Candida glabrata at 10 μg/mL (15.4 μM). The fungicidal effect was observed at 3 to 4 h after exposure to cells. Cytological and morphological studies revealed that OPS affects the budding patterns of treated yeast cells with a significant increase in the number of cells with single small buds. In addition, this budding morphology was found to be sensitive in the presence of OPS. Moreover, the number of cells with single medium-sized buds and cells with single large buds were decreased significantly, indicating that fewer cells were transformed to these budding patterns, suggestive of inhibition of polarized growth. OPS was also observed to disrupt the organized actin assembly in C. glabrata, which correlates with inhibition of budding and polarized growth. It was also demonstrated that phytosphingosine (PHS) reversed the antifungal activity of oceanapiside. We quantified the amount of long chain-bases (LCBs) and phytoceramide from the crude extracts of treated cells using LC-ESI-MS. PHS concentration was elevated in extracts of cells treated with OPS when compared with cells treated with miconazole and amphotericin B. Elevated levels of PHS in OPS-treated cells confirms that OPS affects the pathway at a step downstream of PHS synthesis. These results also demonstrated that OPS has a mechanism of action different to those of miconazole and amphotericin B and interdicts fungal sphingolipid metabolism by specifically inhibiting the step converting PHS to phytoceramide.Oceanapiside (OPS), a marine natural product with a novel bifunctional sphingolipid structure, is fungicidal against fluconazole-resistant  Candida infections has led to increased clinical emergence of fluconazole-resistant, non-Candida albicans Candida species, primarily Candida glabrata  stain. FUN1 is a nucleic-acid binding dye that was found to give rise to the formation of cylindrical intravacuolar structures  in metabolically active cells ed cells . The incC. glabrata by LCMS analysis of crude cell-extracts. The concentrations of key long-chain bases in the extracts were measured by LC-ESI-MS. Two other antifungal agents of differing modes of action, miconazole (inhibition of 14α-demethylase in ergosterol biosynthesis) [N-fatty acyl derivatives, e.g., phytoceramide [From the foregoing results in nthesis)  and amphnthesis) , were usceramide .Fusarium moniliforme that are also sphingoid base analogues [The exceptional activity of OPS is likely attributed to the “two-headed” sphingolipid structure and associated multivalency, as reported earlier in our earlier structure-activity study of OPS and related long-chain α,β-diamines and synthetic mono-aminoalkanols . Future nalogues .These results may have significant implications in the ecological role of oceanapiside and related two-headed sphingolipids in the context of antibiosis against marine-borne fungi and fitness of the host sponges that produce them within the marine environment. The role of antifungal aminoalcohols in these invertebrates has been the subject of speculation . Howeverw/w) was separated from methanol extracts of the sponge, Oceanapia phillipensis (Dendy), and purified according to our published procedures [w/w) was derived from OPS by acid-promoted methanolysis , and purified by silica-gel chromatography (12:18:1 methanol/NH4OH/chloroform) according to our published procedures [Oceanapiside  was used in the assays. Yeast cells were grown on sabouraud dextrose (SD) agar  or sabouraud liquid medium  and incubated at 37 °C.A fluconazole-resistant clinical isolate of 600 of 0.10 to 0.120, and OPS (1 to 30 μg/mL) was added. The cell culture was incubated at 37 °C, and growth rates were determined by measuring the optical density (OD) at 600 nm as a function of time using a Molecular Devices  Spectramax Plus plate reader. An aliquot of the OPS-treated cells was taken hourly, re-plated onto fresh SD agar, and incubated for 24 h at 37 °C to check for cell viability.An overnight culture of cells was diluted with fresh SD medium to an OD600 of 0.10 to 0.120. OPS(10 μg/mL) was added and the cells were incubated for 2 h. The cells were harvested and stained with yeast live/dead kit according to the manufacturer’s instructions . Briefly, the cells were resuspended in 1 mL of sterile water containing 2% d-(+)-glucose, 10 mM Na-HEPES (pH 7.2), and 5 μM of FUN1 dye. The mixture was incubated at 30 °C in the dark for 30 min and was counterstained with 25 μM Calcofluor White M2R. Five μL of stained cells were immobilized on a clean glass slide and cover slip in preparation for microscopy.An overnight culture of cells was diluted with fresh SD medium to an ODt = 0 to 9 h after incubation. From these samples, at least 500 cells were observed and classified according to the following budding patterns: A, unbudded cells; B, cells with a single small bud; C, cells with a single medium-sized bud; and D, cells with a single large bud. To determine the viability of different budding patterns in OPS-treated cells, a 100 μL of the culture was prepared in triplicates and mixed with an equal volume of methylene blue . The mixture was incubated for 5 min at room temperature and observed under the microscope.The morphology of yeast cells treated with OPS (10 μg/mL) was viewed with a photomicroscope (Nikon) using a 100× objective. Triplicate 100 μL aliquots were taken at time points, C. glabrata was transferred to 1 mL of fresh SD medium and incubated at 37 °C to low log (5 × 106/mL). The cell culture was then combined with 10 μg/mL OPS, or 1% DMSO, and incubated for 2 h. After incubation, the cells were fixed in the medium with 4% formaldehyde  for 10 min. The cells were harvested (9000 × g for 5 min) and resuspended in 4% formaldehyde for 1 h with frequent mixing, followed by washing (× 2) with phosphate buffered solution, PBS (pH 7.5). The fixed cells were resuspended in 100 µL of PBS and stained with rhodamine-phalloidin, R415 . The cells were incubated in the dark for 1 h with constant mixing, washed five times with 1 mL PBS, and resuspended in 500 µL of PBS. The mixture was incubated in the dark for 30 min with frequent agitation and washed five times with 1 mL PBS. Rhodamine-phalloidin stained cells (2 μL) were immobilized between a clean glass slide and cover slip for microscopic examination.The effect of marine natural products on the organization of actin assembly was determined by using fluorescent probes to visualize the actin cytoskeleton. Briefly, a 100 μL of an overnight culture of Stained cells were viewed with an Olympus IX70 Deltavision Microscope using a 60× 1.4 N.A. objective and illuminated with a 100 W mercury lamp. The following excitation wavelengths were used for actin and nucleus staining: DAPI, 360 and Rhodamine, 555. Yeast cells stained with FUN1 and Calcofluor White M2R used multi-pass filter sets with the following excitation wavelengths: λ 360, 490, and 555 nm. Images were collected in 0.2 µm sections. Two- and three-dimensional light microscopy data collection and computational removal of out-of-focus information were achieved using an integrated, cooled CCD-based, fluorescence light microscopy data collection, processing, and visualization workstation  in the Molecular and Cellular Biology Imaging Facility, University of California, Davis. Three-dimensional data sets were processed using Delta Vision’s iterative, constrained three-dimensional deconvolution method.600 of 0.10 to 0.120. The cells were treated with a mixture of OPS (10 μg/mL) and ketodihydrosphingosine (3 μg/mL), OPS (10 μg/mL), and dihydrosphingosine (3 µg/mL), or OPS (10 µg/mL) and phytosphingosine (3 µg/mL), then incubated at 37 °C. Growth rates were measured after 24 h by measuring the optical density at 600 nm using a Molecular Devices Spectramax Plus plate reader. An aliquot of the treated cells was streaked onto fresh SD agar and incubated for 24 h at 37 °C to determine viability and fungicidal versus fungistatic concentrations. Ketodihydrosphingosine, dihydrosphingosine, and phytosphingosine were obtained from Matreya LLC .An overnight culture of cells was diluted with fresh SD medium to an ODC. glabrata cells was diluted with fresh SD medium to an OD600 of 0.40 to 0.410 to make a 1 mL culture. The cells were treated with OPS (10 μg/mL), miconazole (15 μg/mL), and amphotericin B (1 μg/mL) and incubated for 3–4 h at 37 °C. The treated cell extracts were prepared as described previously [17-DHS (C17H37NO2), prepared by synthesis in our laboratory as a non-natural surrogate internal standard (23H41NO4S) as a secondary synthetic internal standard  were prepared by dissolving an appropriate amount of each compound in methanol. Serial dilution of these authentic samples was prepared in acetonitrile containing 5% THF. Protein content of the lysate was determined with the bicinchoninic acid (BCA) assay, as described previously [An overnight culture of eviously . Brieflystandard . The mixdard IS; . Stock seviously . Quantif18 100 Å column  on an Agilent 1100 Series liquid chromatography system comprising of a quaternary pump with automatic sampling injector. The gradient components were as follows: H2O containing 0.05% formic acid (solvent A), methanol with 0.05% formic acid (solvent B), and 10% THF in methanol with 0.05% formic acid (solvent C). The gradient program was as follows: 0–2 min 50:50 A/B, 2–20 min 3:97 A/B, 20–25 min 50:50 B/C, 25–32 min 50:50 B/C, 32–35 min 90:10 A/B, and 35–38 min 50:50 A/B. The flow rate was set at 0.4 mL/min and the injection volume was 5 μL. The ESI-MS analyses were conducted on a Surveyor MSQ™ MS detector system with an ESI interface . Data acquisition, and qualitative and quantitative analyses were carried out using Xcalibur™ software . For electrospray ionization, the probe temperature was set at 350 °C with cone voltage set at 90 V and a nitrogen flow setting of 4–5 bar. The mass spectrometer was operated in the selected ion monitoring (SIM) mode with positive ion detection (M + H)+ of ketodihydrosphingosine, KDS (m/z 300.28), dihydrosphingosine, DHS (m/z 302.30), phytosphingosine, PHS (m/z 317.55), C24-phytoceramide, PHC (m/z 668.7), C17-DHS (m/z 288.2), and C17-DHS phenylsulfone (m/z 428.2) at a total scan duration of 0.4 s. Quantitative evaluations were based on the peak area ratios of each analyte to that of an internal standard, and the calculated amount was normalized based on the known amount of the surrogate standard.Analysis of sphingolipids and LCBs was carried out using a method we developed for the purpose using LC-ESI-MS of crude cell extracts. A reversed-phase chromatographic separation of the crude cell extracts was carried out on a Luna 3 μm C3 or CD3OD, 99.8 atom%, Cambridge Isotope Laboratories, Tewksbury, MA, USA). 1H NMR and 13C NMR spectra were acquired on a Varian Inova-400  operating at frequencies of 399.770 and 100.531 MHz, respectively, using a basic ‘1pulse’ sequence: ~45° π/4 excitation pulse, delay time, d1 = 1.5 s, and broad-band 1H decoupling for 13C NMR. Data (FIDs) were processed using native software . 1H and 13C NMR chemical shifts, δ, are referenced to residual CHCl3 (7.26 ppm) and CDCl3, (77.16 ppm), respectively.NMR samples were prepared in specified deuterated solvents (CDClCandida glabrata. Supporting evidence from treatment of C. glabrata cell cultures with OPS includes inhibition of polarized growth and budding, disorganization of actin assembly, reversal of antifungal activity by phytosphingosine, and accumulation of phytosphingosine. To date, OPS is the first bifunctional sphingolipid antifungal marine natural product described to inhibit sphingolipid synthesis, specifically at the step converting phytosphingosine to phytoceramide. Future study involving cytotoxicity testing could be undertaken to validate the safety of OPS as an antifungal candidate, but, in all likelihood, synthetic analogues should be pursued for more “drugable” therapeutics.In summary, our findings demonstrate that oceanapiside targets the sphingolipid pathway in the pathogenic yeast


