Phyllostachys edulis) were separated, precisely identified, and qualitative analysis using NMR (nuclear magnetic resonance) and quantitative analysis using UPLC-Q-TOF-MS (ultra-performance liquid chromatography with high-resolution quadrupole time-of-flight mass spectrometer), respectively. Potentially harmful levels of added excessive preservatives, including benzoic acid, ethylparaben, and sorbic acid, were found in bamboo juice oral liquid. Carbohydrates were determined to be the major components of bamboo juice, with contents as high as 191.13 g L−1, far higher than those of other compounds. The result indicated that the cough relief activity of bamboo juice oral liquid may be related to their high levels of added preservatives.Bamboo juice is a traditional Chinese drink and herbal medicine, and bamboo juice oral liquids are widely sold for the treatment of cough and phlegm in China. In this study, 26 main compounds of bamboo juice ( Phyllostachys glauca), moso bamboo (Phyllostachys edulis), and other bamboo species1. In Chinese culture, bamboo juice, which is known as “zhuli,” is consumed as an herbal medicine and natural beverage. Additionally, bamboo juice oral liquids (BJOL) are well known for as treatments for cough and phlegm in China, and can be bought in almost any pharmacy2. Numerous bamboo juice oral liquids are sold as herbal medicines in the form of a decoction in the Chinese market5.Bamboo juice is the sap from fresh poles of Gramineae plants such as pink green bamboo  include phenols, amino acids, inorganic elements, and organic acidsTherefore, this study focused on the qualitative and quantitative analysis of the main components of fresh bamboo juice and bamboo juice oral liquid, with the aim to discover the activity compounds from bamboo juice to treat coughs. We have analysed nine of the most common BJOL products that are classified as “fresh bamboo juice oral liquid” with the National Medical Products Administration of China. Surprisingly, bamboo juice oral liquids on the Chinese market were found to contain levels of preservatives that might be potentially harmful to the human body. Considering the important role of BJOLs in daily Chinese life, these potentially harmful levels of preservatives represent a potential food safety problem. Additionally, the carbohydrate species and content of bamboo juice and BJOLs were analysed, and the potential function and utilization of bamboo juice is discussed.1)10, β-hydroxypropiovanillone (2)11, 1-(4-hydroxy-3-methoxyphenyl)-glycerol (3)12, 3,5-dimethoxy-4-hydroxydihydrocinnamaldehyde (4)13, syringate (5)14, syringic aldehyde (6)15, p-E-coumaric acid (7)16, triandrin (8)17, ferulaldehyde (9)18, coniferol (10)19, β-arbutin (11)20, tachioside (12)21, 4-hydroxy-3,5-dimethoxyphenyl-β-d-glucopyranoside (13)22, 3,4′-dihydroxy-propiophenone-3-β-d-glucopyranoside (14)23, β-hydroxypropiovanillone (15)24, (+)-balanophonin (16)25, (+)-5-methoxybalanophonin (17)25, 2,6-dimethoxy-p-benzoquinone (18)26, 5-(hydroxymethyl)-2-furaldehyde (19)27, adenosine (20)28, 2-hydroxy-5-methoxyphenyl-β-d-glucopyranoside (21)29, (+)-lyoniresinol-3α-O-β-d-glucopyranosyl (22)30, (+)-5′-methoxyisolariciresinol-3α-O-β-d-glucopyranoside (23)30, (−)-lyoniresinol-3α-O-β-d-glucopyranoside (24)30, 7S,8R,8′R-5, 5′-dimethoxyariciresinol-4-O-β-d-glucopyranoside (25)31, and syringaresinol-4′-O-β-glucopyranoside (26)32. Purified UV images, NMR, and MS data for all compounds are shown in the Supplementary information.Twenty-six compounds were obtained by column chromatography Fig. . The com33. The recoveries of different compounds were also tested and are presented in Table The LOD, LOQ, accuracy, and intraday and inter-day precision data are presented in Table 13 was 20.71 ± 0.11 mg L−1. In fresh bamboo juice diluted 100 times, the content of compound 12 was 35.33 ± 0.10 mg L−1, and that of compound 22 was 47.15 ± 0.06 mg L−1.A typical chromatogram obtained from the mixed standards is presented in Fig. A standard full dose of bamboo juice for an adult is about 60 mL per day; the corresponding average doses of each of the main compounds would thus be about 2.8 mg or less than 3 mg, which could be speculated that the dose is low to provide cough relief activity. Moreover, these compounds do not appear to show good anti-inflammatory activity.