the spread of resistant strains. In addition, medication adherence supervision and management should be increased in order to improve the effectiveness of antiviral drugs.


Cancer is the leading cause of deaths around the world, especially in low- and middle- income countries. Pirarubicin (THP) is an effective drug for treatment of cancer, however, there still exists cardiotoxic effects of THP. Rutin is a kind of antioxidative compound extracted from plants, and might be a protective compound for cardiomyocytes. Phosphatidylinositol 3-hydroxy kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway is critical for cellular survival, proliferation and metabolism, and thus we speculated rutin might perform a protective role in cardiomyocytes via PI3K/AKT/mTOR signaling pathway. And in this experiment, we first established a cardiotoxicity model of THP in mice model and cell models, and then found that rutin treatment could increase the proliferation of cells at low concentration. Then we explored the possible mechanism of the protective effect of rutin using Western blotting, quantitative polymerase chain reaction (qPCR) and ELISA methods, and found that the activation of PI3K/AKT/mTOR/nuclear factor-κB (NF-κB) signaling pathway was increased, and expression of downstream molecules involved in antioxidative stress were also increased. We further noticed that concentration of angiogenesis promoting factors were also increased in medium of cultured cells. Thus, we speculated that rutin could increase the activation of PI3K/AKT/mTOR signaling pathway, further decrease the oxidative stress level via increasing the expression of antioxidative stress enzymes with the increasing concentration of angiogenesis promoting factors, resulting in the protective role in cardiomyocytes and cardiac function. Among them, more than a half of these cases will occur in low- and middle-income countries . Rutin c homolog . PhosphoH-DMEM (10569010) and FBS (10100147) were purchased from Thermo . THP (72496-41-4) and rutin (153-18-4) were purchased from Dahua Pharmaceutical Co., Ltd . Total RNA Extraction Kit (R1200-100T) and TaqMan One Step RT-qPCR Kit (T2210-200T) were purchased from Solarbio Science and Technology Co., Ltd . Anti-HIF-1α (hypoxia inducible factor-1α) (ab1), ROS1 (ab108492), SOD1 (ab13498), Thioredoxin (TRX) (ab26320), Peroxiredoxin1 (PRX1) (ab15571), TGF-β1 (ab64715), p-AKT (ab38449), AKT (ab179463), p-mTOR (ab109268), mTOR (ab2732), p-NF-κB (nuclear factor-κB) p65 (ab222494), NF-κB (ab207297), eNOS (ab76198) antibodies were purchased from Abcam. Angiopoietin 1 , FGF1 , VEGF (ab222510) and TGF-β  ELISA kits were purchased from Abcam. H9c2 (GNR 5) and Ea.hy926 (GNHu39) cells were purchased from Cell Resource Center of the Chinese Academy of Sciences.Twelve male wild-type (WT) C57BL/6 mice (20-week-old) were obtained from Laboratory Animal Center of the Academy of Military Medical Sciences. All mice were raised in a steady environment, which was at 25°C (temperature) and 40–70% humidity with a 12-h light and dark cycle. Mice were divided into four groups, including THP treatment group (TP), blank control group (NC), THP treatment with low-dose rutin group (TL) and THP treatment with high-dose rutin group (TH). Detailed information on mice is listed in 2 atmosphere. Cells were seeded into a 100-mm plate at a concentration of 1 × 106, then cultured for 24 h before performing the following experiments. Cells were seeded into each well of a 96-well plate, and cultured for 24 h. According to previous study, H9c2 and Ea.hy926 cells were first treated with 5 μM THP for 24 h [Sample − ODBlank)/(ODControl − ODBlank) × 100%. Each experiment was repeated for three times independently.H9c2 and Ea.hy 9926 cells were cultured in H–DMEM containing 10% FBS with a humid, 37°C supplied with 5% COCardiac function of mice was measured using echocardiography (Vevo 2100). Mice in each group were first sedated with 1.5% isoflurane anesthesia through nose cone, until successful anesthesia. Then, chest hair was removed, and cardiac function was detected using a 15-MHz probe which was placed in the parasternal, short-axis orientation. Parameters acquired under M mode were analyzed for left ventricular (LV) short axis shortening (LVFS%), LV ejection fraction (LVEF%) and LV in LV short axis (SAX) and LV long axis (PSLAX) views.RNA extraction was performed according to the protocol. Briefly, cells were lysed with lysis buffer, and incubated at room temperature for 5 min. Then, samples were mixed with chloroform with acute shaking for 15 s and then incubated at room temperature for 5 min. After incubation, samples were centrifuged at 12000 rpm for 10 min at 4°C, samples in water phase were collected after centrifugation. Samples were absorbed into adsorption column after centrifugation, and eluted with elution buffer after washing with washing buffer. Concentration of RNA was determined using Nano-drop 2000. Equal amount of RNA was used to perform quantitative polymerase chain reaction (qPCR) assay. Briefly, reaction mixture was made according to the protocol, and primers used are listed as follows: for H9c2 cells: caspase-3: Forward: 5′-GAGCTTGGAACGCGAAGAAA-3′, Reverse: 5′-AGTCCATCGACTTGCTTCCA-3′; caspase-9: Forward: 5′-CCACCTTCCCAGGTTTTGTCT-3′, Reverse: 5′-TCTCAGAAACAGCATTGGCGA-3′; Bax: Forward: 5′-GTTTACCTACTCGTCTCTGGTAC-3′, Reverse: 5′-CCTTATCCCAATACGTGTCGACATCAT-3′; Bcl-2: Forward: 5′-TGACGAGACCTCTATGCCGACTC-3′, Reverse: 5′-ACTTCTCATCGTACTCCCCTG-3′. And primers for Ea.hy 926 cells: caspase-3: Forward: 5′-AAATACCAGTGGAGGCCCGACTT-3′, Reverse: 5′-AAGCTTGTCGGCATACTGTTTCA-3′; caspase-9: Forward: 5′-TCTGGAGGATTTGGTGATGTC-3′, Reverse: 5′-CATTTTCTTGGCATCAGGTC-3′; Bax: Forward: 5′-CGACTACCACTTCGCCGAC-3′, Reverse: 5′-CCGTTCTGTCACTAACTTATGTTTT-3′; Bcl-2: Forward: 5′-GCTTCCCAGTCAGTCCAACA-3′; Reverse: 5′-TAACTGGTGCCTGTAGTATC-3′.CT) was calculated with normalized fluorescence signal after generation of real-time amplification. Relative analysis on expression of each gene was performed using 2−ΔΔCq method according to previous study [The reaction was performed under following conditions: reverse transcription: 50°C for 20 min; denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 30 s, extension at 72°C for 45 s, these three steps were repeated for 45 cycles. The threshold cycle  on ice with ultra-sonication for 10 min. Lysis buffer was centrifuged at 12000 rpm (4°C) for 10 min, then supernatant was collected and removed into a new tube. Concentration of proteins was measured using BCA assay.Sixty micrograms of protein samples were used to perform 10% SDS/PAGE electrophoresis. After electrophoresis, proteins were transferred on to a 