The Boletaceae family species  were collected from the Midu County  in 2018. The activity concentrations of 137Cs in the caps of dried raw mushrooms were in the range 14 ± 1 Bq kg−1 dry biomass (db) (R. griseus) to 34 ± 2 Bq kg−1 db , and in stems from 16 ± 1 Bq kg−1 db (B. bicolor and B. bainiugan) to 23 ± 1 Bq kg-1 db . The mean activity concentration in the whole fruiting bodies in all six species was 18 ± 4 Bq kg-1 db. The activity concentrations of 137Cs were roughly the same in both dehydrated materials, stir-fried, and raw mushrooms, while the contents of 40K and stable K were around 2- to 3-fold smaller in stir-fried than raw product. The raw and stir-fried mushrooms on a whole (wet) weight basis showed activity concentrations of 137Cs in the range from 1.2 to 3.2 Bq kg−1 ww (mean 1.9 ± 0.6 Bq kg−1 ww) and 6.0 to 9.4 Bq kg−1 ww (mean 7.0 ± 1.2 Bq kg−1 ww), respectively. Evidently, when expressed on a whole (wet) weight basis, the cooked mushrooms showed on average around 3.5-fold greater activity concentration of 137Cs when compared with raw mushrooms. The 137Cs, 40K, and total K enrichment in stir-frying (in a whole (wet) weight basis for the meal), confronted with the results for dehydrated raw and fried mushrooms, show the direct correlation with loss of mass (largely moisture) during the cooking procedure but not much of 137Cs and 40K. Edible wild mushrooms from Yunnan were little contaminated with radiocaesium. As assessed, the mean radioactivity dose from natural 40K in around 9.3-fold exceeded the dose obtained for artificial 137Cs from stir-fried mushroom meals, which both were very low doses.The parallel batches of the same species and geographical origin mushrooms both raw and stir-fried were investigated to get an insight into the content and intake of  Historically, the accident at the Chernobyl nuclear power plant in 1986 caused high and long-lasting pollution over significant parts of continental Europe. This included contamination of forest soils with 137Cs in many regions, and consequently also of the wild mushrooms that grow in these forests  sampled from the Changning localization (ca. 200 km west of Midu County) in Yunnan in 2016 showed the 137Cs activity concentration at level 4.9 ± 0.6 Bq kg−1 dry weight  and slow migration down to soil depths that are relevant for incorporation in fungal biological cycles  status of the mushroom  can also influence the activity concentration  for humans , and total Potassium (K) content and their intake from such stir-fried mushrooms meals, are not available in the literature. We investigated the potential effect of stir-frying in a moderate volume of oil using traditional Chinese cooking practice, on the content of 137Cs, 40Potassium (40K), and total Potassium (K) in dishes made of six species of mushrooms widely foraged in Yunnan.In a number of Asian cuisines, stir-frying of foods including mushrooms, in a wok type pan, is a popular method of cooking. Information on the possible effect of stir-frying mushrooms in a wok, on the Baorangia bicolor (Kuntze) G. Wu, Halling & Zhu L. Yang  and the sample whole (fresh) weight was 433 g (n = 21) for uncooked and 402 g (n = 22) for stir-fried fruiting bodies; Boletus bainiugan Dentinger  weighing 280 g (n = 5) and 292 g (n = 6); Butyriboletus roseoflavus  weighing 299 g (n = 5) and 342 g (n = 5); Retiboletus griseus (Frost) Manfr. Binder & Bresinsky  weighing 366 g (n = 8) and 341 g (n = 8); Rugiboletus extremiorientalis (Lj.N. Vassiljeva) G. Wu & Zhu L. Yang ) weighing 440 g (n = 12) and 312 g (n = 12), and Sutorius magnificus (W.F. Chiu) G. Wu & Zhu L. Yang  weighing 154 g (n = 6) and 312 g (n = 7)  located in the west-central region of Yunnan province in China in 2018  were separated. All specimens not subjected for stir-frying were sliced and dried for 24 h to a constant mass at 65 °C in a food dehydrator , and ground to a fine powder using a clean porcelain mortar and pestle and stored dry in sealed polyethylene bags until analysis.All specimens subjected for stir-frying were sliced, separately caps and stems, pooled accordingly and stir-fried using one of the traditional Yunnan methods in moderate amount of hot vegetable oil (15 to 55 g and depending on the sample size) in a wok pan for 10 min. After cooking, the excess oil was drained away and the fried mushrooms were cooled and transferred into fresh polyethylene jars , weighted, deep frozen (− 20 °C), freeze-dried for 72 h, then reweighed , homogenized using a blender with steel-less blades and plastic bowl, and kept tightly closed in screw capped polycarbonate jars which were packed individually into sealed polyethylene bags and stored in a refrigerator. Immediately prior to the instrumental analysis, all the fungal materials were deep frozen (− 20 °C) and freeze-dried for 72 h , so that the activity levels of nuclides were determined in fully dehydrated materials.137Cs and 40K were determined using a gamma spectrometer with a coaxial HPGe detector and with a relative efficiency of 18% and a resolution of 1.9 keV at 1.332 MeV (with associated electronics). Quantitation was carried out using the equation:Activity concentrations of Ni is the number of counts after background correction, ε(E) is the detector efficiency for photons with energy E, y is the emission probability, and t is the measurement time in seconds.where −1 dry biomass (db). The equipment was calibrated using a multi-isotope standard, and the method was fully validated , and background counts were subtracted (using the GENIE 2000 program). The lower limit of detection was at 0.10 Bq kg3(liquid) with different heights: 3, 6, 9, 15, and 25 mm, which allows the selection of the appropriate calibration for samples of different thickness layer. All numerical data obtained were recalculated for dehydrated fungal material (freeze-dried), and all data were decay-corrected back to the time of sampling. Concentrations of total (stable) K  in natural K, which is in the range 27.33 to 31.31 Bq g−1 of K  was used for reference samples during equipment calibration. Calibration was carried out using standards with a density of approximately 1 g cm137Cs in pooled samples of caps of the species were in the range 14 ± 1 Bq kg−1 db in Retiboletus griseus to 34 ± 2 Bq kg−1 db in Rugiboletus extremiorientalis. Values of 137Cs in the pooled samples of stems were from 16 ± 1 Bq kg−1 db in Baorangia bicolor and Boletus bainiugan to 23 ± 1 Bq kg−1 db in Rugiboletus extremiorientalis. The mean activity concentration in the whole fruiting bodies in all six species was 18 ± 4 Bq kg−1 db  has been found in the shaggy  chanterelle Gomphus floccosus (Schv.) Sing in one study  Earle ex Giachini and Castellano  to 1100 ± 93 Bq kg−1 db (Retiboletus griseus) and in stems 500 ± 110 Bq kg−1 db (Sutorius magnificus) to 1000 ± 110 Bq kg−1 db (Retiboletus griseus). The whole fruiting bodies showed activity concentrations of 40K in the range from 740 Bq kg−1 db (Baorangia bicolor) to 1100 Bq kg−1 db (Retiboletus griseus), and the mean value for all six species was 840 ± 120 Bq kg−1 db. The distribution of 137Cs and 40K between morphological parts of the fruiting bodies (QuotientCap/Stem) was similar (QC/S ~ 1) in Boletus bainiugan, Rugiboletus extremiorientalis, and Retiboletus griseus, but favored the caps (mean QC/S 1.6) in Baorangia bicolor, Butyriboletus roseoflavus, and Sutorius magnificus. The overall mean distribution ratios for all the fruiting bodies sampled were QC/S 1.1 ± 0.2 for 137Cs and QC/S 1.5 ± 0.3 for 40K.−1 db (Baorangia bicolor) to 40,000 ± 3300 mg kg−1 db (Retiboletus griseus) in the caps, and 19,000 ± 3000 mg kg−1 db (Baorangia bicolor and Sutorius magnificus) to 37,000 ± 3800 mg kg−1 db (Retiboletus griseus) in the stems. Soil mushrooms, including species of the family Boletaceae, are rich in K. In the present study, the total K contents in the whole fruiting bodies were in the range 26,000 ± 3200 mg kg−1 db (Baorangia bicolor) to 39,000 ± 3500 (Retiboletus griseus) mg kg−1 db, and the overall mean was 30,000 ± 4400 mg kg−1 db.The total K content determined was in the range of 32,000 ± 3400 mg kgB. edulis that grows in European forests, which contained the element in the range 25,000 ± 4000 to 29,000 ± 3000 mg kg−1 db (caps) and 16,000 ± 4000 to 20,000 ± 3000 mg kg−1 db (stems)  for six species was 16 ± 4 Bq kg−1 db (range 11 ± 2 to 22 ± 2 Bq kg−1 db), and of 40K, was 320 ± 92 Bq kg−1 db (range 190 ± 80 to 480 ± 110 Bq kg−1 db). The overall mean content of K in stir-fried mushrooms was 11,000 ± 4600 mg kg−1 db, and the range for all species was 7000 ± 2800 to 18,000 ± 3600 mg kg−1 db   in the course of stir-frying. This may be due to the difference in the distribution of Cs and K in cell structures and their binding sites. Potassium is a major monovalent element that is found in high concentrations in mushrooms and is a key component of the cellular protoplasm. Breakout from cell walls due to the effects of high-temperature cell shrinkage during the course of stir-drying can favor the release of K, but can have a lower effect on Cs. Also a preferable leaching of K from cells into the residual oily phase is possible. Stir-frying causes partial dehydration of cooked foodstuffs and resulting in an increase in the proportion of dry matter in a mushroom meal but also of some less volatile or non-volatile compounds of the metallic elements, e.g., mercury  mushrooms 137Cs by 70 %  but without information on the mushroom species studied or on the intake of the nuclide based on the whole weight of the fried mushroom meal.In an earlier study, Kenigsberg et al.  reported137Cs, 40K, and K in raw mushrooms and mushroom meals were recalculated for appropriate meal portions for stems and caps, and are expressed for the whole fruiting bodies on a whole weight basis  and from 6.0 to 9.4 Bq kg−1 ww (mean 7.0 ± 1.2 Bq kg−1 ww). Evidently, when expressed on a whole (produce) weight basis, the stir-fried meals showed on average around 3.5-fold greater activity concentration of 137Cs when compared with raw mushrooms.The raw mushrooms and stir-fried mushroom meals on a whole weight showed activity concentrations of 40K, the activity concentration in raw mushrooms was in the range of 82 to 91 Bq kg−1 ww (mean 87 ± 4 Bq kg−1 ww), and in the stir-fried mushroom meals, in the range of 100 to 200 Bq kg−1 ww (mean 140 ± 32 Bq kg−1 ww). Analogically, the content of stable K in fresh mushrooms was in the range from 2700 to 3200 mg kg−1 ww (mean 3100 ± 200 mg kg−1 ww) and in cooked produce, from 3800 to 7700 mg kg−1 ww (mean 5000 ± 1300 mg kg−1 ww).In the case of 40K and total K) were slightly enriched in the whole weight stir-fried mushroom meals when compared with the whole weight raw mushrooms  was 12 ± 2 mBq, 230 ± 52 mBq, and 8.3 ± 2.2 mg respectively, and the corresponding weekly intake was 82 ± 14 mBq, 1600 ± 360 mBq, and 58 ± 15 mg respectively  in around 9.3-fold exceeded the dose obtained for artificial 137Cs (0.055 μSv), which both are very low.The average effective daily and weekly doses of gamma radioactivity received per capita from Adequate potassium daily intake for adults has been set for 4700 mg NIH , and a s137Cs were roughly the same in both dehydrated samples, stir-fried, and raw mushrooms, while the contents of 40K and stable K were around 2- to 3-fold smaller in stir-fried than raw produce. The stir-fried mushroom meals on a whole (wet) weight exhibited on average around 3.5-fold greater activity concentration of 137Cs when compared with raw mushrooms. The 137Cs enrichment in cooking, and less of 40K (in a whole (wet) weight basis for the meal), confronted with the results for dried raw and fried mushrooms show the direct correlation with loss of mass (largely moisture during the cooking procedure) but not much of 137Cs and 40K; therefore, activity concentrations of both nuclides in stir-fried mushrooms increase. Hence, exposure to radiocaesium from meals made of the stir-fried mushrooms substantially contaminated with this nuclide can be a more significant source than speculated earlier. However, edible wild mushrooms from Yunnan are little contaminated with radiocaesium. An assessed, the mean radioactivity dose from natural 40K in around 9.3-fold exceeded the dose obtained for artificial 137Cs from stir-fried mushroom meals in Yunnan, which both were very low doses. The effect of the stir-frying process on 137Cs and other mineral constituent content in mushroom meals has to be clarified from the ongoing studies.Examination of the parallel batches of the same species and geographical origin both uncooked and stir-fried mushrooms showed that the activity concentrations of