−1, 471.44 mg L−1, and 1797.48 mg L−1; these values were much higher than those of the main components in fresh bamboo juice. This implies that these three preservatives were artificially added; moreover, the observed levels of these preservatives are potentially harmful to the human body34.The quantitative results for the BJOLs available on the Chinese market are shown in Table 35. However, the BJOLs from the Chinese market contained unacceptably high levels benzoic acid or other preservatives. The results could be surmised the cough relief activity of the BJOLs may be related to these preservatives.Benzoic acid was used as a cough suppressant in the nineteenth century because of its anti-microbial function−1, and those of the BJOLs were in the range of 10,967.70–33,230.78 mg L−1 . The calibration curve was established by analysing six different concentrations. Good linearity was established for all tested authentic samples. The linear relationships observed are presented in Table In ancient China, patients usually could not obtain enough sugar to defeat colds and bacterial infections. Considering that the contents of carbohydrates were far greater than those of other compounds in BJOLs, Chinese people in antiquity may have been able to easily acquire carbohydrates by drinking bamboo juice to boost their immunity.12, 13, and 22, with contents of 35.33 ± 0.10 mg L−1, 20.71 ± 0.11 mg L−1, and 47.15 ± 0.06 mg L−1. These ingredients may have potential biological activity, but they are not known to have anti-inflammation activity, which may because of the low contents. Moreover, guaiacol was not detected in bamboo juice or BJOLs.Twenty-six compounds were isolated from fresh bamboo juice and identified for the first time. The main chemical constituents in fresh bamboo juice were found to be compounds Subsequently, qualitative and quantitative analyses of fresh bamboo juice and BJOLs showed that they had distinct compositions. This may have been due in part to differences in the bamboo material selection and production methods, but the results also indicated that most of the BJOLs had been diluted from fresh bamboo juice. Due to the differences in composition, it was difficult to determine whether fresh bamboo juice had cough relief ability.−1; ethylparaben: 5–10 mg kg−1; sorbic acid: 0–25 mg kg−1). Thus, the use of these BJOLs with high preservative levels as cough medicines could pose a potential hazard to human health.No preservatives were found in fresh bamboo juice. However, excessively high doses of preservatives were found in the BJOLs from the Chinese market, including benzoic acid, ethylparaben, and sorbic acid. The results indicated that these preservatives were deliberately added, as confirmed by the manufacturers of the BJOLs. However, the preservative contents greatly exceeded the relevant FAO/WHO regulations  . Chemical shifts (δ) are given in ppm, with TMS as an internal standard, and coupling constants (J) are in Hz. High-resolution electrospray ionisation-mass spectrometry (HRESIMS) spectra were obtained using an Agilent 6540 ultra-performance liquid chromatography (UPLC) with high-resolution quadrupole time-of-flight (Q-TOF) mass spectrometer , using an RRHD Eclipse Plus C18 column  36.Ultraviolet spectral were obtained on a Waters 2695 high performance liquid chromatography (HPLC) instrument with a photo diode array detector . Preparative HPLC was carried out on a Shimadzu LC-6AD instrument  with a UV–Vis detector (SPD-20A), using a YMC-Pack-ODS-A column  . The carbohydrates were measured by ion chromatography using an amperometric detector  with a Hamilton RCX-30 column  . Nuclear magnetic resonance (NMR) spectra were obtained using Bruker AV-500 spectrometers  was deposited at the International Centre for Bamboo and Rattan.The liquid juice of Nine varieties of fresh bamboo juice oral liquid (BJOL) were purchased from the Chinese market; the sample identification codes used in this study and National Medical Products Administration (NMPA) numbers for the products are as follows: BJOL-1 (NO. Z51021002), BJOL-2 , BJOL-3 , BJOL-4 (NO. Z36021620), BJOL-5 (NO. Z36021539), BJOL-6 (NO. Z33021079), BJOL-7(NO. Z36020335), BJOL-8 (NO. 36020282), and BJOL-9 (NO. Z36021127).18 , and Sephadex LH-20 . All reagents were purchased from Beijing Chemical Works  unless otherwise specified. HPLC-grade methanol (MeOH) and acetonitrile (ACN) were purchased from Fisher Scientific .Column chromatography was performed with a macroporous resin , RP-CP. edulis sample (100.00 kg) was concentrated under reduced pressure to obtain a residue (3.80 kg). The residue was then sequentially extracted with ethyl acetate and water-saturated n-butanol, yield a ethyl acetate-soluble fraction (50.48 g), and an n-butanol–soluble fraction (194.12 g), respectively37.Liquid juice of n-butanol extract was ultrasonically dissolved in 200 mL of distilled water and subjected to gradient elution through an HP-20 type macroporous adsorption resin column. The eluting solvents were distilled water, 10% ethanol, 30% ethanol, 50% ethanol, and subsequently 95% ethanol. All the fractions were eluted separately on a LH-20 gel column using distilled water as the eluent. The samples were collected in fractions of 50 mL, and the fractions were combined according to the results of HPLC with a diode array detector (HPLC–DAD). The merged samples were then concentrated and lyophilised. Subsequent purification by C18 reverse-phase preparative liquid chromatography column gave compound 3 (17.9 