0.22-µm nitrocellulose membrane using a semi-dry electroblotter. Membranes were incubated with primary antibodies (1:1000) overnight at 4°C, and then incubated with secondary antibody for 1 h at room temperature (1:5000). Concentration of proteins was detected using ECL immunoblotting reagent. Gray values of bands were quantitated using Scion Image software. GAPDH was used as an internal control. Each experiment was repeated for three times independently.P-value <0.05 was set as a statistically significant difference.Data were presented as means ± S.E.M., each experiment was repeated for three times. Differences between groups were analyzed using one-way ANOVA followed with Tukey–Kramer post hoc test. P<0.05), while not significantly changed in TH group, and compared with NC group, the LVFS% and LVEF% was significantly decreased in TH group (P<0.05) both in SAX and PSLAX views. Compared with TP group, the LV mass/body weight was significantly decreased in other three groups (P<0.05), and compared with NC group, the ratio was significantly increased in TL and TH groups (P<0.05).As shown in P<0.05), and 50 μM rutin treatment could reduce the viabilty rate of H9c2 and Ea.hy 926 cells while only significantly of Ea.hy 926 cells. According to the result of MTT assay, we decided to use 50 μM as low concentration of rutin and 70 μM rutin as high concentration of rutin to perform following experiments. As shown in P<0.05), and low concentration of rutin treatment could significantly incerase the proliferation of cells while high concentration of rutin treatment could significantly decerase the proliferation of cells (P<0.05).In order to screen the suitable concentration of THP and rutin for H9c2 and Ea.hy926 cells, we first performed the MTT assay on THP and rutin alone. As shown in P<0.05), and compared with NC group, the expression was significantly decreased in TH group (P<0.05). The ratio of p-mTOR/mTOR in these groups were 0.32 ± 0.02, 0.88 ± 0.07, 1.22 ± 0.09, and 0.89 ± 0.07. The ratio of p-mTOR/mTOR was significantly increased in all groups compared with TP group (P<0.05), and was significantly increased in TL group compared with NC group (P<0.05). The ratio of p-NF-κB p65/NF-κB in these groups were 0.90 ± 0.07, 1.42 ± 0.11, 1.60 ± 0.12, and 0.87 ± 0.07. Compared with TP group, the ratio of p-NF-κB p65/NF-κB was significantly increased in NC and TL groups (P<0.05), and was significantly decreased in TH group compared with NC group (P<0.05). And the expression of eNOS were 0.63 ± 0.05, 1.23 ± 0.09, 1.47 ± 0.11, and 1.37 ± 0.11. Compared with TP group, the expression of eNOS were significantly increased in all group (P<0.05). And in Ea.hy 926 cells, the ratio of p-AKT/AKT in TP, NC, TL, and TH groups were 0.66 ± 0.04, 0.96 ± 0.06, 0.87 ± 0.06, and 0.66 ± 0.04. Compared with TP group, the ratio of p-AKT/AKT was significantly increased in NC and TL groups (P<0.05), and was significantly decreased in TH group compared with NC group (P<0.05). The ratio of p-mTOR/mTOR in these groups were 0.15 ± 0.01, 0.81 ± 0.05, 0.86 ± 0.06, and 0.82 ± 0.05. Compared with TP group, the ratio of p-mTOR/mTOR was significantly increased in other three groups (P<0.05). The ratio of p-NF-κB p65/NF-κB in these groups were 0.39 ± 0.03, 0.85 ± 0.06, 1.03 ± 0.07, and 0.46 ± 0.03. Compared with TP group, the ratio of p-NF-κB p65/NF-κB was significantly increased in other three groups (P<0.05), and compared with NC group, the ratio was significantly increased in TL group (P<0.05) and significantly decreased in TH group (P<0.05). And the expression of eNOS were 0.52 ± 0.03, 0.93 ± 0.06, 1.08 ± 0.07, and 0.76 ± 0.05. Compared with TP group, the expression of eNOS was significantly increased in other three groups (P<0.05), and compared with NC group, the expression of eNOS was significantly increased in TL group (P<0.05) and significantly decreased in TH group (P<0.05).As shown in P<0.05), and compared with NC group, the expression was significantly decreased in TL group (P<0.05) and significantly increased in TH group (P<0.05). The expression of ROS1 in these groups were 0.50 ± 0.04, 0.99 ± 0.08, 0.94 ± 0.07, and 0.80 ± 0.06. Compared with TP group, the expression of ROS1 was significantly increased in all groups (P<0.05), and compared with NC group, the expression of ROS1 was significantly decreased in TH group (P<0.05). The expression of TRX in these groups were 0.40 ± 0.03, 0.84 ± 0.06, 1.14 ± 0.09, and 0.81 ± 0.06. Compared with TP group, the expression of TRX was significantly increased in all groups (P<0.05), and compared with NC group, the expression of TRX was significantly increased in TL group (P<0.05). The expression of PRX1 in these groups were 0.52 ± 0.04, 0.91 ± 0.07, 0.99 ± 0.08, and 0.87 ± 0.07. And the expressions of PRX1 were significantly increased in all groups compared with TP group (P<0.05). And in Ea.hy 926 cells (P<0.05), and compared with NC group, the expression was significantly decreased in TL group (P<0.05) and significantly increased in TH group (P<0.05). The expression of ROS1 in these groups were 0.46 ± 0.03, 0.93 ± 0.06, 1.20 ± 0.08, and 0.55 ± 0.04. Compared with TP group, the expression of ROS1 was significantly increased in other three groups (P<0.05), and compared with NC group, the expression level was significantly increased in TL group (P<0.05) and significantly decreased in TH group (P<0.05). The expression of TRX in these groups were 0.34 ± 0.02, 0.65 ± 0.04, 0.86 ± 0.06, and 0.28 ± 0.02. The expression level of TRX presented a similar trend as ROS1. The expression of PRX1 in these groups were 0.48 ± 0.03, 0.92 ± 0.06, 0.94 ± 0.06, and 0.56 ± 0.04. The expression of PRX1 was significantly increased in other three groups compared with TP group (P<0.05), and the expression of PRX1 was significantly decreased in TH group (P<0.05) compared with NC group.In H9c2 cells , the exp26 cells , the expP<0.05), while the expression of these molecules were not significantly changed between NC and TL groups.In H9c2 cells, the expression levels of Bcl-2 in these groups were 0.34 ± 0.02, 0.82 ± 0.06, 0.76 ± 0.04, and 0.51 ± 0.03 A. The exP<0.05), and compared with NC group, the concentration of these factors were significantly decreased in TL and TH groups (P<0.05).In H9c2 cells, the concentrations of VEGF in cultured medium in TP, NC, TL, and TH groups were 158.8 ± 13.2, 242.3 ± 16.2, 233.3 ± 14.8, and 193.5 ± 14.1 pg/ml A. The coAccording to previous study, more than a quarter people will be affected by cancer worldwide, and surgical removal remains a mainstay in most solid cancers . MicroenAKT is known as serine/threonine kinases, which belongs to general class of AMP/GMP kinase and PKC subfamily