Recently, III-V semiconductor nanowires have been widely explored as promising candidates for high-performance photodetectors due to their one-dimensional morphology, direct and tunable bandgap, as well as unique optical and electrical properties. Here, the recent development of III-V semiconductor-based single nanowire photodetectors for infrared photodetection is reviewed and compared, including material synthesis, representative types (under different operation principles and novel concepts), and device performance, as well as their challenges and future perspectives. In the last decades, there has been an increased demand for semiconductor infrared (IR) photodetectors, due to their broad defense and civilian applications such as imaging, night vision, free space optical communication, search and rescue, surveillance, missile tracking, material inspection and identification, scientific instrumentation , and astronomy ,2. For eIlight/Idark ratio for infrared photodetection. In addition, surface passivation . InG. InG0.564 × 105% . Ren et o 3.5 µm . A respo.52P0.48 . It was x = 0.52 . This imanowires ,79.2O3 can be used to significantly reduce charge trap density with an order of magnitude increase in field-effect mobility (µ), leading to a PPC in InAs nanowires [5, responsivity of 2.8 × 105 A/W, and specific detectivity of 9.1 × 1015 Jones at λ = 0.83 µm were measured [3 A/W and detectivity of ∼8.5 × 1010 Jones (cm·Hz1/2W−1) at 77 K [Single horizontal nanowires are usually fabricated on thermally oxidized Si substrates with electrodes on the two ends of the nanowire for drain and source contacts, as shown in anowires . Zheng eanowires . This bemeasured . More remeasured ,82. The Single InAs nanowire-based FETs have also been successfully developed as highly sensitive room temperature THz detectors with a detection frequency range from 0.3 to 2.8 THz ,84,85,8610 Jones, and fast response time of 80 µs at λ = 2 µm at a low bias voltage of 0.1 V [4 A/W at MWIR wavelength of 5.5 µm [Fabrication of proper metal contacts or implementing doping can be used to form Schottky metal-semiconductor, homo- or heterojunction in nanowires for photodetection. A Schottky barrier tends to be formed when some noble metals such as Au and Ag make contact with a semiconductor such as InP and GaAs . The junof 0.1 V . Kuo et f 5.5 µm .Ilight/Idark ratio of 500 at λ = 1 µm by integrating a horizontal single InAs nanowire on a vertically stacked graphene layer as shown in 3 A/W, EQE of 104 and response time of 2 ms at the optical communication wavelength of 1.55 µm were obtained by applying a high forward bias (+1 V). Wang et al. demonstrated a GaAsSb/InAs p-n heterojunction-based photodetector that showed a wide spectral photoresponse ranging from 0.488 to 1.8 µm [In addition to the aforementioned configurations, p-n or p-i-n nanowire photodiodes have been employed to operate under photovoltaic mode at zero bias  or photoo 1.8 µm . A respoo 1.8 µm .When a photodiode operates as an avalanche photodetector (APD) at a high reverse bias condition, impact ionization leads to multiplication of holes and electrons created by the initial photon excitation, enabling large photocurrent gain and sensitivity. The intrinsic small photon collection area and active volume of nanowire-based APDs make them especially attractive device structures to enable photocurrent gain and sensitivity even down to the single photon level. Linear mode APDs have been reported in ensemble InGaAs/GaAs core–shell nanoneedles , exhibit+-i-n+ structure implemented for the single InP nanowire THz detectors, leading to enhancement of signal-to-noise ratio, ~2.5 times of the undoped InP nanowire detector [GaAs ,95 and Idetector . The fundetector .Plasmonic structures could be combined with nanowires to enhance the detector performance and polarization sensitivity. Casadei et al. demonstrated a nanowire/nanoantenna hybrid structure that could be used to engineer the polarization response of nanowires by embedding a GaAs horizontal nanowire in a bow-tie antenna array, as shown in Although III-V semiconductor nanowires present unique and excellent optical and electrical properties and have been demonstrated to have promising performance for room temperature infrared photodetection, there are still many technological challenges to overcome. Despite good device performance, the output current of the single nanowire photodetectors is still too low for practical applications. Therefore, the fabrication technique for large-scale and controlled assembly of single nanowires into horizontal arrays is highly desirable . There hAnother limitation of III-V semiconductor nanowire photodetectors is their detection wavelength that are determined by the material bandgap, especially for the relatively easy synthesis but wider bandgap binary materials such as GaAs and InP. To enable light detection of photon energies well below the semiconductor bandgap, Knight et al. proposed a novel idea of active optical antennas, in which hot carriers are generated and injected into the semiconductor material  through the antenna–semiconductor Schottky barrier, contributing to a detectable photocurrent response at SWIR wavelengths up to 1.6 μm . To furtSingle III-V semiconductor nanowires have been extensively explored in the past decade as room temperature high-performance infrared photodetectors with broad detection wavelength range covering UV, VIS, IR, and THz regimes. In this article, we present a review on their recent development in materials, structures, and comparative performances, as well as the existing challenges and possible future directions.


FGFR1 (fibroblast growth factor receptor 1) gene mutations and to evaluate the effect of gonadotropin or pulsatile gonadotropin-releasing hormone (GnRH) therapy on spermatogenesis.  The aim of this study was to investigate the clinical characteristics of patients diagnosed with congenital hypogonadotropic hypogonadism (CHH) caused by FGFR1 mutation group and the mutation-negative group.  A retrospective study was conducted on CHH patients admitted to Peking Union Medical College Hospital from January 2012 to March 2020. Clinical features and laboratory results were recorded. Testicular volume and sperm count responding to gonadotropin and pulsatile GnRH therapy were compared between the FGFR1 mutation group included 14 patients who received sperm-induction therapy, and the mutation-negative group enrolled 25 CHH patients. (2) The incidence of cryptorchidism was 50.0% (7/14) and 12.0% (3/25) in the FGFR1 group and the mutation-negative group, respectively (p=0.019). The baseline testicular volume of the FGFR1 mutation group was smaller than that of the mutation-negative group, 1.6 (0.5–2.0) mL vs. 2 (1.75–4) mL (p=0.033). The baseline luteinizing hormone (LH), Follicle-stimulating hormone (FSH), and testosterone levels were similar between the two groups. (3) Using the Kaplan–Meier and log-rank tests for the analysis of spermatogenesis, it was found that there was no significant difference in the first sperm appearance between the FGFR1 mutation group and the mutation-negative group . The median time of spermatogenesis in the FGFR1 mutation group was longer than that in the mutation-negative group, 16 months vs. 10 months, respectively. The cumulative spermatogenesis success rate at 12 months in the FGFR1 mutation group (35.71%) was lower than that in the mutation-negative group (68.75%) (p=0.047). The sperm concentration in the mutation-negative group was more easily achieved for different thresholds compared with that in the FGFR1 mutation group, but no significant difference was observed (p > 0.05) between the two groups. The last follow-up examination showed that the testicular volume was 7.00 (4.75–12.00) mL and 10.56 ± 4.82 mL (p=0.098), the ejaculate volume of sperm was 2.20 (1.40–2.26) mL and 3.06 ± 1.42 mL (p=0.175), and the sperm concentration was 7.19 (1.00–9.91) million/mL and 18.80 (4.58–53.62) million/mL (p=0.038) in the FGFR1 mutation and mutation-negative groups, respectively, while the sperm motility  was similar for the two groups . The testosterone level during treatment was 366.02 ± 167.03 ng/dL and 362.27 ± 212.86 ng/dL in the FGFR1 mutation and mutation-negative groups, respectively (p=0.956).  (1) FGFR1 mutations have a higher prevalence of cryptorchidism and smaller testicular volume. Although patients with FGFR1 mutations have a similar rate of success for spermatogenesis compared to that of the mutation-negative patients, a longer treatment period was required and a lower sperm concentration was achieved. Patients with  Male congenital hypogonadotropic hypogonadism (CHH) is a secondary testicular hypofunction caused by gonadotropin-releasing hormone (GnRH) secretion or action defects. The incidence in males is approximately 1/5,000–8,000 [ANOS1 (anosmin 1), FGFR1, FGF8 (fibroblast growth factor 8), PROKR2 (prokineticin receptor 2), PROK2 (prokineticin 2), CHD7 (chromodomain helicase DNA binding protein 7), WDR11 (WD repeat domain 11), TACR3 (tachykinin receptor 3), TAC3 (tachykinin 3), KISS1R (KISS1 receptor), NSMF , HS6ST1 (heparan sulfate 6-O-sulfotransferase 1), SOX2 (SRY-box transcription factor 2), and SEMA3A (semaphorin 3A) [FGFR1 mutation [In recent years, a number of gene mutations have been found to cause CHH, including mutation .FGFR1 is an important cause of CHH and previous studies have suggested that FGFR1 mutations may cause more severe damage to the hypothalamic-pituitary-testis axis [FGFR1 expressed on the cellular membranes of various cell types is a member of the fibroblast growth factor receptor (FGFR) family [β (TGF-β) and Adenosine 5′-monophosphate (AMP) activated protein kinase (AMPK) pathways. Overactivation of FGFs and the FGFR1 system can cause multiple tumorigeneses [FGFR1 gene mutations are expressed in a variety of cells throughout the body and are widely involved in the proliferation and differentiation of various cell types [Mutation of tis axis . The pro) family , 7. Ther) family . When thigeneses , whereasigeneses . FGFR1 gll types .FGFR1 mutations. This study aimed to evaluate the effects of spermatogenesis in patients with FGFR1 mutations by comparing the disparity between the FGFR1 group and the mutation-negative group.Currently, there is insufficient research conducted on sperm-inducing therapy in patients with CHH patients who were admitted to the Peking Union Medical College Hospital from January 2012 to March 2020 were recruited, and the clinical data were retrospectively collected.FGFR1 mutation group (FM group): FGFR1 gene mutations were identified by high-throughput next-generation sequencing, then verified by Sanger sequencing, and the pathogenicity was determined by the American College of Medical Genetics and Genomics (ACMG) guidelines [Inclusion criteria: (1) CHH males were diagnosed if the patient fulfilled all the following conditions: no pubertal development after 18 years old, blood testosterone ≤100 ng/dL with a low or inappropriately normal level of serum gonadotropins, normal secretion of other anterior pituitary hormones, and normal magnetic resonance (MR) imaging in the seller region; (2) sperm-inducing therapy was administered to patients; (3) idelines ; (4) theExclusion criteria: (1) patients with any other gene mutations that were pathogenic or likely pathogenic genes of CHH by the ACMG guidelines; (2) patients with uncompleted medical records; (3) patients who did not receive sperm-inducing treatment. Written informed consent was obtained from all patients. The patient inclusive procedure is shown in FGF8 and its receptor FGFR1, GNRH1 and its receptor GNRHR, PROK2 and its receptor PROKR2, TAC3 and its receptor TACR3, LEP and its receptor LEPR, and CHD7, ANOS1, KISS1R, NSMF, WDR11, LHB, FSHB, and PCSK1 [FGFR1 mutations. Sanger sequencing was performed on patients and their parents to verify and track FGFR1 mutations .Pathogenic genes were screened by a next-generation sequencing panel, which included (but not limited to) nd PCSK1 –16. The FGFR1 gene mutation and MN patients undergoing spermatogenesis treatment included the following information: age; body mass index (BMI); clinical characteristics outside the gonadal axis; age for starting spermatogenesis treatment; a history of cryptorchidism; testis volume before and after spermatogenesis treatment; a history of testosterone replacement therapy before spermatogenesis treatment; the ejaculate volume and concentration in the follow-up duration; sperm motility, which was classified as fast progressive sperm (A), slow progressive sperm (B), and nonprogressive sperm (C); LH, FSH, and testosterone levels during the treatment. For LH, FSH, and testosterone, the average values of the last two visits were taken for data analysis. The ejaculate volume, sperm concentration, and sperm motility at the last follow-up were analyzed.Clinical data collected from Testicular volume was measured by two gonadal clinical specialists. During the follow-up examination, testis volume was measured by direct comparison with the orchidometer. If there were bilateral testicles, the average value was taken. If the testis was unilateral (cryptorchidism was on the other side), the descended testis volume was taken for statistical analysis. The average testicular size of the last two visits was defined as the testicular size after therapy for data analysis. The first sperm was detected under the microscope, and semen was centrifugated if necessary.