mg), compound 7 (6.2 mg), compound 8 (5.3 mg), compound 11 (230 mg), compound 12 (248.2 mg), compound 13 (52.3 mg), compound 14 (10 mg), compound 15 (8.7 mg), compound 18 (5.3 mg), compound 20 (9.7 mg), compound 21 (94.3 mg), compound 22 (228.2 mg), compound 23 (7.8 mg), compound 24 (5.9 mg), compound 25 (17.8 mg), and compound 26 (21.9 mg).The 18 reverse-phase preparative liquid chromatography column gave compound 1 (5.3 mg), compound 2 (10.2 mg), compound 4 (6.3 mg), compound 5 (5.5 mg), compound 6 (13 mg), compound 9 (5.7 mg), compound 10 (6.4 mg), compound 16 (5.2 mg), compound 17 (5.1 mg), and compound 19 (8.3 mg).The ethyl acetate fraction was eluted on a silica gel column with dichloromethane/methanol (from 80:1 to 1:1), and 7 fractions were obtained by HPLC analysis. The samples were collected as 5 mL fractions, and the fractions were combined according to the HPLC–DAD test results. The merged samples were then concentrated and lyophilised. Subsequent purification by C1–26 were isolated and purified to 98% or higher purity and used as the reference standards. Compounds 5, 6, 7, 9, 15, 16, 17, and 18 were formulated with a concentration of 1000 μg mL−1, compounds 1, 3, 4, 8, 10, 19, 20, 23, and 24 with a concentration of 2000 μg mL−1, and the remaining compounds 2, 11, 12, 13, 14, 21, 22, 25 and 26 with a concentration of 5000 μg mL−1. Each single-standard solution was precisely measured and placed in a 2-mL flask. The mixed standard solution was obtained by combining the standard solutions in chromatography-grade methanol. Acetonitrile (B)-formic acid water (A) was used as the mobile phase for elution . The flow rate was set to 0.25 mL min−1, column temperature was set at 40 ℃, and the positive ion mode was adopted. A series of concentrations of the mixed standard solution were prepared to assess the linear relationship, and each of the different concentrations of solution was injected using the stated chromatographic conditions to generate the corresponding regression equations31.Quantitative analysis was carried out using UPLC-Q-TOF-MS. Compounds 3, 4, 5, 6, 7, 8, 9, 10, 13, 15, 16, 18, 19, 23 were calculated using the peak area after tenfold dilution, and the other compounds 1, 2, 11, 12, 14, 17, 20, 21, 22, 24, 25, 26 were calculated using the peak area of 100-fold times dilution.To prevent overloading, the 26 compounds were divided into two groups according to the response peaks of the compounds. Compounds 33. The LOD and LOQ for the main constituents were measured by duplicate injections of standard solutions based on S/N ratios of 3 and 10, respectively. The precision of the method was studied through six replicate injections of the mixed standard solutions and was expressed as relative standard deviation (RSD). The intraday precision was studied by injecting the mixed standard solutions six times on the same day , and the inter-day precision was evaluated by injecting the mixed standard solutions six times on four consecutive days. Results are expressed as RSD. The accuracy was based on a standard addition recovery test of the method, which was performed by diluting fresh bamboo juice 10 times and 100 times and adding three concentrations of the mixed standard solution. The three replicate spiked samples were analysed using UPLC-Q-TOF-MS. Subsequently, the recovery rates of the 26 components were calculated.UPLC-Q-TOF-MS analysis was performed to determine the limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy−1 and a 250 × 20 mm C18 analytical column was used. The LC-6AD was used for the repeated injection of the BJOL samples from the Chinese market, and the monomers a (5 mg), b (5 mg), and c (5 mg) were obtained.1H NMR  δ 12.94 , 7.95 , 7.60 , 7.49 , 3.75 , 3.17 ; 13C NMR  δ 167.8 (s), 133.3 (s), 129.8 (s), 128.9 (s).a: 1H NMR  δ 10.27 , 7.83 , 6.87 , 4.22 , 1.26 ; 13C NMR  δ 166.18 (s), 162.55 (s), 131.92 (s), 121.28 (s), 115.84 (s), 60.56 (s), 14.71 (s).b: 1H NMR  δ 12.09 , 7.14 , 6.21 , 5.75 , 1.79 ; 13C NMR  δ 168.25 (s), 144.97 (s), 139.44 (s), 130.25 (s), 120.40 (s), 18.81 (s).c: MeOH-formic acid water was used as the mobile phase for elution. The flow rate was set to 8 mL min18 column. The flow rate was 1 mL min−1, and the column temperature was set at 25 °C. The injection volume was 10 μL. The UV response was monitored at 228 nm (λmax for benzoic acid), 255 nm (λmax for ethylparaben), and 259 nm (λmax for sorbic acid). A series of mixed standard working solutions of benzoic acid, ethylparaben, and sorbic acid were sequentially injected into the liquid chromatograph to determine the corresponding peak areas. The standard curve was constructed using the concentration as the abscissa and the peak area as the ordinate. The samples BJOL-1 to BJOL-9 were injected into the liquid chromatograph to obtain the peak area and concentration of benzoic acid, ethylparaben, or sorbic acid in the solutions according to the standard