of proteins (AGC) kinases . Cell gr2O2 through the alternate reduction. Redox homeostasis is determined by the balance between ROS production and scavenging capacity, and dysregulation in redox homeostasis would cause cellular damage, including lipoperoxidation, nucleic acid, and structure of proteins, leading to the occurrence of neurodegenerative and cardiovascular diseases. Peroxiredoxin, thioredoxin, and thioredoxin reductase form the mammalian thioredoxin system play an important role in protecting against oxidative stress injury. Prx1 is non-seleno peroxidase, which could reduce hydrogen peroxide, organic hydroperoxides and possibly peroxynitrite, forming critical antioxidants [As is well known, oxidative stress response is the main target of rutin, thus we detected the expression of critical proteins in oxidative stress response. HIF-1α is the first identified mediator response to hypoxia in cells under oxidative stress . ROS1 isoxidants . TRX is oxidants ,34. Besioxidants . In thisc from the mitochondria, further activates caspase-9 [Balance between the members of Bcl-2 family proteins is critical for the survival of cells, changing in the expression of Bcl-2 family proteins could lead to the occurrence of apoptosis. Proteins in Bcl-2 family were divided into three groups: anti-apoptotic proteins , pro-apoptotic proteins  and BH-3 only proteins . In this experiment, the increase in expression of Bcl-2 and decrease in the expression of Bax could increase the survival of cardiomyocytes, presenting the protective role of low-concentration rutin treatment. Caspase cascade kinases and Bcl-2 family proteins play important role in apoptosis process. Caspase-3 is the major executive caspase in apoptosis, especially in mitochondria-dependent apoptosis pathway . Caspaseaspase-9 , activataspase-9 . In thisVEGF is a well-known endothelial-specific mitogen, which could induce the physiological and pathological angiogenesis and is controlling the regulation of vascular permeability , besidesin vivo and in vitro experiment, and found that low concentration of rutin treatment could increase the proliferation. Then we found that the activation of PI3K/AKT/mTOR/NF-κB signaling pathway was increased, and expression of antioxidative stress were also increased using Western blotting analysis. We further noticed that concentration of angiogenesis promoting factors were also increased in medium of cultured cells. Thus, we speculated that rutin could increase the activation of PI3K/AKT/mTOR signaling pathway, further decrease the oxidative stress level via increasing the expression of antioxidative stress enzymes with the increase in concentration of angiogenesis promoting factors, resulting in the protective role in cardiomyocytes and cardiac function.In the present study, we first established a cardiotoxicity model of THP using


In humans, three major metabolites were observed: two glucuronides and one sulfate. By contrast, only one major glucuronide was observed in rats. This strong hepatic clearance of ε-viniferin in human and rat could explain its poor bioavailability and could help to characterize its active metabolites.ε-Viniferin is a resveratrol dimer that possesses antioxidant or anti-inflammatory activities. However little is known about the metabolism of this oligostilbene. This study was thus undertaken as a first approach to identify and characterize the metabolites of ε-viniferin and to describe the kinetic profile of their appearance in humans and rats. The glucuronides and sulfates of ε-viniferin were first obtained by chemical hemi-synthesis and were fully characterized by UPLC-MS and NMR spectroscopy. Then, ε-viniferin was incubated with human or rat S9 liver fractions that led to the formation of four glucuronoconjugates and four sulfoconjugates. In both species, ε-viniferin was subjected to an intense metabolism as 70 to 80% of the molecule was converted to glucuronides and sulfates. In humans, the hepatic clearance of ε-viniferin (V Stilbenes are naturally-occurring phenolic compounds mainly found in grapes and wine. One of them, resveratrol, has been widely studied. It has been reported to act preventively against human diseases ,2,3,4, pO-glucuronide and resveratrol-3-O-sulfate − at m/z 533, corresponding to the ε-viniferin mono-sulfate.First, the glucuronide and sulfate metabolites of ε-viniferin were hemi-synthesised according to the material and methods section. We chose this approach for several reasons. First, it allowed us to obtain all the complete metabolites likely to be produced in vivo and, second, it allowed us to obtain a sufficient quantity of metabolites for their identification at a lower cost than by using classical biosynthesis with S9 liver fractions. These metabolites were then purified and identified through mass spectrometry and NMR spectroscopy. 1, corresponding to MG1, shows NOEs between the anomeric proton (H-1′) and the H-12b and H-14b. In addition, the H-12b and H-14b protons were shifted upfield compared to that of ε-viniferin. These data indicate that glucuronic acid is attached to the C-13b carbon. Concerning 2 (MG2), NOE correlation between the anomeric proton and H-3b/5b protons indicated that glucuronic acid is attached to the C-4b carbon. This hypothesis was confirmed by the chemical shift variations of H-2a/6a and H-3b/5b protons. The NOESY spectrum of 3 (MG3) showed a correlation between the anomeric proton and H-3a/5a, indicating the position of glucuronic acid. Finally, the NOEs between H-1′ and H-10a/H-12a protons and the chemical shift variations allowed the identification of 4 (MG4). Like the glucuronide derivatives, the sulfate metabolites 5, 6, 7, and 8  were based on chemical shift variations of ε-viniferin. The chemical structures of ε-viniferin and its metabolites are shown in After incubation of ε-viniferin (50 µM) for 40 min at 37 °C with either human a. These In humans, incubation of ε-viniferin with UDPGA showed the presence of four mono-glucuronides, MG1, MG2, MG3, and MG4. In rat, incubation of ε-viniferin with UDPGA revealed the presence of four mono-glucuronides, namely MG1, MG2, MG3 and MG4 . In humans, however, the Vmax/Km values for glucuronidation and sulfation were quite similar .To further understand the contribution of glucuronidation or sulfation metabolic pathways to ε-viniferin in vitro clearance, kinetic studies were performed using the liver S9 fraction. The kinetic parameters are summarized in max) for the glucuronides was observed with MG1 in both species  of 1:15 in rats and 1:1.5 in humans. This interspecies difference was already observed in the formation of resveratrol glucuronides. Indeed, it has been shown that