μg/90 min. The dosage was adjusted according to the levels of LH and testosterone, and the level of testosterone was maintained at 200–400 ng/dL. Follow-up was conducted at an interval of 3–6 months. The seminal test, sex hormone, and testis volume were measured during each visit.Patients discontinued androgen therapy (if used) for at least 3 months before starting gonadotropins therapy. Human chorionic gonadotrophin/human menopausal gonadotropin (HCG/HMG) treatment included intramuscular injection of 2000–3000 IU of HCG and 75 IU of HMG twice a week. The HCG dose was adjusted according to the testosterone level, which was maintained at 200–400 ng/dL. Pulsatile GnRH treatment included a subcutaneous injection of pulsatile gonadorelin, starting at a dosage of 10 FGFR1 gene mutations who received spermatogenic therapy. Normally distributed data are expressed as the mean ± SD, and nonnormally distributed data are expressed as the median (quartiles). The nonpaired t-test was used to compare data between two groups, such as the plasma testosterone and testicular volumes, if the data were normally distributed. If the two groups of data were not normally distributed, nonparametric tests were used to test the significance of the difference between the two groups. The Kaplan–Meier and log-rank tests were used for the analysis of spermatogenesis. The chi-square test was used to compare the rates between groups. When p < 0.05, it was considered statistically significant.SPSS version 23.0 was used for analyzing the difference between CHH patients with and without FGFR1 gene mutation. 17 among the 37 patients received sperm-inducing treatment. Among the 17 patients, 14 had complete medical records. A total of 14 patients with FGFR1 gene mutations were included. 25 patients without mutations who matched the FGFR1 group with a complete medical record and matched the age of spermatogenesis, preuse, and use time of testosterone were selected as the control group , the testicular volume was 1.6 (0.5–2.0) mL and 2.0 (1.75–4) mL (p=0.033), the LH level was 0.20 (0.10–0.70) IU/L and 0.35 (0.20–0.72) IU/L, and the baseline testosterone was 24.1 (18.58–37.25) ng/dL and 32.70 (20.65–46.5) ng/dL (measured after stopping testosterone replacement therapy for at least 3 months) in the FM group and the MN group, respectively  years, while in the MN group, 12 patients had been treated with testosterone for 1 (0.5–4) years. The age for starting spermatogenic treatment was 20.00 (18.75–24.75) years and 24.12 ± 5.75 years (ectively .FGFR1 group (50.0%): three were unilateral and four were bilateral. There were three cases of cryptorchidism in the MN group (12.0%): one was unilateral and two were bilateral   .χ2 = 1.974, p=0.160) , and the testosterone level was 366.02 ± 167.03 and 362.27 ± 212.86 (n = 24) ng/dL (p=0.956) in the FM and MN groups, respectively  after induced spermatogenesis therapy. Analysis of spermatogenesis using the Kaplan–Meier and log-rank test showed that there was no difference in the overall spermatogenesis rate between the two groups (p=0.160) . The sucectively . At the ectively .μg/90 min, the LH level increased to 8.46 ± 3.31 IU/L, the FSH level increased to 9.77 ± 6.20 IU/L, and the testosterone level increased to 286.95 ± 130.11 ng/dL. Among these patients, one switched from 12-month gonadotropin therapy to pulsatile GnRH therapy. Ten patients in the FM group received gonadotropin therapy. The final adjusted dosage was 2600 ± 516.40 (2000–3000) IU for HCG and 75 IU for HMG, twice weekly. The testosterone level increased to 397.65 ± 189.78 ng/dL  78 ng/dL .µg/90 min. The other 11 patients received gonadotropin treatment and the testosterone level increased to 431.76 ± 247.42 ng/dL. The final adjusted dosage was 2500 ± 527.05 (2000–3000) IU for HCG and 75 IU for HMG, twice weekly  e weekly .p=0.175), and the sperm concentration was 7.19 (1.00–9.91) million/mL and 18.80 (4.58–53.62) million/mL, respectively (p=0.038) .Analysis of attaining different sperm concentration thresholds  using Kaplan–Meier and log-rank tests showed that there was no significant difference in overall spermatogenesis rate between the FM and MN groups . The medp=0.038) , while tSix patients failed to produce sperm after two years of treatment, two were in the FM group and four were in the MN group. Among these patients, three had a history of cryptorchidism. During the follow-up examination, the testosterone mean level was as low as 79.65 ng/dL (32.8–148 ng/dL), and four patients had testis size smaller than or equal to 5 mL .FGFR1 gene mutations who underwent sperm-inducing treatment. We found that the FM patients had a higher incidence of cryptorchidism  and smaller testis size  compared to patients without positive gene mutations. After a year of sperm promoting therapy, the MN group had a higher rate of achieving successful spermatogenesis than the FM group . After two years of therapy, 75.9% of the patients in the FM group achieved successful spermatogenesis, similar to that of patients without gene mutations (82.14%). The median time needed for first sperm achievement in the FM gene group was longer compared to that of the MN group (16 months vs. 10 months). At the last follow-up, the sperm concentration in the FM group was lower than that in the MN group (7.19 million/mL vs. 18.80 million/mL), although the sperm mobility was similar for the two groups.Our research focused on the clinical characteristics and spermatogenesis of patients with FGFR1 was speculated as pathogenic and disease-causing, based on the following evidence: (1) in this study, patients in the FM group were single-gene mutant; (2) according to the criteria of the ACMG guidelines [FGFR1 gene mutations (11/12) were de novo (Supplementary Table 2), suggesting that the mutated FGFR1 is most likely pathogenic in this group.In our study, mutated idelines , half ofFGFR1 mutations have a more severe impairment of the reproductive phenotype, mainly manifested by a high incidence of cryptorchidism and smaller testis volume during spermatogenesis treatment. Our study found that the incidence of cryptorchidism in patients with FGFR1 gene mutations was 50%, which is consistent with previous studies [FGFR1, ANOS1, and CHD7 mutations is 50–60% [FGFR1 is expressed in Leydig cells, Sertoli cells, and spermatogonia, its function is important for testicular development and spermatogenesis [FGFR1 gene mutations.Patients with  studies , 17. Thes 50–60% , 38.1–66s 50–60% , 0, and s 50–60% , respectogenesis –26. Therp=0.032). In these patients, a longer time was needed to achieve the first appearance of sperm [The presence of cryptorchidism is not only a reflection of severe reproductive axis damage but also a harmful predictive factor of spermatogenesis , 28. It of sperm . Severalof sperm . Higher of sperm . SecondlFGFR1 and MN groups was similar, the median time for first sperm appearance was longer in the FM group.In our study, after two years of sperm-inducing treatment, the success rate of the FM and MN groups was 75.89% and 82.14%, respectively. According to the meta-analysis results from several studies, the overall success rate is 74–90% in patients receiving pulsatile GnRH therapy  and 69.2FGFR1 mutation, some presented CHH, while others showed delayed puberty [PROKR2 mutations also have variable reproductive conditions [There is no study on the evaluation of spermatogenic effects on patients with different genotypes. The possible reasons are as follows: 1) it is difficult to make a gene diagnosis on patients with CHH, as many patients do not have gene variations, while some have multiple gene mutations . (2) Pat it is di puberty . Patientnditions . These vFGFR1 may impair spermatogenesis at the hypothalamus, pituitary, and testis levels. It was found that 25.6% (23/90) of CHH patients had a poor testicular response to gonadotropins and 11.1% (10/90) had a poor response to pulsatile GnRH therapy, indicating defects in the pituitary gland and testicular levels [FGFR1 gene mutations may result in a weaker response of Leydig cells and spermatogonia to gonadotropins. Therefore, patients need longer treatment time to promote testicular volume and spermatogenesis.r levels . FGFR1 gFGFR1 gene mutations and cryptorchidism history had a failure of spermatogenesis after two years of treatment. In patient 1  In this study, the MN group was defined by having no known CHH related genes. Other gene mutations causing CHH could not be excluded. (3) Pulsatile GnRH and gonadotropin therapies have different mechanisms to induce sperm. Due to the small sample size, the disparity between the two therapeutic methods could not be further analyzed. (4) The mutation pathogenicity was determined based on the ACMG guidelines, not by in vitro verification.The shortcomings of this study are as follows: (1) the sample size is too small to accurately reflect the entire possible spermatogenic outcomes in the FGFR1 mutations, presenting a higher incidence of cryptorchidism, smaller testicular size, and longer time required for sperm production. However, the rate of successful spermatogenesis in different sperm concentration thresholds was similar between patients with FGFR1 mutations and nonmutation patients, providing valuable evidence and confidence for sperm promoting therapy for these patients.In conclusion, gonadal axis defects were more severe in CHH patients with