curve39.HPLC–UV analysis was conducted using a Waters 2695–2996 system. The optimal mobile phase for analysis was a binary gradient elution system consisting of solvent A (MeOH) and solvent D (water containing 0.5% acetic acid). The gradient was programmed as 5% (solvent A) to 95% (solvent A) over 30 min. The column was a YMC-PACK ODS-AQ C33. The fresh bamboo juice and BJOLs were diluted 1000 times as test sample solutions. The sample solutions (10 μL) were treated with 72% H2SO4 and ultrapure water in a brown bottle at 105 °C for 2.0 h. Once cooled, the acidolysis solution was neutralised to pH 7.0 using 2.0 mM NaOH, and a portion of the sample (5 mL) was added to 10 mL of ultrapure water and then filtered using a membrane filter with a pore size of 0.22 mm. The filtrate (10 mL) was then tested. The detection of carbohydrates  and analyses of the carbohydrates were conducted using a mobile phase consisting of ultrapure water with 2.0 mM NaOH and 0.5 mM NaAc at a rate of 1 mL min−1 for 60 min. The injection volume was 2 mL. Five standard monosaccharide stock solutions (0.5 mg mL−1) were prepared by mixing in a 10-mL volumetric flask and diluted to six different concentrations  with ultrapure water to examine the linear relationship41.Analysis of the composition of polysaccharides was based on the sulfuric acid acidolysis method with some modificationsThe test results were expressed as mean ± SD (n = 3). The software used to analyse the test data was SPSS 16.0, and analysis of variance was used for the significance study.Supplementary Information.


In this work, CuS/Cu1.8S nanocomposites were firstly prepared simultaneously by the one-pot synthesis method at a relatively higher reaction temperature 200 °C. The CuS/Cu1.8S nanocomposites anodes exhibited a high initial discharge capacity, an excellent reversible rate capability, and remarkable cycle stability at a high current density, which could be due to the nano-size of the CuS/Cu1.8S nanocomposites and the assistance of Cu1.8S. The high electrochemical performance of the CuS/Cu1.8S nanocomposites indicated that the CuxS nanomaterials will be a potential lithium-ion battery anode.CuS and Cu A serieications ,32,33,34ications ,30,31,32 S cm−1) ,41,42,43xS nano/micro-morphologies have been prepared by many kinds of methods [−1 [−1 at 2 A g−1 after 1000 cycles, and 603.5 mAh g−1 at 200 mA g−1 after 300 cycles [In order to solve the volume expansion and to shorten the diffusion length of lithium ions, various types of Cu methods ,46,47,48 methods ,45, nano methods , hollow  methods , mircrof methods , nanotub methods , nanoshe methods ,40,44, h methods , and oth methods ,42,43. Zhods [−1 . Du and 0 cycles . Jiao an0 cycles .1.8S has also been paid significant attention in sodium-ion batteries and lithium-ion batteries owing to its crystallographic stability and narrow band gap of 1.2 eV  [1.8S/C-500 nanowire composites exhibited the best performance, and showed a specific capacity up to 220 mAh g−1 after 200 cycles [1.8S-C/C core/shell anode structure with a high surface area achieved a specific capacity of 372 mAh g−1 after 110 cycles with a performance of ~93% capacity retention [1.8S/C@MoS2 nanocomposite exhibited good stability, outstanding rate performance, and high reversible capacity, because of the assistance of Cu1.8S/C [Recently, Cuctivity) ,50,51. Zetention . Chen anCu1.8S/C .2S nanocomposites [2S/Cu1.8S nanocomposites [1.8S have not been prepared and studied. It was reported that CuS could be reduced to Cu1.8S at a high temperature and a high pressure [1.8S nanocomposites by a simple one-pot synthesis method at a correspondingly higher reaction temperature 200 °C. The CuS/Cu1.8S nanocomposites anodes exhibited high initial discharge capacity (1555 mAh g−1 at 100 mA g−1 and 1130 mAh g−1 at 267 mA g−1), excellent cycle stability at high current density , and excellent rate performance, which indicated that the CuxS nanomaterials will be a potential lithium-ion battery anode.Graphene and carbon nanotubes were added to CuS to improve the electrochemical properties in many articles ,46,47,48mposites  and Cu2Smposites ,52,53 ha1.8S nanocomposites is shown in 2·2H2O were sequentially added into 70 mL ethylene glycol. After1 h of magnetic stirring, the dissolved solution was put into two Teflon-sealed autoclaves (50 mL), which were heated to 200 °C and maintained for 24 h. When naturally cooled down to room temperature, the black precipitates were rinsed by ethanol and deionized water, in turn, many times by centrifugation (10000 rpm 10 min) until the solution is clear. Finally, the obtained samples were dried in a vacuum oven at 70 °C for 12 h.The schematic illustration of the preparation of CuS/CuScanning electron microscope  and X-ray diffraction  were used to characterize the structure, morphology, and composition of the samples. The XRD measurements were performed in the rage of 20° to 80° at a measuring rate of 3°/min using a Cu Kα radiation.