resveratrol can be metabolized by human and rat liver microsomes into resveratrol-3- in rats . We founRecent studies reported that the oral bioavailability of δ-viniferin and ε-viniferin are 2.3% and 0.77%, respectively ,24. Thusε-Viniferin is known to have a variety of pharmacological effects, such as anti-inflammatory , anti-oxOne could question whether the native form of viniferin or its metabolites could have pharmacological properties given its extremely low bioavailability. The beneficial health effects of polyphenols have been demonstrated for several years both in human and animal studies. The discrepancy between the beneficial health effects and the poor bioavailability due to extensive biotransformation may be accounted for the pharmacological action of the metabolites. For resveratrol, there is evidence that its conjugates could elicit biological activities. Indeed, sulfoconjugates can induce quinone reductase, inhibit cyclooxygenase ,38 and a2 were purchased from Sigma-Aldrich . Human liver S9 and rat liver S9 fractions were purchased from Biopredic International . Acetonitrile and methanol were purchased from Thermo Fisher Scientific .ε-Viniferin was produced and purified in our laboratory . Pyridine, chlorosulfonic acid, acetobromo-glucuronic acid methyl-ester, sodium hydroxide (NaOH), uridine 5′-diphospho-glucuronic acid trisodium salt (UDPGA), alamethicin, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), dithiothreitol (DTT), trifluoroacetic acid (TFA), formic acid (FA), and MgClTo a chilled (−5 °C) solution of ε-viniferin  in 5 mL of pyridine, chlorosulfonic acid  was added dropwise. When the addition was complete, the reaction was placed at room temperature for 1 h. Then acidified water (0.5% FA) was added in order to reach a pH of 3 and the mixture was evaporated. The solid obtained was then re-dissolved in water (2 mL) prior to purification by semi-preparative HPLC-DAD to furnish after freeze-drying MS1 , MS2 , MS3 , and MS4 .O-glucuronidation of the previously purified ε-viniferin using acetobromo-glucuronic acid methyl ester in alkaline conditions. ε-Viniferin  was dissolved in dry ethanol (5 mL) and NaOH  was added. The solution was then placed at 50 °C for 30 min before the addition of acetobromo-glucuronic acid methyl ester  also dissolved in dry ethanol (5 mL). The reaction mixture was kept at 50 °C and in the dark for 4 h. Then after cooling down, acidified water (0.5% FA) was added in order to reach a pH of 3 and the mixture was evaporated. The solid obtained was then re-dissolved in water (2 mL) prior to purification by semi-preparative HPLC-DAD to furnish after freeze-drying MG1 , MG2 , MG3 , and MG4 .Monoglucuronides of ε-viniferin were obtained by chemical Samples from chemical hemi-synthesis were separated by HPLC equipped with a binary pump, an UV-VIS detector , and Prontosil C18 column . Detection was carried out at 320 nm.2O 0.025% TFA); solvent B (acetonitrile 0.025% TFA); gradient program 0–4 min, 10% B; 4–9 min, 10–20% B; 9–13 min, 20–30% B; 13–17 min, 30% B; 17–21 min, 30–35%; 21–30 min, 35–60%; 30–38 min, 60–100%; 38–44 min, 100%  and solvent B (acetonitrile 0.1% FA) by the following gradient program: 0–1.7 min, 10% B; 1.7–3.4 min, 10–20% B; 3.4–5.1 min, 20–30% B; 5.1–7.8 min, 30% B; 7.8–8.5 min, 30–35% B; 8.5–11.9 min, 35–60% B; 11.9–15.3 min, 60–100% B; 15.3–17 min, 100% B; 17–17.3 min, 100–10% B. The flow rate was set to 0.4 mL/min and the UV detector was set at the wavelength 320 nm. Total ion chromatograms were obtained using negative mode with a range of m/z 130–1400. The parameters were: capillary voltage, +4 kV; nebulizer pressure, 40 psi; dry gas, 10 L/min; dry temperature, 365 °C. Data analysis was performed with Bruker Data Analysis 3.2 . Metabolite concentrations were expressed as an equivalent of the ε-viniferin standard curve.Samples from either chemical hemi-synthesis or metabolic reactions were carried using a 1290 Infinity UPLC . The UPLC was coupled to an Esquire 3000 Plus ion trap mass spectrometer using an ESI source . 2 µL were injected into an Agilent SB-C18 column . Samples were eluted with solvent A (Hd6) at 2.05 ppm. Assignments were performed with COSY and ROESY experiments.NMR experiments were performed on a Bruker Avance III 600 MHz  spectrometer equipped with a 5-mm triple-resonance probe. Chemical shifts were calibrated using residual solvent signal (acetone-2 (5 mM) in a final volume of 100 µL. After 40 min at 37 °C, the reaction was stopped with 100 µL of methanol to precipitate the protein. Glucuronidation was investigated by incubating human or rat liver S9 fractions (2 mg/mL) with ε-viniferin at different concentrations (5 to 300 µM) in Tris-HCl buffer , in the presence of UDPGA (1 mM), alamethicin (25 µg/mL), and MgCl2 (5 mM) in a final volume of 100 µL. After 40 min at 37 °C, the reaction was stopped with 100 µL of methanol to precipitate the protein. Samples from sulfation and glucuronidation were then centrifuged for 30 min , and the resultant supernatants were analyzed using UPLC-DAD-MS as described above. The kinetic model that was used to analyze the results was substrate inhibition. Kinetic parameters, i.e., Vmax and Km were obtained using GraphPad Prism software . Results are expressed as mean ± sem of three independent experiments.The incubation conditions were optimized by preliminary experiments to ensure the formation rates of metabolites were linear over the incubation time and at the concentration of protein added. Sulfation was investigated by incubating human or rat liver S9 fractions (2 mg/mL) with ε-viniferin at different concentrations (5 to 300 µM) in Tris-HCl buffer , in the presence of PAPS (1 mM), DTT (10 mM), MgClmax: maximal velocity; Km: substrate affinity constant; [S]: substrate concentration; Ki: substrate inhibition constant.VIn conclusion, we provide evidence of a strong in vitro hepatic clearance of ε-viniferin and have characterized its metabolite profile in humans and rats. Glucuronidation and sulfation are both involved in humans to a similar degree whereas glucuronidation is the main pathway in rats. This suggests that rats are not the best animal model to study ε-viniferin metabolism in humans. Nevertheless, pharmacokinetic studies play a pivotal role in drug development and rat studies are essential. The beneficial health effects of polyphenols in vivo are now well known despite their low bioavailability, which might be partly due to their intense metabolism. Therefore, our findings underline the need to characterize the active metabolites of ε-viniferin, because this might explain differences in its beneficial health effects between species according to its metabolism.