Aconiti Lateralis Radix Praeparata to reduce alkaloids-related toxicity of the latter. Such herb-pairing also results in better therapeutic effect in heart failure, as compared to the singular use of either herb. The purpose of this study was to investigate the effect of Aconiti Lateralis Radix Praeparata and its combination with red ginseng on the activities of CYP450 enzymes in rats. A sensitive and reliable HPLC-MS/MS method was established and validated for the simultaneous determination of eight probe drugs, phenacetin (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), dapsone (CYP3A4), 7-hydroxycoumarin (CYP2A6), bupropion (CYP2B6), and amodiaquine (CYP2C8), in rat plasma using diazepam as internal standard (IS). The chromatographic separation was performed on a Waters XBridge™ C18 column  using a gradient elution with the mobile phase consisting of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.3 mL/min. The method was successfully applied in evaluating the effect of Aconiti Lateralis Radix Praeparata and red ginseng on the activities of CYP450 enzymes. The pharmacokinetic results of the eight probe drugs suggested that Aconiti Lateralis Radix Praeparata may inhibit the activity of CYP2A6, CYP2C19, CYP2B6, CYP1A2, CYP3A4, and CYP2C9 enzymes in rats. Comparison between the two groups, Aconiti Lateralis Radix Praeparata combined with red ginseng and Aconiti Lateralis Radix Praeparata, indicated that red ginseng may inhibit the activity of CYP2D6 and CYP2B6 enzymes while inducing the activity of CYP1A2, CYP3A4, and CYP2C9 enzymes.Red ginseng is often combined with  Aconiti Lateralis Radix Praeparata (“Fuzi” in Chinese), recorded in Chinese Pharmacopoeia (2015 edition), is the lateral root of Aconitum carmichaelii Debx., belonging to family Ranunculaceae. The major chemical components of Aconiti Lateralis Radix Praeparata are alkaloids including diester diterpenoid alkaloids, monoester diterpenoid alkaloids, and lipid alkaloids. Aconiti Lateralis Radix Praeparata has multiple pharmacological properties, including antiarrhythmia, anti-inflammatory, antitumor, and antiaging properties, but it has very narrow therapeutic range due to its severe toxicity [Aconiti Lateralis Radix Praeparata is commonly prescribed with red ginseng  for treating heart failure [Aconiti Lateralis Radix Praeparata is able to reduce the contents of diester diterpenoid alkaloids (hypaconitine and deoxyaconitine) and increase the contents of monoester diterpenoid alkaloids (benzoylmesaconine and benzoylhypaconine) in Aconiti Lateralis Radix Praeparata, thus strengthening the therapeutic effect of Aconiti Lateralis Radix Praeparata while decreasing its toxicity [Aconiti Lateralis Radix Praeparata combined with red ginseng is still unclear in vivo.opoeia 205 editiontoxicity . Clinicatoxicity , 7. HoweAconiti Lateralis Radix Praeparata-red ginseng herbal formulation. Compounds such as Ginsenosides Rc, Rf, and Rb2 induced CYP3A4 metabolic enzymes [Aconiti Lateralis Radix Praeparata were identified as substrates of CYP3A4 metabolic enzymes [Aconiti Lateralis Radix Praeparata and its combination with red ginseng on CYP450 enzymes.CYP450 family is the most important drug-metabolizing system, playing a vital role in the metabolism of endogenous and exogenous compounds , 9. It i enzymes , 16, whi enzymes . HoweverAconiti Lateralis Radix Praeparata and its combination with red ginseng on CYP450 enzymes. The results would be significant in explaining the mechanism of toxic-reduction and therapeutic-enhancing effects of Aconiti Lateralis Radix Praeparata and red ginseng, which would provide a theoretical basis for rational usage of red ginseng and Aconiti Lateralis Radix Praeparata in clinical.Cocktail approach has become one of the basic analytical tools to assess the impact of DDIs on CYP450 enzymes in vivo. This method not only provides information of multiple CYP450 enzymes activities in a single experiment but also minimizes the intersubject and intrasubject variability –20. CompAconiti Lateralis Radix Praeparata were made in the laboratory, both of which were extracted with 70% ethanol. The extraction rates of red ginseng and Aconiti Lateralis Radix Praeparata were 43.36% and 15.38%, respectively.Acetonitrile (chromatographic purity) and methanol (chromatographic purity) were purchased from Merck Co., Ltd. Formic acid (chromatographic purity) was obtained from ROE Co., Ltd. Ultrapure water was prepared with a Milli-Q water purification system . Omeprazole, dextromethorphan, bupropion, 7-hydroxycoumarin, and amodiaquine were purchased from the China National Institutes for Food and Drug Control. Phenacetin, tolbutamide, and diazepam were obtained from Tianjin Vientiane Hengyuan Technology Co., Ltd. Dapsone was purchased from J&K Scientific Ltd. The extracts of red ginseng and μm) maintained at 30°C. Mobile phases consisting of 0.1% formic acid in water (A) and acetonitrile (B) were used in the following gradient elution method: 0–2 min, 10%–30% B; 2–7 min, 30%–80% B; and 7–10 min, 80%–80% B. The flow rate was 0.3 mL/min, and the injection volume was 5 μL. All data were analyzed by Mass Hunter workstation software .The HPLC-MS/MS system consists of an Agilent 1200 high-performance liquid chromatography coupled with an Agilent 6430 series triple quadrupole mass spectrometer with an electrospray ionization (ESI) source. The chromatographic separation was performed on a Waters XBridge™ C18 column  flow rate, 11 L/min; and capillary temperature, 350°C. The precursor-product ion pairs used for the MRM detection and MS parameters are shown in The mass spectrometer was carried out in positive ionization multiple reaction monitoring (MRM) mode. The optimum MS values were maintained as follows: nebulizing gas pressure, 25 psi; drying gas , the dosage of red ginseng extract was 2.34 g/kg. Appropriate amounts of Aconiti Lateralis Radix Praeparata and red ginseng were weighed, prepared with 0.5% CMC-Na suspension solution, and stored at 4°C.The dosage of In order to make the stock solutions (1 mg/mL), phenacetin, tolbutamide, omeprazole, dextromethorphan, dapsone, 7-hydroxycoumarin, bupropion, amodiaquine, and diazepam  were separately weighed and dissolved in methanol. The working solutions of the eight probe drugs were prepared by mixing and diluting the primary stock solutions with methanol to obtain concentrations of 50–25000 ng/mL for 7-hydroxycoumarin; 10–5000 ng/mL for phenacetin, bupropion, and amodiaquine; 5–2500 ng/mL for dextromethorphan and dapsone; 30–15000 ng/mL for tolbutamide; and 20–10000 ng/mL for omeprazole. The working solution of diazepam (IS) was prepared at a concentration of 1000 ng/mL in methanol.μL of working solutions and 20 μL of IS solutions to 100 μL blank plasma to give nominal concentration range of 10–5000  ng/mL for 7-hydroxycoumarin; 2–1000  ng/mL for phenacetin, bupropion, and amodiaquine; 1–500  ng/mL for dextromethorphan and dapsone; 6–3000  ng/mL for tolbutamide; and 4–2000  ng/mL for omeprazole.The calibration standard solutions were prepared by adding 20 Quality control (QC) samples at three concentrations  were made up of appropriate mixed standard solutions with blank plasma and IS solutions as calibration standard solutions to meet the required concentrations. All the solutions were stored at 4°C.μL plasma sample, 20 μL IS , and 20 μL methanol were added to a centrifuge tube and mixed by vortexing. Liquid-liquid extraction was performed by adding 1 mL ethyl acetate to the centrifuge tube. The mixture was then vortexed for 3 min and centrifuged at 14,000 rpm for 10 min. The supernatant was transferred into a new tube and evaporated to dryness under a nitrogen stream. The residue was reconstituted with 100 μL methanol and vortexed for 3 min, followed by centrifugation at 14,000 rpm for 10 min. 5 μL supernatant was injected into the LC-MS/MS system for analysis.100 The method was validated with respect to specificity, linearity, accuracy, precision, extraction recovery, matrix effect, and stability according to bioanalytical method validation.The specificity of the method was investigated by comparing chromatograms of blank plasma samples from six different rats, blank plasma spiked with the eight probe drugs and IS, and a rat plasma sample after the tail vein administration of cocktail probes.y) versus the concentration of analyte in spiked plasma samples (x), using 1/x2 as a weighing factor. The lower limit of quantification (LLOQ) was determined at a signal-to-noise ratio of about 10 by analyzing the standard plasma samples.Calibration curves were achieved using a series of calibration standard samples and constructed by plotting the peak area ratio analyte to IS , while the accuracy was determined by the relative error (RE).The extraction recovery and matrix effect of eight analytes were calculated at three concentration levels, each in six replicates. The extraction recovery was calculated using the ratio of peak area of the extracted samples and that of postextraction spiked samples. The matrix effect was obtained by comparing the peak areas of the postextraction spiked samples versus standard solutions.The stability of eight probe drugs was analyzed in six replicates at three concentration levels. The QC samples were prepared for 4 h at room temperature, three freeze-thaw cycles, 12 h in autosampler, and 14 days at −70°C.Aconiti Lateralis Radix Praeparata extract by oral administration for 7 consecutive days and then dosed 10 mL/kg cocktail solution through the tail vein on the eighth day in group B. The rats of group C were administered 0.415 g/kg Aconiti Lateralis Radix Praeparata extract and 2.34 g/kg red ginseng extract by oral administration for 7 consecutive days and dosed 10 mL/kg cocktail solution through the tail vein on the eighth day. Blood samples of each rat were collected from the fossa orbitalis vein at time points 0.03, 0.08, 0.17, 0.25, 0.33, 0.50, 0.75, 1, 2, 4, 6, 8, 10, 12, and 24 h after tail vein administration. The blood samples (200 μL) were transferred into heparinized microcentrifuge tubes and centrifuged at 6000 rpm for 10 min. All plasma samples were stored at –70°C. The animal studies described in this paper were approved and conducted in accordance with the guidelines of Laboratory Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (TCM-LAEC20180057).Male Sprague-Dawley rats (190–230 g) were obtained from Beijing HFK Experimental Animal Technology Co., Ltd. The rats were fed with standard diet and water in a stable environment, with temperatures between 23 and 26°C and relative humidity of 40–60%. After a week of acclimation, the animals were fasted 12 h before the PK experiment and allowed free access to water during the experiment. 18 rats were randomly divided into three groups. The rats were given 10 mL/kg cocktail solution  through the tail vein in group A. The rats were administered 0.415 g/kg Data were presented as mean ± SD. Pharmacokinetic parameters were calculated by the computer program “Drug and Statistics 1.0”  and analyzed with one-way-ANOVA by SPSS 17.0 statistical software.Acetonitrile was selected as the mobile phase as it has strong elution ability. The responses of eight probe drugs and IS were improved after adding 0.1% formic acid to water. The analytes have good retention time, peak symmetry, and appropriate ionization in the mobile phase with gradient elution.m/z 346.2/136.1 for omeprazole, 249.1/92.1 for dapsone, 272.2/147.1 for dextromethorphan, 180.1/110.1 for phenacetin, 271.1/91.0 for tolbutamide, 240.1/184.1 for bupropion, 356.2/283.1 for amodiaquine, 163.0/107.1 for 7-hydroxycoumarin, and 285.0/193.0 for diazepam (IS) were chosen for the quantification studies.The eight probe drugs were monitored in positive and negative ion modes, and it was discovered that ions in positive mode had more stable and stronger MS signal than in negative mode. The transitions The representative chromatograms of a blank plasma (A), a rat plasma sample after the tail vein administration of cocktail probes at 10 mL/kg (B), and blank plasma sample spiked with probe drugs and IS (C) are shown in r > 0.991) in the calibration ranges of each probe drug in rat plasma. The regression equations, linear ranges, correlation coefficients, and LLOQ are displayed in The curves were linear , and AUC(0-∞) of 7-hydroxycoumarin, omeprazole, bupropion, phenacetin, dapsone, and tolbutamide in group B were increased, and MRT(0-24) of phenacetin, dapsone, and tolbutamide were significantly added (P < 0.05 or P < 0.01). The results suggested that Aconiti Lateralis Radix Praeparata may inhibit the activity of CYP2A6, CYP2C19, CYP2B6, CYP1A2, CYP3A4, and CYP2C9 enzymes in rats. The pharmacokinetic parameters of dextromethorphan and amodiaquine in group B had no significant differences when compared to group A, implying that Aconiti Lateralis Radix Praeparata showed little alteration on CYP2D6 and CYP2C8 activity in vivo.Pharmacokinetic profiles of eight probe drugs after max, AUC(0-24), and AUC(0-∞) of dextromethorphan and bupropion in group C were significantly increased (P < 0.01), while the half-life (t1/2) of dextromethorphan and bupropion was markedly reduced (P < 0.01). The results showed that metabolism of dextromethorphan and bupropion in group C was slowed. But Cmax, AUC(0-24), and AUC(0-∞) of phenacetin, dapsone, and tolbutamide were significantly decreased (P < 0.05 or P < 0.01), indicating that red ginseng may enhance the metabolism of phenacetin, dapsone, and tolbutamide. Thus, these pharmacokinetic parameters suggested that red ginseng may inhibit the activity of CYP2D6 and CYP2B6 enzymes but induce the activity of CYP1A2, CYP3A4, and CYP2C9 enzymes in rats.Alteration in CYP450 enzymes activity caused by red ginseng was estimated by comparing the pharmacokinetic results of eight probe drugs in rats by different treatment methods. Compared with group B, CAconiti Lateralis Radix Praeparata is mainly attributed to its diester diterpenoid aconitines such as aconitine, hypaconitine, and mesaconitine [Aconiti Lateralis Radix Praeparata are substrates of CYP3A4 metabolic enzymes [Aconiti Lateralis Radix Praeparata and its combination with red ginseng (max and AUC(0-∞) of aconitine, hypaconitine, and mesaconitine of Aconiti Lateralis Radix Praeparata-red ginseng were decreased as compared to those of Aconiti Lateralis Radix Praeparata administration. Therefore, red ginseng may accelerate the metabolism of toxic components in Aconiti Lateralis Radix Praeparata by inducing the activity of CYP3A4 enzymes. This experiment provides a theoretical basis for explaining the interaction mechanism of Aconiti Lateralis Radix Praeparata combined with red ginseng, which would provide reference for rational usage of red ginseng and Aconiti Lateralis Radix Praeparata in clinic.The toxicity of conitine , 24. Acc enzymes . Further ginseng . Cmax anAconiti Lateralis Radix Praeparata and its combination with red ginseng on rat CYP450 enzymes. The results indicated that Aconiti Lateralis Radix Praeparata may inhibit the activity of CYP2A6, CYP2C19, CYP2B6, CYP1A2, CYP3A4, and CYP2C9 in rats. Comparison between the two groups of Aconiti Lateralis Radix Praeparata with or without red ginseng showed that red ginseng may inhibit the activity of CYP2D6 and CYP2B6 while inducing the activity of CYP1A2, CYP3A4, and CYP2C9 enzymes.In this study, a sensitive and rapid LC-MS/MS method was developed and validated for the simultaneous determination of eight probe drugs in rat plasma to evaluate the effect of