−2.The working anodes were mixed with as-prepared black powders, carbon black, and carboxymethyl cellulose (CMC) dissolved in deionized water with a weight ratio of 7:2:1. The obtained slurry was evenly coated on a copper foil and dried in a vacuum oven at 70 °C for 12 h. And then, the obtained coated foil was punched into disks with a diameter of 12 mm. The weight of each disk sheet was measured to calculate the mass of active materials. The mass density of the active material was calculated at about 0.4 mg cmThe electrochemical measurements were carried out on CR-2032 coin-type cells. The half-cells were assembled in an argon glove box. The concentrations of oxygen and moisture in the argon glove were lower than 1 ppm. The Celgard 2250 film and lithium metal disk were selected as diaphragm and counter electrode, respectively. The electrolyte was an organic electrolyte of 1 M LiPF6, which is dissolved in a mixture of dimethyl ethyl carbonate (DEC) and ethyl carbonate (EC) with a volume ratio of 1:1.−1 and in a potential range between 0.01 V and 3.0 V. The electrochemical impedance spectroscopy (EIS) was also obtained by the CHI660E electrochemical workstation with a frequency range of 10−2 Hz–105 Hz. All the cells measured were set more than 24 h to ensure total penetration of the electrolyte into the diaphragm, and all measurement results were obtained at room temperature.Land-ct2001A battery measuring system was used to test the charge-discharge characteristics under different current densities with a potential range of 0.01 V–3.0 V. CHI660E electrochemical workstation was used to get the cyclic voltammetry (CV) measurements with a scanning rate of 0.1 mV s1.8S), demonstrating that the as-prepared samples were composed of CuS and Cu1.8S. No other characteristic peaks for impurities were observed. The sharp diffraction peaks in the patterns indicated the good crystallinity of our samples. Different peaks at 27.8°, 29.4°, 31.9°, 33.0°, 48.1°, 52.9°, and 59.4° corresponded to the (101), (102), (103), (006), (110), (108), and (116) crystal plane of CuS [1.8S [1.8S could overlap to 27.8°. The average sizes of crystal particles can be obtained by the Scherrer Equation [λ is the wavelength of the X-ray applied in the measurement, k is a constant fact 0.9, θ is the diffraction angle, and b is the full width at half maximum (FWHM). The sizes of CuS/Cu1.8S particles were estimated to be 23 nm (D(110)) and 22 nm (D(220)), which are nearly equal to each other.e of CuS ,51,52,53uS [1.8S ,51,52. TEquation ,54:(1)D(1.8S was measured by Energy Dispersive Spectrometer (EDS). The EDS results are shown in The morphology of as-prepared samples was further investigated using SEM, as is shown in 1.8S nanocomposites, the cyclic voltammetry (CV) curves were measured with a scanning rate of 0.1 mV s−1 and in a potential range between 0.01 V and 3.0 V. As shown in xCuS and the conversion of LixCuS to Cu and Li2S, respectively. During the first anodic sweep (delithiation), two obvious oxidation peaks at 1.9 V and 2.4 V were observed, which denoted the reversible process related to the cathodic reactions [1.8S nanocomposites at 267 mA g−1. In the first discharge process, two potential plateaus appeared at about 2.1 V and 1.6 V, and in the first charge process, two potential plateaus appeared at around 1.9 V and 2.3 V. These are matched with the redox peaks (redox reactions) of the first CV curve very well [−1 and charge capability of 707 mAh g−1 [1.8S [−1 and 580 mAh g−1, which are also higher than the theoretical capability 560 mAh g−1 of CuS. This extra capacity has been widely observed transition metal compounds [1.8S nanocomposites.In order to further understand the electrochemical process of CuS/Cueactions ,47,48,54eactions ,47,55. F−1 [1.8S ,42,43,44ompounds , the intompounds , and the1.8S nanocomposites at 267 mA g−1. As shown in −1 to about 450 mAh g−1. The values of specific capacity and the corresponding coulombic efficiency almost maintained to 1000 cycles with a small range of fluctuation, which demonstrated excellent cycling stability and relatively high lithium storage capacity. A fluctuation of the capacity can be seen in 1.8S nanocomposites in our work has good lithium-storage capability, high initial capacity, and good cycling stability. The CuS/Cu1.8S nanocomposites electrode could maintain the reversible capacity of 440 mAh g−1 after 100 cycles, 450 mAh g−1 after 500 cycles, 470 mAh g−1 after 700 cycles, and about 325 mAh g−1 after 1000 cycles. During the 1000 cycles, the average discharge capability and charge capability were 425 mAh g−1 and 420 mAh g−1 respectively. The high electrochemical performances of CuS/Cu1.8S nanocomposites electrode in our work could be attributed to the nano-size of the composites and the assistance of Cu1.8S [The electrochemical performances of CuS-based electrodes as anode