Leucetta sp., led to the discovery of a pair of solvolysis adducts, leucettazoles A1 (1a) and B1 (2a), as artifacts of an unprecedented family of 15-membered macrocyclic alkaloid dimers featuring a pair of imino bridged 2-aminoimidazoles, together with a putative monomeric precursor, leucettazine A (3). The dimeric alkaloids 1a and 2a, and monomer 3, were identified by detailed spectroscopic analysis, supported by chemical transformations, analytical mass spectrometry, and biosynthetic considerations. Global natural product social networking (GNPS) molecular analysis of crude sponge extracts and solvent partitions, supported by single ion extraction (SIE) and diagnostic MS/MS fragmentations, revealed the associated natural products, leucettazoles A (1) and B (2). This study highlights that the study of natural product artifacts can be useful, and can on occasion serve as a pathway to discover cryptic new classes of natural products.Chemical analysis of a southern Australian sponge,  Marine sponges are a remarkable source of structurally diverse natural products. For example, over several decades, our investigation of southern Australian marine sponges revealed a wealth of natural product classes, many unique to marine sponges. These include the terpenyls luffarins  and trunHalichondria/Geodia sp. sponge complex ..3) dimerD) measurements were acquired on a JASCO P-1010 polarimeter in a 100 × 2 mm cell at room temperature (22 °C). Ultraviolet–visible (UV–Vis) spectra were obtained on a Cary 50 UV–visible spectrophotometer. NMR spectra were obtained on a Bruker Avance DRX600 spectrometer equipped with either a 5-mm PASEL 1H/D-13C Z-Gradient probe or 5-mm CPTCI 1H/19F-13C/15N/DZ-Gradient cryoprobe, controlled by TopSpin 2.1 software. Chemical shifts (ppm) were referenced internally against residual solvent signals . High-resolution electrospray ionization mass spectra (HRESIMS) were obtained on a Bruker micrOTOF mass spectrometer by direct infusion in MeCN at 3 μL/min using sodium formate clusters as an internal calibrant. HPLC–MS data were acquired using an Agilent 1100 series separation module equipped with a diode-array multiple wavelength detector coupled to an Agilent 1100 series LC/mass selective detector (MSD), operating in positive and negative modes using electrospray ionization (ESI) mode. Ultra-high-performance liquid chromatography (UHPLC) was performed on an Agilent 1290 infinity UHPLC system composed of a 1290 infinity quaternary pump, thermostat, autosampler, and photodiode-array detector. Analytical, semi-preparative, and preparative HPLC analyses were performed on Agilent 1100 series LC modules, with corresponding detectors and fraction collectors. All solvents used for HPLC separation and purification were chromatographic grade.Specific optical rotation ([α]Leucetta sp. (CMB-01047), was collected in 1990 by hand (SCUBA) from submerged shallow water rock platforms off Flinders, Victoria, Australia. The sponge material was cooled to 0 °C shortly after collection, and transported to the laboratory, where it was documented, diced, and steeped in EtOH at −20 °C for long-term storage. The taxonomic description of the specimen is as follows: Leucetta sp. ; growth form—irregular massive; texture—harsh friable; oscules—small scattered; surface—translucent porous; spicules—triactines regular, equiangular, size range (rays 10–120 μm); ectosome—indistinct from choanosome; choanosome—leuconoid, densely spiculose, lacunose, little collagen, exclusively free spicules, solid body; color in EtOH—brown. A voucher sample was deposited with Museum Victoria (Registry No. MVF222666).A specimen of marine sponge, Leucetta sp. CMB-01047 was decanted, concentrated in vacuo, and partitioned between H2O and n-BuOH. The n-BuOH-soluble material (731.2 mg) was concentrated in vacuo, subjected to sequential solvent trituration, and concentrated in vacuo to yield materials soluble in n-hexane (42.0 mg), CH2Cl2 (21.9 mg), and MeOH (270.4 mg). The MeOH solubles were subjected to preparative HPLC  for 3.0 min) to afford leucettazole A1 (1a) , leucettazole B1 (2a) , and a mixture containing leucettazole A (1) and leucettazine A (3) (3.5 mg). Fractionation of the latter mixture by semi-preparative HPLC  yielded pure leucettazine A (3) . Note that purification using reverse-phase HPLC utilizing TFA as a modifier yielded compounds as TFA salts. The percentage yields were calculated as the estimated weight/weight percentage of each compound in the crude EtOH extract.A portion (300 mL) of the crude aqueous EtOH extract of Leucettazole A (1): light-yellow powder, as a mixture with 3; UV (MeCN) λmax 318 nm, 284 nm; 1D and 2D NMR (DMSO-d6), see m/z 439.1361 .Leucettazole A1 (1a): light-yellow powder; [α]D22 0.0 ; UV (MeOH) λmax 318 nm (ε 56293), 284 nm (ε 4335); 1D and 2D NMR (DMSO-d6), see m/z 467.1677 .Leucettazole B1 (2a): light-yellow powder; [α]D22 0.0 ; UV (MeOH) λmax 318 nm (ε 56295), 284 nm (ε 4725); 1D and 2D NMR (DMSO-d6), see m/z 481.1832 .Leucettazine A (3): light-yellow powder; 1D and 2D NMR (DMSO-d6), see m/z 220.1084 .Leucetta sp. (CMB-01047) were concentrated in vacuo, re-dissolved in MeOH, and heated overnight in sealed vials at 40 °C and 60 °C. MS/MS data were acquired by subjecting an aliquot of CMB-01047 crude extract (1 μL) to UHPLC-QTOF analysis. UHPLC conditions involved a 0.5-mL/min gradient elution from 10% CH3CN/H2O to 100% CH3CN over a period of 4.5 min, with constant 0.1% formic acid, through an Agilent SB-C8 1.8-μm, 2.1 × 50 mm column with column compartment temperature set at 40 °C. The source parameters were as follows: electrospray positive ionization; mass range of m/z 50–1700; MS scan rate, 3× per second; MS/MS scan rate, 3× per second; fixed collision energy, 40 eV; source gas temperature, 325 °C; gas flow, 10 L/min; and nebulizer, 20 psig. The scan source parameters were as follows: VCap 4000; fragmentor 180; skimmer 45; and octopole RF Peak 750. SIE at m/z 453.1500 (peak width ± 0.05 amu) revealed a species eluting at a retention time of 0.82 min (m/z 453.1516) with a molecular formula , consistent with the proposed structure for the methanolysis adduct leucettazole A2 (1b). MS/MS analysis of 1b revealed diagnostic fragmentation consisting of the loss of OMe (m/z 421.1235)  of EtOH crude extract of 0.82 min . Further21.1235) .1a) recovered from Leucetta sp. (CMB-01047) were dissolved in either MeOH, MeCN, or 0.02% TFA/H2O and heated (12 h) in sealed vials at 40 °C and 60 °C. An aliquot was subjected to UPLC-QTOF analysis under the method mentioned above. A new peak attributed to leucettazole A2 (1b) was observed when 1a was treated with MeOH (1) was observed when 1a was treated with 0.02% TFA/H2O  of leucettazole A1 ( TFA/H2O .Leucetta sp. (CMB-01047) was concentrated in vacuo, reconstituted in 50% MeCN/H2O, and subjected to UHLPC-QTOF-MS/MS analysis with UHPLC conditions as mentioned previously. A UHPLC-QTOF analysis using SIE at m/z 453.1500 (peak width ± 0.05 amu) detected a species isomeric with leucettazole A2 (1b), but eluting at a different retention time  . MS/MS analysis of 2 revealed diagnostic fragmentation consisting of the loss of OH (m/z 436.1249)  of crude EtOH extract of 36.1249) .n-butanol solubles (1 μL) to UHPLC-QTOF analysis. UHPLC conditions involved a 0.5-mL/min gradient elution from 