Lactuca sativa L.) “Rouxai” in a growth room at 23°C and under a 20-h photoperiod created by warm-white (WW), blue , green , red , and/or far-red  light-emitting diodes. From day 0 to 11, plants received six static lighting treatments with the same total photon flux density (400–800 nm): WW180, R180, B20R160, B20G60R100, B20R100FR60, or B180 (subscripts denote photon flux densities in μmol⋅m–2⋅s–1). On day 11, plants grown under each of the six treatments were transferred to all treatments, which created 36 temporal spectrum alternations. Plant growth, morphology, and coloration were measured on days 11 and 25. Increasing B radiation from 0 to 100% in static treatments decreased shoot fresh and dry weights and increased foliage redness of seedlings and mature plants. Compared to B20R160, B20R100FR60 increased shoot fresh weight, but not dry weight, on both days. However, other phenotypic responses under static treatments changed over time. For example, leaf length under B180 was 35% lower on day 11 but similar on day 25 compared to that under R180. In the B20 background, substituting G60 for R radiation did not influence shoot weight on day 11 but decreased it by 19% on day 25. When plants were switched from one treatment to another on day 11, the treatments applied before day 11 influenced final shoot weight and, to a lesser extent, leaf length and foliage coloration on day 25. In comparison, effects of the treatments applied after day 11 were more pronounced. We conclude some phenotypic responses to light quality depend on time and sequential light quality treatments had cumulative effects on lettuce growth. The temporal complexity of spectral responses is critical in photobiological research and creates opportunities for time-specific spectrum delivery to optimize crop characteristics.Lighting is typically static for indoor production of leafy greens. However, temporal spectrum differentiation for distinct growth phases can potentially control age-specific desirable traits. Spectral effects can be persistent yet dynamic as plants mature, necessitating characterization of time-dependent responses. We grew red-leaf lettuce ( The spectral composition of lighting in controlled environments can regulate a wide range of commercially relevant crop traits such as harvestable yield, morphology, coloration, and nutritional quality . Red R;. In contArabidopsis thaliana (L.) Heynh.], K+ efflux antiporter 3 mediated H+/K+ antiport to facilitate rapid restoration of photosystem II quantum efficiency after plants were transferred from high to low light or from darkness to low light  is unnecessary for seedlings but desirable for mature plants at harvest. It can be induced rapidly by ≥4 days of end-of-production supplemental lighting from B and/or R LEDs (Brassica napus L. var. sabellica) seedlings, showing strong plant plasticity in response to spectral changes (Lactuca sativa L. var. longifolia) compared to simultaneous B + R radiation  promoted flowering when plants were finished in a greenhouse environment  radiation and created a wide array of lighting treatments shifted temporally between the seedling and mature phases of indoor lettuce production. The objectives of this study were (1) to investigate how spectral treatments for lettuce seedlings influence phenotypes of mature plants grown under different spectra; (2) to compare lettuce growth under single wavebands, combinations of two or three wavebands, and WW radiation; and (3) to find temporal spectral combinations for desirable lettuce growth and morphology. We postulated that (1) the spectral effects during the seedling stage would persist through the mature phase, regardless of the finishing spectral environment; (2) substituting G radiation for R radiation would increase lettuce growth during the seedling stage but have little influence on growth of mature plants; and (3) B radiation alone would inhibit leaf expansion and dry weight during the seedling phase but promote them during the mature phase.2O5–16K2O RO Hydro FeED; JR Peters, Inc., Allentown, PA, United States), and magnesium sulfate  to achieve a pH of 3.9 and an electrical conductivity of 1.6 mS⋅cm–1. The nutrient solution contained the following nutrients (in mg⋅L–1): 125 N, 42 P, 167 K, 73 Ca, 49 Mg, 39 S, 1.7 Fe, 0.52 Mn, 0.56 Zn, 0.13 B, 0.47 Cu, and 0.13 Mo. Seed trays were covered with transparent humidity domes and placed under six different lighting treatments, each at a total photon flux density  of 180 μmol⋅m–2⋅s–1 with a 20-h photoperiod. Air temperature was set at 20°C from April 28 to 30, 2018 and increased to 23°C for the remainder of the experiment. From day 1 to 11, seedlings were subirrigated as needed using the same nutrient solution with a pH of 5.8 adjusted with potassium bicarbonate. The humidity domes were removed on May 3, 2018 for both blocks.This experiment was performed in a refrigerated walk-in growth room of the Controlled-Environment Lighting Laboratory . We chose lettuce to study because it is the most widely grown hydroponic crop in indoor vertical farms for its short stature, fast growth rate, and high value. Our previous studies showed generally similar growth responses of green- and red-leaf lettuce, so we studied red-leaf lettuce because of its unique foliage coloration in response to spectral alternations . Seeds o–1): 150 N, 50 P, 200 K, 88 Ca, 58 Mg, 47 S, 2.1 Fe, 0.63 Mn, 0.68 Zn, 0.15 B, 0.56 Cu, and 0.15 Mo. It was oxygenated with a circular air stone  connected to a 60-W air pump . The pH, electrical conductivity, and temperature of the nutrient solution for each lighting canopy were measured daily using a portable pH and electrical conductivity meter   floating in flood tables  on three-tier racks . Plants were spaced 20 cm apart horizontally and 15 cm apart diagonally. The recirculating nutrient solution was mixed as described for seedlings to provide the following nutrients (in mg⋅L States) . Potassi2 transmitter , and a relative humidity and temperature probe . All sensors were connected to a datalogger  with a multiplexer , which recorded environmental parameters every 10 s and logged hourly averages using computer software . The air temperature, canopy temperature, CO2 concentration, and relatively humidity throughout the experiment (mean ± standard deviation) were 22.5 ± 1.0°C, 24.1 ± 0.9°C, 392 ± 31 ppm, and 44 ± 8%, respectively.Temperature in the growth room was regulated with an industrial ventilation and air-conditioning unit  connected to a wireless thermostat . The deep-flow hydroponic system was equipped with two light quantum sensors , two thermocouples , two infrared sensors , a CO180, R180, B20R160, B20G60R100, B20R100FR60, or B180 LEDs , where the subscript following each LED type indicates its photon flux density (in μmol⋅m–2⋅s–1). The peak wavelengths of WW, B, G, R, and FR LEDs were 639, 449, 526, 664, and 733 nm, respectively. The outputs of seven color channels, including five used in this study, in each LED fixture were independently controlled with software . The specifications, layout, and positioning of the LED fixtures were as described by Seedlings were grown under WWL∗, a∗, and b∗ indicate foliage brightness (ranging from 0 for black to 100 for diffuse white), greenness–redness (corresponding to negative–positive directions), and blueness–yellowness (corresponding to negative–positive directions), respectively. Subsequently, plants were dried in an oven  at 60°C for ≥5 days followed by dry weight measurements with the same analytical balances as for shoot fresh weight.Shoot fresh and dry weights, leaf morphology, and coloration data were collected on ten young lettuce plants per block grown under each of the six static lighting treatments on day 11 and on eight mature lettuce plants per block grown under each of the 36 temporal lighting combinations on day 25. Shoot fresh weight was measured with an analytical balance  for young plants and a different one  for mature plants based on capacities. Length of the fifth most mature true leaf was measured to quantify extension growth. The International Commission on Illumination Lab color space analysis was conducted on a representative leaf per plant using a colorimeter . n = 10), assuming fixed block effects. Data from alternate treatments were analyzed as a strip-split-plot design with two blocks, six whole-plot levels (post-transplant lighting treatments), six subplot levels (pre-transplant lighting treatments), and subsampling (n = 8), assuming fixed block effects. The split-plot design included whole plots arranged in a randomized complete block design.Data on young and mature lettuce plants were analyzed with the PROC MIXED procedure and Tukey’s honestly significant difference test (α = 0.05) in SAS . Data from static treatments were analyzed as a randomized complete block design with two blocks (using opposite racks of the growth room), six static lighting treatments, and subsampling (180 than under WW180 and R180 (20R160 with 60 μmol⋅m–2⋅s–1 of G radiation (B20G60R100) or FR radiation (B20R100FR60) did not influence shoot dry weight, whereas the substitution with FR radiation increased shoot fresh weight by 18%. Substituting 20 μmol⋅m–2⋅s–1 of B radiation for R radiation (B20R160 versus R180) decreased shoot dry weight by 15%, but not shoot fresh weight. On day 25, increasing substitution of R radiation with B radiation decreased shoot fresh and dry weights  was the greatest under R180, followed by WW180 and B20R100FR60  and yellowest (highest b∗) under R180, followed by WW180 and B20R100FR60, and the reddest and least yellow under B180  and eventual (applied day 11–25) lighting treatments on lettuce harvested on day 25, data of plants grown under the same initial treatments were pooled for initial treatment analysis, whereas data of plants grown under the same eventual treatments were pooled for eventual treatment analysis. The effects of the six lighting treatments on final shoot fresh and weight weights, leaf length, and color parameters were different when applied day 0–11 versus day 11–25 ,6.180, R180, and B20R100FR60, followed by B20R160 and B20G60R100, and the lowest under B180  were the greatest under WWder B180 . In addiand B180 . Leaves der B180 . Otherwi180, followed by WW180 and B20R100FR60, and the lowest under B180  and cherry tomato (Solanum lycopersicum L. var. cerasiforme) seedlings and lettuce “Grand Rapids”  seedlings but did not influence shoot fresh or dry weight  subsided. This could explain why leaf length under B20R100FR60 had similar shoot dry weight and 17–22% higher shoot fresh weight (partly due to increases in moisture content) compared