materials for lithium-ion batteries were listed in f Cu1.8S ,50.1.8S nanocomposites anodes was further tested at a series of current densities. Despite an obvious fading of capacity during the initial several cycles, the CuS/Cu1.8S nanocomposites electrodes exhibited extraordinary stability and reversible capabilities in subsequent cycles. The reversible capacities had an obvious stepwise trend with the changing of current densities. The capacities were 1555 mAh g−1, 485 mAh g−1, 275 mAh g−1, and 195 mAh g−1 at 100 mA g−1, 200 mA g−1, 500 mA g−1, and 800 mA g−1, respectively. When the current densities came back to 200 mA g−1 and 100 mA g−1, the capacities could recover to 470 mA g−1 and 530 mA g−1, respectively, which implied the outstanding stability and reversibility [As shown in sibility ,55,61.1.8S nanocomposites electrode, electrochemical impedance spectroscopy (EIS) was measured. The Nyquist plot (black dots) measured before cycling and the relative fitting line (red line) were shown in Rs) of the electrode and electrolyte [Rcf) [Rct) induced by the diffusion of lithium ions between electrode and electrolyte. The slope of the straight line in low frequency corresponds to Warburg impedance (Zw) relating to the diffusion of lithium ions in CuS/Cu1.8S nanocomposites electrode [Rs, Rcf and Rct are 1.8 ohm, 34.3 ohm and 175 ohm, respectively. The EIS measured after 1000 cycles and the fitting line were shown in Rs, Rcf and Rct increased after 1000 discharge-charge cycles, and the comparisons of Rs, Rcf and Rct before cycling and after 1000 cycles can be seen in ω is the angular frequency in the low frequency region, σ is the Warburg coefficient, C is the concentration of lithium ions, n is the number of transferred electrons, F is the Faraday constant, T is the measuring temperature, A is the surface area of the electrode, and R is the gas constant [σ was obtained by linear fitting of Zreal versus ω−1/2 Equation (3), and then the diffusion coefficient of Li-ions  ,64. The lectrode ,55,61,62lectrode ,55,61,62f Cu1.8S ,51,52,53iffusion ,55. The ensities ,44,50,511.8S nanocomposites half-cell after 1000 cycles illuminating an electronic watch and a LED lamp. The test demonstrated the high electrochemical performance and potential application of CuxS nanomaterials in lithium-ion batteries.In addition, as shown in 1.8S nanocomposites were firstly prepared simultaneously by the one-pot Synthesis method at a relatively high reaction temperature 200 °C. The CuS/Cu1.8S nanocomposites anodes exhibited a high initial discharge capacity (1555 mAh g−1 at 100 mA g−1 and 1130 mAh g−1 at 267 mA g−1), excellent cycle stability at high current density (average 425 mAh g−1 at 267 mA g−1 during 1000 cycles), and remarkable reversible rate performance, which could be attributed to the nano-size of the CuS/Cu1.8S nanocomposites and the assistance of Cu1.8S. Though the more suitable ratio of CuS to Cu1.8S needs to be further investigated, the high electrochemical performance of the CuS/Cu1.8S nanocomposites indicated that the CuxS nanomaterials will be a potential lithium-ion battery anode.In this work, CuS/Cu


On day 5 (acute), capsules containing placebo or caffeine (6 mg∙kg−1) were ingested 60 min before completing a 16 km time-trial (TT). CC and PC showed improvements in time  and in output power  (p < 0.05) compared to CP and PP conditions, respectively. These effects were accompanied by increased heart rate , minute volume , expired O2 fraction , lactate blood concentration , 36.01% ES = 0.84; 31.22% ES = 0.81), and reduction in expired CO2 fraction . In conclusion, these results indicate that caffeine, when ingested by cyclists in a dose of 6 mg∙kg−1 for 4 days, does not induce tolerance to the ergogenic effects promoted by acute intake on physiological, metabolic, and performance parameters.The present study investigated whether the caffeine supplementation for four days would induce tolerance to the ergogenic effects promoted by acute intake on physiological, metabolic, and performance parameters of cyclists. A double-blind placebo-controlled cross-over design was employed, involving four experimental trials; placebo (4-day)-placebo (acute)/PP, placebo (4-day)-caffeine (acute)/PC, caffeine (4-day)-caffeine (acute)/CC and caffeine (4-day)-placebo (acute)/CP. Fourteen male recreationally-trained cyclists ingested capsules containing either placebo or caffeine (6 mg∙kg The search for licit ergogenic resources is growing by cyclists in an attempt to minimize muscle fatigue generated in training sessions/competitions. In cyclists, muscle fatigue is manifested by the inability to repeatedly produce muscle strength or power over some time . The erg1, A2A, A2B, and A3) , carbohydrates , protein , and lipids .Food intake, which did not exhibit significant main effects in treatment 24 h before the trials, is shown in p = 0.0001] for the main effect. There were no differences