10% CH3CN/H2O to 100% CH3CN over a period of 4.5 min, with constant 0.1% formic acid, through an Agilent SB-C8 1.8-μm, 2.1 × 50 mm column. The source parameters were as follows: electrospray positive ionization; mass range of m/z 50–1700; scan rate, 10× per second; MS/MS scan rate, 3× per second; fixed collision energy, 40 eV; source gas temperature, 325 °C; gas flow, 10 L/min; and nebulizer, 20 psig. The scan source parameters were as follows: VCap 4000; fragmentor 100; skimmer 45; and octopole RF Peak 750. The acquired MS/MS data were converted from an Agilent MassHunter data file (.d) to the mzXML file format using the software MS-Convert [MS/MS data were acquired by subjecting an aliquot of CMB-01047 -Convert . A minim-Convert  and visu4–105 colony-forming units (CFU)/mL. The compounds to be tested were dissolved in DMSO and diluted with H2O to give a 600 µM stock solution (20% DMSO), which was serially diluted with 20% DMSO to give concentrations from 600 µM to 0.2 µM in 20% DMSO. An aliquot (10 µL) of each dilution was transferred to a 96-well microtiter plate and freshly prepared microbial broth (190 µL) was added to each well to give final concentrations of 30–0.01 µM in 1% DMSO. The plates were incubated at 37 °C for 24 h, and the optical density of each well was measured spectrophotometrically at 600 nm using POLARstar Omega plate . Each test compound was screened against the Gram-negative bacteria Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 10145 and the Gram-positive bacteria Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633. Rifampicin was used as a positive control (40 µg/mL in 10% DMSO). For Pseudomonasaeruginosa, a mixture (1:1) of rifampicin and ampicillin (40 µg/mL in 10% DMSO) was used as a positive control. The IC50 value was calculated as the concentration of the compound or antibiotic required for 50% inhibition of the bacterial cells using Prism 7.0 . The antibacterial results are presented in The bacterium to be tested was streaked onto a tryptic soy agar plate and was incubated at 37 °C for 24 h. One colony was then transferred to fresh tryptic soy broth (15 mL) and the cell density was adjusted to 10Candida albicans ATCC 10231 was streaked onto a Sabouraud agar plate and was incubated at 37 °C for 48 h. One colony was then transferred to fresh Sabouraud broth (15 mL) and the cell density adjusted to 104–105 CFU/mL. Test compounds were dissolved in DMSO and diluted with H2O to give a 600 µM stock solution (20% DMSO), which was serially diluted with 20% DMSO to give concentrations from 600 µM to 0.2 µM in 20% DMSO. An aliquot (10 µL) of each dilution was transferred to a 96-well microtiter plate, and freshly prepared fungal broth (190 µL) was added to each well to give final concentrations of 30–0.01 µM in 1% DMSO. The plates were incubated at 37 °C for 24 h and the optical density of each well was measured spectrophotometrically at 600 nm using POLARstar Omega plate . Amphotericin B was used as a positive control (30 µg/mL in 10% DMSO). Where relevant, IC50 values were calculated as the concentration of the compound or antifungal drug required for 50% inhibition of the fungal cells using Prism 7.0 . The antifungal results are presented in The fungus l-glutamine (2 mM), penicillin (100 unit/mL), and streptomycin (100 μg/mL), in a humidified 37 °C incubator supplied with 5% CO2. Briefly, cells were harvested with trypsin and dispensed into 96-well microtiter assay plates at 3000 cells/well after which they were incubated for 18 h at 37 °C with 5% CO2 . Test compounds were dissolved in 20% DMSO in phosphate-buffered saline (PBS) (v/v), and aliquots (10 μL) were applied to cells over a series of final concentrations ranging from 10 nM to 30 μM. After 48 h of incubation at 37 °C with 5% CO2, an aliquot (20 μL) of 3--2,5-diphenyltetrazolium bromide (MTT) in PBS (5 mg/mL) was added to each well , and microtiter plates were incubated for a further 4 h at 37 °C with 5% CO2. After final incubation, the medium was aspirated, and precipitated formazan crystals dissolved in DMSO (100 μL/well). The absorbance of each well was measured at 580 nm with a PowerWave XS Microplate Reader from Bio-Tek Instruments Inc. . IC50 values were calculated using Prism 7.0 , as the concentration of analyte required for 50% inhibition of cancer cell growth (compared to negative controls). Negative controls comprised 1% aqueous DMSO, while positive controls used vinblastine as the test sample. All experiments were performed in duplicate. The cytotoxicity results are presented in Adherent human colorectal carcinoma (SW620) and human embryonic kidney (HEK293) cells were cultured in Roswell Park Memorial Institute (RPMI) medium 1640. All cells were cultured as adherent monolayers in flasks supplemented with 10% foetal bovine serum, 1) and B (2) is an intriguing late addition to our understanding of the natural product scaffolds embodied within southern Australian marine sponges, and the proposed biosynthesis provides useful insights into a prospective biomimetic synthesis. Significantly, our discovery of the leucettazoles showcases three important aspects of modern natural product science. The first is the importance of valuing and exploring the potential of artifacts, such as leucettazoles A1 (1a) and B1 (2a), as a window into the world of otherwise reactive (and cryptic) natural products. The second is the remarkable capacity of modern UHPLC-QTOF methods, supported by techniques such as GNPS and SIE visualization, and MS/MS analysis, to give access to new and exciting chemical diversity, overlooked by earlier generations of researchers. The third is the potential relationship between natural products, artifacts, and biological activity. The fact that the leucettazole ethanolysis adducts 1a and 2a did not exhibit cytotoxic properties against a panel of bacterial, fungal, and mammalian cells is perhaps not surprising, and should not discount the possibility that natural products 1 and 2 are bioactive. Much as is the case with Michael acceptors and other bioactive molecules that form covalent bonds with biological targets, it is possible that 1 and 2 are bioactive due to their innate solvolytic reactivity,. If correct, the solvolysis products 1a and 2a could be viewed as inactivated adducts, much as Michael adducts deactivate Michael acceptors. While the ecological purpose and pharmacological potential of the leucettazoles remains a mystery, we contend that this scaffold is deserving of further investigation. That said, given their rarity in nature, and inherent chemical reactivity, further exploration will likely require the input of synthetic and medicinal chemists.Knowledge of leucettazoles A (