to that under B20R160, although B20R100FR60 was 32% lower in the PPFD and 26% lower in the YPFD. The similar TPFDs across all lighting treatments cannot explain differences in shoot dry weight. In addition, 20R100FR60 versus B20R160) was likely the product of the reduced YPFD (74% of that for B20R160) and increased light interception (141–142% of that under B20R160). This suggests that changes in shoot dry weight can be predicted by multiplying percentage changes in the YPFD (to account for the changing instantaneous photosynthetic rate and quantum efficiency) and percentage changes in leaf size . The YPFD is a better predictor of plant biomass than the PPFD because it accounts for relative quantum efficiency and the contribution of FR radiation to net photosynthesis, albeit less significant than B, G, or R radiation. Lastly, light interception may be better estimated with leaf area instead of leaf length.In contrast, FR radiation was a stronger shade signal than G radiation at the same photon flux density  and cons–2⋅s–1 increased anthocyanin concentration of lettuce “Red Sails” in a dose-dependent manner; however, the inclusion of G radiation reduced anthocyanin accumulation in lettuce “Red Sails” and arabidopsis  by additional FR radiation applied during the seedling phase (When lettuce “Rouxai” was grown under different initial treatments day 0–11 but the same eventual treatments day 11–25, initial light quality had a residual effect on final shoot fresh and dry weights, responses of which generally resembled those under static treatments. For example, for plants transferred to the same Rtransfer . Contrarperiment . Such unay 17–45 . Increasay 17–45 . Althougay 17–45 . The latng phase , 2018.Although light quality during the seedling phase modified shoot fresh and dry weights of mature lettuce, the magnitude of this modification was less pronounced than that by light quality in the mature phase. As the plant underwent the exponential growing phase, light interception increased drastically with leaf development, which likely led to greater impacts of eventual treatments on photosynthesis and morphology. In addition, leaf length and coloration of mature lettuce were primarily controlled by eventual lighting treatments and negligibly affected by initial ones. The greater influence of eventual light quality on foliage coloration could at least partly be attributed to rapid anthocyanin accumulation in lettuce under light stresses within days . In geneThe following conclusions are in response to the three original hypotheses. First, effects of light quality applied during the seedling phase persisted into the mature phase, although they were less pronounced than those applied during the mature phase. Second, substituting substantial G radiation for R radiation did not influence growth of seedlings but decreased growth of mature lettuce. Third, B radiation alone decreased lettuce shoot weight during both the seedling and mature phases. However, compared to R radiation, B radiation alone suppressed leaf elongation during the seedling phase but promoted it during the mature phase. In addition to testing hypotheses, we conclude temporally alternating light quality improved precision of phenotype control over static lighting. Thus, differential lighting treatments could be delivered at various developmental stages to optimize crop growth and quality attributes. Our results suggest that lettuce biomass can be maximized with WW, R, or B + R + FR radiation during propagation, followed by R radiation during production. End-of-production B radiation can be used to induce anthocyanin accumulation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.QM conceived and performed the experiment, collected and analyzed data, and wrote the first draft of the manuscript. ER was the recipient of funds and reviewed experimental design. Both authors contributed to the manuscript revision and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Eucalyptus radiata, Laurus nobilis, and Myristicafragrans EOs displayed inhibitory capacities comparable to that of the positive control . A bio-guided fractionation approach was adopted to isolate and identify the active fractions/compounds of Eucalyptus radiata and Myristica fragrans EOs. The bio-guided fractionation revealed that EOs α-amylase inhibitory activity is often the result of antagonist, additive, or synergistic interactions among their bioactive constituents and led to the identification of 1,8-cineole, 4-terpineol, α-terpineol, α-pinene, and β-pinene as bioactive compounds, also confirmed when they were tested singularly. These results demonstrate that EO oils are a promising source of potential α-amylase inhibitors.Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by unpaired blood glycaemia maintenance. T2DM can be treated by inhibiting carbohydrate hydrolyzing enzymes (α-amylases and α-glucosidases) to decrease postprandial hyperglycemia. Acarbose and voglibose are inhibitors used in clinical practice. However, these drugs are associated with unpleasant gastrointestinal side effects. This study explores new α-amylase inhibitors deriving from plant volatile specialized metabolites. Sixty-two essential oils (EOs) from different plant species and botanical families were subjected to α-amylase in vitro enzymatic assay and chemically characterized using gas chromatography coupled to mass spectrometry. Several EOs were found to be potential α-amylase inhibitors, and  The term "diabetes mellitus" groups together a series of metabolic disorders characterized by hyperglycemia that is caused by the impairment of insulin secretion, action, or both. If not treated, chronic hyperglycemia, in association with diabetes, leads to long-term damage, dysfunction and organ failure especially in the eyes, kidneys, heart, nerves, and blood vessels . DiabeteIpomoea batatas (L.) Poir. (Convolvulaceae) and Pharbitis nil (L.) Choisy (Convolvulaceae), and polyphenols from Pinus pinaster Aiton (Pinaceae), have effective inhibitory activity against intestinal α-glucosidase and α-amylases from human salivary glands [Medicinal plants have proven to be potentially promising in the treatment of T2DM. Hydroalcoholic and aqueous extracts that are rich in phenolic compounds, such as anthocyanins from y glands ,6,7. Mentha x piperita and Carum carvi EOs) [Mentha x piperita EO) [Relatively less attention has been paid to volatile compounds  and to essential oils (EOs). They are complex mixtures of volatiles that are either obtained by steam distillation, dry distillation, or by mechanical means from a single plant species. Terpenoids are the most important group of specialized metabolites in EOs, and include, in particular, hemiterpenoids (C5), monoterpenoids C10), and sesquiterpenoids C15), whose volatility is compatible with the steam distillation process . In addirvi EOs) , irritab, and sesrita EO) ,12, gastrita EO) , and therita EO) ,15. To t, whose vrita EO) ,17,18. This study deals with the preliminary screening of 62 hydrodistilled EOs from different botanical families and species, in order to evaluate their potential hypoglycemic activity . Bio-guided fractionation, based on an in vitro α-amylase inhibition assay, was also performed to isolate the bioactive fractions and/or to identify the EO components with the most significant activity. Gas chromatography coupled to mass spectrometry (GC-MS) was used to identify the compounds that are characteristic of both the EOs and the isolated fractions, and that are responsible for their α-amylase inhibition activity.n = 5) and pH variation negligible (from 7.00 to 7.13) and ethyl acetate was therefore selected as the “bridge” solvent. The required EO final concentration in the reaction mixture was set at 0.670 mgmL−1 to match the half maximal inhibitory concentration (IC50) of acarbose (0.601 ± 0.0673 mg mL−1). To minimize the amount of ethyl acetate in the reaction mixture, thus avoiding partial interference with the enzymatic activity, 10 μL of a 200 mg mL−1 solutions in ethyl acetate of each investigated EO were added to the enzymatic assay reaction mixture.EOs are poorly soluble in water, and, as in vitro enzymatic tests are carried out in an aqueous buffer solution, the choice of the solvent to be used as a “bridge” to solubilize the EO in the mixture test is the first aspect to be optimized, while avoiding interference with 1) enzymatic activity, 2) UV absorption, and 3) pH variation. Three solvents were tested as solubilizing solvents: ethyl acetate was the only one giving a clear solution when adding 50 μL of the EO solution, while methanol, and ethanol gave opalescent solutions. Absorbance at 540 nm and the pH variation of a 1 mL of buffer solution containing 50 μL of ethyl acetate were measured to evaluate any possible interference. Absorbance was found to be very low . A 4.0 mL vial was used for all of the in vitro inhibition experiments as the total volume of the reaction mixture was 3.0 mL.Eucalyptus radiata, Myristica fragrans, and Laurus nobilis, which are characterized by inhibition activities higher than, or very similar to, that of acarbose, 65%, 59%, and 51%, respectively. Within the Lauraceae family, in addition to laurel EO, Cinnamomum camphora (20%) showed interesting activity, although it was lower than that of acarbose. Similar considerations can be made for other botanical families, with several species presenting interesting inhibition activity, i.e., Myrtaceae , Lamiaceae , and Compositae (Artemisia vulgaris 48% and Matricaria chamomilla 32%).The inhibitory activity against α-amylase of the EOs listed in 2 = 0.9985, 95% confidence interval for the slope 1.3 < β < 1.5; SEb = 0.10, t0.005(2), 3 = 3.18). The results obtained using the two approaches were compared, in particular for the three most active EOs, adopted as reference, i.e., laurel, nutmeg, and eucalyptus  in function of maltose concentration. Knowing the final absorbance at 540 nm, the effective amount of maltose liberated during the enzymatic reaction was determined by interpolation from the regression line of A 540nm on maltose concentration , fennel and clove, among others. Twenty-three EOs belonging to different botanical families were found to be inactive in inhibiting α-amylase at the tested concentration under the applied experimental conditions: All EOs, both active and inactive, were analyzed using gas chromatography, with flame ionization (FID) and mass spectrometry (MS) detectors, to characterize their composition and evaluate the relative percentage abundance of hydrocarbons and oxygenated compounds for correlation with the α-amylase inhibition activity. Cinnamomum camphora, Citrus limon, Melaleuca alternifolia, Myrtus communis, and Origanum majorana. Only eight EOs are characterized by high percentages (above 70%) of hydrocarbons: Citrus limon, Citrus medica, Citrus nobilis, Citrus × aurantium, Cupressus sempervirens, Juniperus communis, Juniperus virginiana, and Myristica fragrans. The others mostly consisted of oxygenated compounds (from 62.2% of Carum carvi to 99.1% of Mentha × piperita). The total composition of all investigated EOs is reported in the Eucalyptus radiata and Myristica fragrans were selected from the most active EOs for further bio-guided assay fractionation; the first is representative of a class of EOs with a composition that is prevalently oxygenated monoterpenes, and the latter of a class of EOs that mainly consists of terpenic hydrocarbons. While inhibiting α-amylase to nearly the same extent, Eucalyptus radiata and Myristica fragrans EOs have significantly different chemical compositions, and their bioassay-oriented fractionation should thus provide good insight into the components that may be responsible for their biological activity.The pure oxygenated and hydrocarbon fractions of the investigated EOs were isolated using automated flash chromatography and were individually subjected to α-amylase inhibitory assays. The amount of the EO (960 mg) to be fractionated was selected so that a suitable amount of each fraction could be recovered for the next biological testing. Myristica fragrans EO, and 744.7 mg and 82.4 mg, respectively, for Eucalyptus radiata. Each isolated fraction was analyzed using GC-MS and GC-FID to assess the chemical composition