between treatments and interaction between treatment and distance traveled in TT (p > 0.05)  .p = 0.000] and percentage of time curve  in the main effect. However, there was no significance observed in the interaction between treatment and percentage of time curve . HR was higher with caffeine in the PC condition (174.01 ± 9.94 vs. 170.6 ± 9.51 bpm)  than that with PP, and higher in the CC condition (176.01 ± 9.59 vs. 171.49 ± 9.47 bpm−1)  than that with CP. No differences were observed between CC and PC (p > 0.05).HR A showed p = 0.7947) in the main effects on VO2 (p = 0.0001] and interaction between treatment and percentage of time curve (p = 0.0005) effects were seen.There was no significant difference  = 38.33, p = 0.001] main effects. However, there was no significance observed in the interaction between treatment and percentage of time curve (p = 0.7184). VCO2 was higher with caffeine in the PC condition (45.06 ± 5.32 vs. 42.04 ± 5.15 mL∙kg−1∙min−1)  than that with PP. No differences were observed between CC and PC (p > 0.05).VCO2 C exhibitE  = 76.07, p = 0.000] main effects. However, a significant interaction between treatment and percentage of time curve (p = 0.070) was not observed. VE was higher with caffeine in the PC condition (86.38 ± 16.50 vs. 74.26 ± 15.62 L∙min−1)  than that with PP, and with caffeine in the CC position (84.62 ± 16.44 L∙min−1 vs. 74.81 ± 15.44 L∙min−1)  than that with CP. No differences were observed between CC and PC (p > 0.05).VE D exhibit2  = 91.09, p = 0.0001] main effects. However, there was no significant interaction between treatment and percentage of time curve (p = 0.9996) observed. FeO2 was higher with caffeine in the PC condition (17.17 ± 0.65% vs. 16.69 ± 0.68%)  than that with PP, and in the CC condition (17.22 ± 0.65% vs. 16.67 ± 0.24%)  than that with CP. No differences were observed between CC and PC (p > 0.05).FeO2 E exhibit2  = 60.46, p = 0.0001] main effects. However, there was no significant interaction between treatment and percentage of time curve (p = 0.9996) observed. FeCO2 was lower with caffeine in the PC condition (4.28 ± 0.64% vs. 4.64 ± 0.63%)  than that with PP, and with caffeine in the CC condition (4.23 ± 0.63% vs. 4.58 ± 0.28%)  than with CP. No differences were observed between CC and PC (p > 0.05).FeCO2 F exhibitp = 0.0001] and percentage of time curve , and an interaction between treatment and percentage of time curve  main effects. Power output was higher with caffeine in the PC condition (250.3 ± 30.24 W vs. 244.1 ± 29.33 W)  than that with PP, and in the CC condition (249.7 ± 27.23 W vs. 242.5 ± 29.51 W)  than that with CP. No differences were observed between CC and PC (p > 0.05).Power output A exhibitp = 0.0001]. TT test completion time was lower with caffeine in both PC (1631 ± 90.45 s vs. 1674 ± 72.88 s)  than that with PP, and in CC (1634 ± 61.29 s vs. 1692 ± 90.47 s)  than that with CP. No differences were observed between CC and PC (p > 0.05).There was a main effect of treatment on TT performance B  main effect. Lactate concentration exhibited significant treatment  and time , and an interaction between treatment and time  main effects. Glucose concentration exhibited significant time (p = 0.0001) main effect , and reduction in expired CO2 fraction were similar in both withdrawal and 4-day supplementation conditions.The results of the present study indicate that caffeine, when ingested by cyclists in a dose of 6 mg∙kg−1 body mass of caffeine) significantly improves exercise performance, regardless of whether a period of withdrawal or consumption (3 mg∙kg−1 body mass of caffeine) for 4 days is imposed on regular users of caffeine.The cyclists in the present study who consumed caffeine acutely (CC and PC) had more output power in the first half of the TT test A. It can−1∙day−1). In another study [−1 or 0.80 mg∙kg−1∙day−1), moderate (143 ± 25 mg∙day−1 or 1.90 mg∙kg−1∙day−1) and high (351 ± 139 mg∙day−1 or 4.91 mg∙kg−1∙day−1) consumers, when administered in cyclists an acute dose of 6 mg∙kg−1 body mass of caffeine. The plausible explanation for this finding indicates that the dose used for acute intake exceeded the average amount of caffeine about the usual consumption of low, moderate, and high consumers [−1 body mass of caffeine exceeded the average amount of usual caffeine consumption. Thus, in the present study, it was suggested that the improvement in performance is related to acute intake, regardless of habitual consumption and caffeine supplementation for 4 days.According to the authors , an expler study , the aut−1 body mass of caffeine could cause saturation in hepatic metabolism and, consequently, the maintenance of caffeine blood concentration for a prolonged time. Therefore, we believe that the improvement in time (−1 body mass) may reduce interindividual metabolic responses [A previous study by Graham and Spriet  conclude in time B observe in time . These