Annona purpurea, known in Mexico as “cabeza de negro” or “ilama”, belongs to the Annonaceae family. Its roots are employed in folk medicine in several regions of Mexico. Taking that information into account, a chemical and biological analysis of the components present in the roots of this species was proposed. Our results demonstrated that the dichloromethane (DCM) extract was exclusively constituted by a mixture of five new acetogenins named annopurpuricins A–E (1–5). These compounds have an aliphatic chain of 37 carbons with a terminal α,β unsaturated γ-lactone. Compounds 1 and 2 belong to the adjacent bis-THF (tetrahydrofuran) α-monohydroxylated type, while compounds 3 and 4 belong to the adjacent bis-THF α,α’-dihydroxylated type; only compound 5 possesses a bis-epoxide system. Complete structure analysis was carried out by spectroscopy and chemical methods. All compounds were evaluated for their antiproliferative activity on three human tumor cell lines . Compounds 1–4 inhibited significantly the growth of HeLa and HepG2 cells, showing GI50 values in the low/subnanomolar range, while 5 was completely ineffective under the tested conditions. The investigation of the mechanism of action responsible for cytotoxicity revealed for the most interesting compound 1 the ability to block the complex I activity on isolated rat liver mitochondria (RLM). The family of Annonaceae is widely distributed in tropical areas. In Mexico, 13 genera and 37 species have been reported. Several species of this family have been used in traditional medicine in many countries and they are characterized by the production of alkaloids, terpenes and acetogenins ,6,7,8,9.With respect to the biological activities of acetogenins, a wide spectrum of effects has been described, including cytotoxicity on cancer cells and anti-inflammatory activities ,11,12. TAnnona purpurea Moc & Sessé ex Dunal is a tree reaching 5–10 m, known in Mexico as “cabeza de negro”, “toreta” or “ilama”. Several parts of this species are traditionally used in Mexico in folk medicine. In particular, its bark is used for the treatment of dysentery and edema, and the roots are employed against kidney diseases. Moreover, seeds and leaves have been used to prepare insecticide solutions ). IR spectra contained absorptions for hydroxyl (3369 cm−1) and α, β-unsaturated lactone (1737–1748 cm−1) functionalities. UV λmax absorptions were observed between 208–212 nm [methanol (MeOH)]. The positive Kedde reaction as well as the comparison of 1H and 13C NMR data of the isolated (1–5) with those reported for known acetogenins, made evident the presence of the terminal α,β-unsaturated γ-lactone  and correlated with the signals at 3.78 to 9.93 ppm in the 1H NMR spectra. A careful analysis of the TMSi (trimethylsilyl) derivatives and the mass spectra revealed that 1–4 had a bis-THF ring system. Unlike the others, the correlations observed in HSQC (heteronuclear single-quantum correlation), COSY (correlated spectroscopy) and HMBC (heteronuclear multiple bond correlation) experiments confirmed that 5 instead of THF units possess an epoxide system with an olefinic bond separated by two methylenes.A close examination of all compounds showed a similar profile in the spectroscopic data (infrared (IR), ultraviolet–visible spectrophotometry (UV), circular dichroism (CD) and nuclear magnetic resonance [NMR (-lactone  22]. Co. Co1H an1–5) were determined according to Gawroński and Wu ; as well as the fragment ions at m/z 535 (cleavage at C-19/20) and m/z 401 (cleavage at C-18/19) in the TMSi mass spectra. The third hydroxyl moiety was placed in C-24 by the presence of the fragment ions at m/z 345 and 425, cleavage between C-23/24 and C-24/25 respectively. A COSY experiment using benzene-6d (C6D6) as solvent allowed identifying a THF system with only one flanking hydroxyl moiety (3.49/3.85 ppm) , guanaconne and glabracin A  comes to lack, even though complex II should be operating, that can lead to cell death by necrosis. The investigation on the mechanism of cell death was performed by incubating HeLa cells in the presence of 1 at 5 nM concentration for 28 h. The obtained results highlighted a decrement of cell viability from 80.4%  to 54.2% in the presence of test compound (1) induces a significant increase of cells in late apoptosis (from 12.9% to 20.6%) and, more notably, an increase of necrotic cells from 1.2 % to 21.4%.The result reporting that compound B. Intere3 and C6D6 as solvents. The chemical shifts are given in δ (ppm) and coupling constants (J) are reported in Hz. ESI mass analysis was carried out employing a direct infusion in a triple quadrupole mass spectrometer Xevo TQD . The fragmentation patterns were obtained under conditions of capillary voltage: 2.5, 3.0 and 3.2 kV, cone voltage: 20, 50 and 120 V, collision energy: 10 to 35 V; a source temperature of 300 °C, cone gas flow 40 L/h and desolvation gas flow was adjusted to 600 L/h. The source parameters were established in positive ion mode (ESI+) with a sample flow of 10 μL/min. The EI-MS analysis of trimethylsilyl derivatives was made in a Varian 3800 Gas Chromatograph equipment coupled with Varian Saturn 2200 Ion Trap Mass Spectrometer. The flow were set at 1 mL/min with a temperature range from 100 °C to 280 °C. High-resolution mass spectra were acquired with an Agilent Technologies ESI-TOF spectrometer. Additional elemental analyses were performed on a Truspec® Micro Analyzer. The purity of each isolate was determined by HPLC analysis performed on a Waters e2695 coupled with a photodiode array detection 2998, using an analytical column XBRIDGE , the mobile phase was MeOH/water (90:10), with a flow of 1 mL/min. The detector was set at 210 nm. The diode array detector was set at 200–600 nm. The Empower 3 software program (Waters) performed control of the equipment, data acquisition, processing and management of chromatographic information.Melting points were obtained in a Fisher Johns fusiometer. Optical rotations were recorded in methanol on a JASCO P-2000 polarimeter. UV data were obtained on an Evolution 300 spectrophotometer in MeOH solution. Circular Dichroism spectra were performed on a JASCO 810 spectropolarimeter at 25 °C in MeOH solution. FTIR spectroscopic data were measured on a Varian 660-IR spectrophotometer. NMR spectra were recorded in a Bruker 400 MHz using CDClAnnona purpurea Moc & Sessé ex Dunal were collected in January 2014, in Minatitlán, Colima, México, . The plant was correctly identified at the “Herbario Nacional de México” (MEXU) of the “Universidad Nacional Autónoma de México (UNAM)” and a voucher specimen was deposited (MEXU 1457308). An additional collection from Santa María, Michoacán, Mexico was also used (MEXU 1054418) to compare the presence of the metabolites in a different area (data not included). Considering the importance of the root in the survival of a tree, the sample taken by individual was an amount that did not represent risk, and after the collection a continuous monitoring of the survival of trees was made.The roots of A. purpurea (3.47 Kg) were cleaned, dried and ground. Then, they were exhaustively extracted with hexane, DCM and methanol by maceration at room temperature (24 h × 5 L) to give three extracts, hexane (10.2 g), DCM (20.1 g) and methanol (103.5 g). The extracts were analyzed by TLC. Kedde´s reagent was employed to detect the presence of lactones in the DCM extract. Separation of the compounds present in the DCM extract was carried out using a chromatographic column of 4Φ × 60 cm packed with silica gel for thin layer chromatography (Macherey-Nagel). The column was loaded with 13 g of the extract, and it was eluted stepwise with solutions of n-hexane: ethyl acetate: methanol of increasing polarity. During the elution procedure, 210 fractions of 25 mL volume were obtained. The composition of the eluent solutions and their total volumes where: 100:0:0 , 90:10:0 , 80:20:0 , 70:30:0 , 50:50:0 , 20:80:0 , 0:100:0 , 0:80:20 , 0:0:100 . These fractions were grouped in 27 pools (A-Z) according to the TLC profile, of which the most abundant fractions were I (fractions 100–115), R (fractions 131–143), W (fractions (147–160), X (fractions 167–170), Z (172–202); showing a positive reaction to Kedde’s reagent. These pools were purified to afford compounds 1 to 5. Compound 1 (500 mg) was obtained from fraction Z by precipitation with a cold mixture of hexane: acetonitrile (1:1). 