of the fraction and its chemical homogeneity. Eucalyptus radiata and Myristica fragrans EO together with those of the corresponding hydrocarbon and oxygenated fractions. Eucalyptus radiata and Myristica fragrans EOs, and of their hydrocarbon and oxygenated fractions, as obtained by flash chromatography.Flash chromatography separations were carried out on pre-packed 50 μm silica gel cartridges using a gradient of 0–20% ethyl-acetate in petroleum ether. All of the hydrocarbons eluted during the isocratic step of the gradient with 100% petroleum ether, and the complete fractionation of the pure material was completed within 20 minutes, regardless of the EO being treated. The yields of the oxygenated and hydrocarbon fractions were 123.6 mg and 718.1 mg, respectively, for the Eucalyptus radiata EO inhibited α-amylase by 40%, while the hydrocarbon fraction was fully inactive. The oxygenated fraction of Eucalyptus radiata mainly consists of 1,8-cineole (68.9%) and α-terpineol (16.0%), which were found to be responsible for its α-amylase inhibitory activity. These compounds inhibited α-amylase by 31 ± 2 (IC50 1.08 ± 0.0432 mg mL−1) and 33 ± 1% (IC50 1.01 ± 0.0221 mg mL−1), respectively, when tested separately at a concentration of 0.670 mg mL−1. These values are quantitatively close to that of acarbose (IC50 0.670 mg mL−1). Considering the relative percentage abundance of 1,8-cineole and α-terpineol in the oxygenated fraction of Eucalyptus radiata EO, and their respective inhibitory activities, the bioactivity of Eucalyptus radiata oxygenated fractions seems to be the result of synergic interactions between the two identified bioactive compounds. The observed bioactivity of 1,8-cineole agrees with the data available in the scientific literature [Cinnamomum camphora, Eucalyptus globulus, Hyssopus officinalis, Laurus nobilis, and Melaleuca viridiflora, can thus partially be ascribed to the presence of 1,8-cineole, which is the major compound in all of the above mentioned EOs. However, linear regression analyses (data not shown) did not reveal a significant positive linear relationship between the amount of 1,8-cineole in an EO and the observed percentage of α-amylase inhibition. This is probably because of combination effects with other EO components having an influence on 1,8-cineole overall activity. The isolated oxygenated and hydrocarbon fractions for both EOs were tested separately for their α-amylase inhibitory activity at the same concentration as the original EO. Only the oxygenated fraction of terature ,19. HoweMyristica fragrans EO inhibited α-amylase by 11.1% and 15.0%, respectively. Unlike Eucalyptus radiata EO, the chemical composition of this EO mainly consists of hydrocarbons, namely sabinene, α-pinene, β-pinene, and limonene, whose relative percentage areas are 27.0%, 23.0%, 13.0%, and 10.0%, respectively, and that are present in the corresponding isolated fraction in similar percentages . When tested as pure standards, at a concentration of 0.670 mg mL−1, sabinene and limonene were inactive, while α- and β-pinene inhibited enzymatic activity by 32 ± 1% (IC50 1.05 ± 0.0252 mg mL−1) and 29 ± 1% (IC50 1.17 ± 0.0233 mg mL−1), respectively. This result suggests that they are responsible for the observed bioactivity of the hydrocarbon fraction, but that their contribution is only partial compared to that of the whole EO. The oxygenated fraction mainly contains 4-terpineol which, similarly to its structural isomer α-terpineol, inhibits α-amylase by 40 ± 2% (IC50 0.838 ± 0.0335 mg mL−1) when tested individually at a concentration of 0.670 mg mL−1. Despite the evidence that has emerged from these experiments, further studies into the mechanism of enzymatic inhibition of both Myristica fragrans EO and its respective bioactive components are required to understand how they influence the overall EO activity.The hydrocarbon and oxygenated fractions of Myristica fragrans EO have provided indications that allow us to, at least partially, understand the inhibition percentage results of other screened EOs. In addition to Myristica fragrans, the investigated EOs that contain high amounts of α and β-pinene are Cupressus sempervirens, Juniperus communis, Pinus mugo, and Pinus sylvestris EOs. However, unexpectedly, only the Myristica fragrans and Cupressus sempervirens EOs significantly inhibited α-amylase, while the others were inactive. In terms of chemical composition, Myristica fragrans and Cupressus sempervirens EOs mainly differ from the others because they are both free from trans-β-caryophyllene. Pearson’s correlation coefficient was thus measured to define whether an association exists between the presence of trans-β-caryophyllene and biological activity. The results suggest that the variables are indeed negatively related (r = −0.4583), and the correlation was sufficiently high to warrant the rejection of the null hypothesis of zero correlation .The results obtained from the bioassay-oriented fractionation of The precision of the in vitro α-amylase inhibition test was evaluated in terms of repeatability (by repeating the enzymatic inhibition assay three times in the same day) and intermediate precision (by repeating the enzymatic inhibition assay three times every four weeks over a period of six months). Results were very satisfactory and never exceeded 8.1% for repeatability and 11.8% for intermediate precision. Mentha x piperita EO (leaves) was obtained from fresh leaves , and submitted to steam distillation. Sixty-one EOs from different botanically authenticated species were supplied by Witt Italia SpA. −1 solutions of tangerine and chamomile EOs were prepared for each investigated solvent. For each solution, 50 μL were added to 1 mL of buffer solution and UV absorption at 540 nm and pH variation were assessed. The enzyme solution was prepared daily at a concentration of 33.3 μg/mL in a 0.02 M phosphate buffer (pH 6.9). The starch solution (1% w/v) was obtained by stirring 1 g of potato starch in the same buffer at 90 °C for 15 min. Then, 200 mL of the 3,5-dinitrosalicylic reagent was prepared as follows: first, 2.18 g of 3,5-dinitrosalicylic acid (DNSA) was dissolved in 40 mL NaOH 2 M and 100 mL of deionized water; 60 g of potassium citrate  was then added and the volume was made up to 200 mL with deionized water; the solution was finally heated to 40 °C to dissolve the potassium citrate. The reactant solution was stored in the dark and protected from CO2 [−1 solution in ethyl acetate was prepared and stored at 4 °C for each investigated EO, each standard sample, each isolated fraction, and for acarbose. The in vitro inhibition test adopted in this study was modified from that of Sigma-Aldrich . Three dfrom CO2 . A 200 m−1 , which corresponds to IC50 for acarbose. Sample mixtures were prepared as follows: 10 μL of each EO solution (corresponding to an absolute amount of 2 mg) was added to 1 mL of enzyme solution (corresponding to 1 unit of α-amylase) and pre-incubated for 5 min at 25 °C under constant stirring. Then, 1 mL of the starch solution was added and incubated for 3 min at 25 °C. Finally, 1 mL of the DNSA solution was added and left to react for 15 min at 90 °C. The mixture was then cooled and diluted with 9 mL of deionized water and the absorbance was measured at 540 nm. The standard test containing the non-inhibited enzyme was prepared as described above, with 10 µL of pure ethyl acetate instead of the EO solution. The final concentration of EOs and acarbose in the reaction mixture was 0.67 mg mLStandardA is the absorbance at 540 nm when the enzyme was not inhibited, Standard blankA is the absorbance of the standard blank solution containing the same reagents as the standard mixture, but with 1 mL of buffer instead of the enzyme, SampleA is the absorbance at 540 nm in the presence of the investigated inhibitor, and Sample blankA is the absorbance of the sample blank solution prepared with the same reagents as the sample mixture, including the 10 uL of the EO solution, but with 1 mL of buffer instead of the enzyme. Each blank underwent the same steps as the corresponding sample. A blank of each EO was performed to account for possible variations in absorbance due to differences in EO chemical composition. Acarbose was used as the positive reference; positive reference inhibition tests were carried out using 10 μL of acarbose solution at a concentration of 200 mgmL−1 in ethyl acetate (corresponding to an absolute amount of 2 mg).The α-amylase inhibition activity of each investigated EO was measured using two different approaches. The first approach (Method A) relies on difference in absorbance and α-amylase inhibition activity is measured by applying the following Equation (1):maltose sampleC is the concentration of maltose produced by α-amylase in the presence of EO or acarbose, and maltose StandardC is the concentration of maltose produced by the non-inhibited α-amylase.The second approach (Method B) measures amylase inhibition activity considering the differences in the amount of maltose generated by α-amylase activity via the reduction of 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid, by applying the following Formula (2):−1. Then, 1.0 mL of each solution was mixed with 1.0 mL of buffer solution and 1 mL of the DNSA solution and the mixture was left to react for 15 min at 90 °C. Once the reaction was finished, the mixture was cooled and diluted with 9 mL of deionized water and the absorbance was read at 540 nm. The maltose calibration curve was built by plotting the absorbance at 540 nm as a function of maltose concentration (mg mL−1).Maltose solutions were prepared in 0.02 M phosphate buffer (pH 6.9) at seven different concentrations: 1.0, 0.75, 0.50, 0.25, 0.20, 0.15, and 0.10 mg mL−1. Linear gradient elution was adopted from 100% of A to 80% of A and 20% of B in 20 min.EO fractionation was carried out on a flash column chromatography system PuriFlash 450 by Sepachrom, equipped with both UV and Evaporative Light Scattering (ELSD) detectors. Stationary phase: Sepachrom silica Daily 50 μm; mobile phase: petrolether (A) and ethyl acetate (B), flow 25 mL min−1; column: Mega SE 52  25 m × 0.25 mm dc × 0.25 μm df, from MEGA. Temperature program: from 50 °C (1 min) to 250 °C (5 min) at 3 °C min−1. MSD conditions: MS operated in EI mode (70 eV), scan range: 35 to 350 amu; dwell time 40 ms, ion source temperature: 230 °C; quadrupole temperature: 150 °C; transfer line temperature: 280 °C. EO markers were identified by comparing both their linear retention indices (LRIs), calculated versus a C9-C25 hydrocarbon mixture, and their mass spectra against those of authentic samples, or from commercially available mass spectral libraries .GC-MS analyses were carried out using a Gerstel MPS-2 multipurpose sampler installed on a Shimadzu 2010 GC unit coupled to a Shimadzu QP2010 Mass spectrometer. GC conditions: injector temperature: 280 °C, injection mode: split, ratio: 1/20; carrier gas: helium, flow rate: 1 mL min−1; column: Mega SE 52  25 m × 0.25 mm dc × 0.25 μm df, from MEGA. Temperature program: from 50 °C (1 min) to 250 °C (5 min) at 3 °C min−1. Relative percentage composition of the analyzed EOs was determined using GC-FID peak areas with response factors [GC-FID analyses were carried out on the previously-described Shimadzu 2010 GC unit. GC conditions: injector temperature: 280 °C, injection mode: split, ratio: 1/20; carrier gas: hydrogen, flow rate: 1 mL min factors ,23.Eucalyptus radiata, Laurus nobilis, and Myristica fragrans) that was comparable or slightly higher than that of acarbose, which was chosen as the positive control. The results showed that the EO components seem to act synergistically, as the total EO percentage inhibition activity is higher than those of both the combined isolated fractions and pure compounds. Moreover, an interesting number of both hydrocarbon and oxygenated compounds. were characterized as good α-amylase inhibitors, with percentage inhibitions of about 30% . These results demonstrate that EO oils are a promising source of potential α-amylase inhibitors.This study explores the α-amylase inhibition ability of 62 EOs of different composition and belonging to different plant families. Under the applied experimental conditions, three EOs showed high inhibitory capacity (