2 (30 mg) and 4 (268 mg) were obtained by a similar method employing the fractions W and X, respectively. Treatment of the fraction R with a heterogeneous system of methanol and hexane (95:5) yielded the purification of a white solid 3 (209 mg). Compound 5 was obtained from fraction I by treatment with methanol until a white wax was obtained; this was subsequently precipitated employing a mixture of hexane and acetonitrile (1:2) to yield 185.8 mg.Roots of 25D +0.34° ; CD (MeOH) ∆ε (nm): −2.09 × 103 (237.5); UV (MeOH) λmax (log ε) 209 (4.00); IR νmax 3369, 2916, 2850, 1742, 1471, 1324, 1080, and 869 cm−1; HRMS (ESI-TOF) m/z: 645.4695 [C37H66O7 +Na]+, calcd. 645.4701 [C37H66O7Na]+, calcd. 622.4809 [C37H66O7]. 1H and 13C NMR spectroscopic data, see Yellow amorphous powder; 0.018% (500 mg); mp 57–60 °C; [α]−3% (30 mg); mp 65–66 °C; [α]25D −0.11° ; CD (MeOH) ∆ε (nm): −1.81 × 103 (237.5); UV (MeOH) λmax (log ε) 212 (4.00); IR νmax 3414, 3370, 2916, 2849, 1737, 1471, 1072 cm−1; HRMS (ESI-TOF) m/z: 645.4700 [C37H66O7 +Na]+, calcd. 645.4701 [C37H66O7Na]+, calcd. 622.4809 [C37H66O7]. 1H and 13C NMR spectroscopic data, see White amorphous powder; 1.30 × 10−3% (209 mg); mp 69–71 °C; [α]25D +0.44° ; CD (MeOH) ∆ε (nm): −2.16 × 103 (237.5); UV (MeOH) λmax (log ε) 209 (4.00); IR νmax 3414, 2915, 2849, 1743, 1471, 1070 cm−1; HRMS (ESI-TOF) m/z: 629.4753 [C37H66O6 +Na]+, calcd. 629.4752 [C37H66O6Na]+, calcd. 606.4859 [C37H66O6]. 1H and 13C NMR spectroscopic data, see White amorphous powder; 6.65 × 10−3% (268 mg); mp 77–78 °C; [α]25D −0.27° ; CD (MeOH) ∆ε (nm): −1.67 × 103 (238.5); UV (MeOH) λmax (log ε) 210 (3.94); IR νmax 3413, 2917, 2850, 1740, 1072 and 1025 cm−1; HRMS (ESI-TOF) m/z: 645.4706 [C37H66O7 +Na]+, calcd. 645.4701 [C37H66O7Na]+, calcd. 622.4809 [C37H66O7]. 1H and 13C NMR spectroscopic data, see White amorphous powder; 8.1 × 10−3% (185.8 mg); mp 55–56 °C; [α]25D −0.43° ; CD (MeOH) ∆ε (nm): −1.68 × 103 (239); UV (MeOH) λmax (log ε) 208 (3.85); IR νmax 2950, 2916, 2848, 1748, 1462, 831 cm−1; HRMS (ESI-TOF) m/z: 595.4693 [C37H64O4+Na]+, calcd. 595.4697 [C37H64O4Na]+, calcd. 572.4804 [C37H64O4]. 1H and 13C NMR spectroscopic data, see White amorphous powder; 5.63 × 101–4 (10 mg) with acetic anhydride/pyridine  and subsequent work-up gave compounds 1a–4a (13–20 mg of yield) as a yellow semisolid  were added. The reaction was mixed with a magnetic stir bar and placed on ice bath, under a N2 gas flow. Subsequently, 30 mg of DCC and 5 mg of DMAP were added. The reaction system was carefully shaken in an ice bath for 5 min, and then at room temperature. After that dicyclohexylurea precipitated. Each reaction was monitored by TLC and dried under vacuum pressure once the reaction had finished. Then, the residue was percolated using Si-gel and employing an ascending polarity system as eluent. Finally, the product was dissolved in CDCl3 and analyzed by 1H NMR spectroscopy. The (S)-MTPA ester of each acetogenin was prepared from (S)-MTPA [(S)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetic acid] following the same method ethanesulfonic acid), 0.11 g/L pyruvate sodium and Nutrient Mixture F-12 [HAM] , respectively. HepG2 were grown in Dulbecco’s Modified Eagle’s Medium . 10% Heat-inactivated fetal bovine serum , 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 μg/mL amphotericin B  were added to all media. The cells were cultured at 37 °C in a moist atmosphere of 5% carbon dioxide in air. Cells (3 × 10g) for 5 min. The supernatant was centrifuged at 12,000× g for 10 min to separate the crude mitochondrial pellets. The resulting pellets were suspended in isolation medium lacking EDTA  sucrose and 3H2O [+ was added at a concentration of 2 μmol L−1 to allow accurate measurements while avoiding toxic effects on the H+-ATPase and Ca2+ [+ electrode was calibrated using the equation: ∆Ψ = (∆Ψ electrode−66.16)/0.92 [−1 ADP) and state 3 (in the presence of 300 μmol L−1ADP) using a Clark oxygen electrode  in a closed, thermostatically controlled vessel with a magnetic stirrer, coupled to a Perkin-Elmer 561 recorder [The ∆Ψ was measured by assessing the distribution of the lipophilic cation tetraphenylphosphonium  were seeded into each cell culture plate. After 24 h of incubation, the test agent was added to the complete medium at the indicated concentration and cells were incubated for a further 28 h. After treatment, cells were centrifuged, resuspended in JC-1 working solution and incubated for 30 min at 37 °C in CO2 incubator. Following incubation, cells were washed twice, suspended in assay buffer and analyzed by a FACSCanto II flow cytometer. Results are presented as percentage of cells with depolarized mitochondrial membrane (JC-1 monomers) [The mitochondrial ΔΨ was evaluated by using the BD™ MitoScreen Kit  containing the membrane-permeable lipophilic cationic fluorochrome 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) . HeLa ceonomers) .5) were seeded into each cell culture plate. After incubation for 24 h the test acetogenin was added to the complete medium at the indicated concentration and the cells were incubated for a further 28 h. After treatment, the cells were centrifuged and suspended at 106 cells/mL in binding buffer. Cell suspensions (100 μL) were added with Annexin V-FITC and propidium iodide (PI) as indicated by the supplier’s instructions and incubated for 15 min at room temperature in the dark. The populations of Annexin V-negative/PI-negative (viable), Annexin V-positive/PI-negative (early apoptosis), Annexin V-positive/PI-positive (late apoptosis) and Annexin V-negative/PI-positive (necrosis) cells were evaluated by FACSCanto II flow cytometer  [To detect phosphatidylserine translocation from the inner to the outer face of plasma membrane a FITC Annexin V Apoptosis Detection Kit I  was used. HeLa cells  .A. purpurea, which is in total agreement with previous studies of its leaves and seeds [1–5), were isolated from roots of Annona purpurea. Their structures and absolute configuration were determined by interpretation of the spectroscopic data of original compounds and some of their derivatives. Compounds 1–4 showed a significant cytotoxic activity on MSTO-211H, HeLa and HepG2 cancer cell lines with GI50 values in the nanomolar range. In particular, annopurpuricin A (1) a bis-THF acetogenin, was the main component of the DCM extract (0.018% yield) and showed a GI50 value notably low on HeLa cells (0.06 nM). The investigation on the mechanism of action of 1 highlighted the ability to inhibit NADH dehydrogenase (complex I) in RLM, similarly to rot. Cytofluorimetric experiments demonstrated that 1 is able to cause the depolarization of mitochondrial membrane in whole cells, and able to induce cell death through late apoptosis and necrosis.Although a previous study reports the presence of an alkaloid , our resnd seeds ,19,20. F