responses .2A/A2B) [Furthermore, evidence shows that an increase in primary caffeine metabolites  is due to caffeine metabolism, which may promote tolerance effects ,17,18. B2A/A2B) . However2A/A2B)  suggeste2max [2max.The effects of acute caffeine intake appear to regulate blood cortisol and glucose concentrations during exercise ,21. From2max , as show+, and Pi) as a result of increased muscle metabolism during exercise. Thus, we attributed the increase in HR to the high blood lactate concentrations observed after TT (T2 and recovery)  . It was ecovery) . Accordi2 to the muscles. Interestingly, Jones et al. [p < 0.05) with the improvement in time to TT. Investigators [Adopting a high pacing strategy at the beginning of TT is the most commonly used form by cyclists expecting to increase fatigue tolerance . Howevers et al.  observedtigators ,28 arguetigators . Thus, oIn future studies, it should be encouraged to test whether the blinding procedure was effective. Asking participants to identify the supplement ingested, to test the efficacy of the blinding method, should be a great way to avoid bias in the analysis of the results . The exp−1 for 4 days, does not induce tolerance to the ergogenic effects promoted by acute intake on physiological, metabolic, and performance parameters. Furthermore, the ergogenic effects observed in the supplementation and withdrawal conditions for 4 days were due to acute intake, verified by a similar blood concentration of caffeine.In conclusion, these results indicate that caffeine, when ingested by cyclists (moderate to high caffeine consumers) in a dose of 6 mg∙kg


Panicum miliaceum. There is a dearth of information about its uses and properties; hence, in this study, the nutritional quality of kabog millet was evaluated. The total starch, % amylose, ash, dietary fibre, proteins, essential amino acid profile, phenolic acids, carotenoids, tocopherols, and the antioxidant properties of its total phenolic acid extracts were compared to four types of rice  and a reference millet, purchased from local Swiss supermarkets. Our analyses showed that kabog millet has higher total dietary fibre, total protein, total phenolic acids, tocopherols, and carotenoids content than white rice. It also performed well in antioxidant assays. Our results indicate that kabog millet is a good alternative to rice. It is hoped that the results of this study will encourage consumers and farmers to diversify their food palette and address food insecurity.Consumption of underutilised ancient crops has huge benefits for our society. It improves food security by diversifying our staple foods and makes our agriculture more adaptable to climate change. The Philippines has a rich biodiversity and many plant species used as staple foods are native to the Philippines. An example of ancient Philippine crops is the kabog millet, an ecotype of  Cultivating ancient crops has huge benefits for our society. It improves food security by diversifying our staple foods, provides a greater range of options to address climate change, optimises land resources by cultivating soils unsuitable for the world’s major crops, promotes access to better nutrition for communities, especially in the developing regions of the world, and generates income opportunities for small- and medium-scale farmers . The PhiPanicum miliaceum L., and the only known proso millet existing in the Philippines. Morphological studies on two kabog millet cultivars grown in Cebu, Philippines, placed them under the Panicum patentissium and Panicum compactum subspecies  using a Hamilton syringe. After, 5 mL of 1-butanol was added to each tube with a glass pipette. The samples were vortexed quickly and were put in a sonicator for 15 min. They were stirred for 30 min on a magnetic stirrer and were sonicated again for 15 min. The samples were stirred for another 30 min, after which they were centrifuged at room temperature for 3 min at 4000 rpm. The supernatant (1 to 1.5 mL) was filtered through a hydrophilic syringe filter   into an amber-coloured high-performance liquid chromatography (HPLC) vial. The samples were stored at −20 °C and light protected until further analysis with HPLC.The method in  was follThe HPLC parameters were as follows: Flow rate was 1.0 mL/min, injection volume was 50 μL, temperature of column was at 35 °C, diode-array detection (DAD) was at 450 nm, and the separation was within 18 min. The samples were run using the HPLC column YMC Carotenoid, S-3µm, 100 × 4.6 mm with the pre-column YMC Carotenoid, S-3µm, 10 × 4.0 mm  in reverse phase high-performance liquid chromatography (RP-HPLC) Agilent 1200 . The composition of the eluent and the gradient program were A: Methanol/MTBE/Milli-Q water, (81:15:4); B: MTBE/methanol (90:10): with a gradient from 100% to 10% A (0–15 min), and from 10% to 100% A (15–18 min).w/v, 50 g KOH in 100 mL MilliQ water) was added to each tube with a magnetic stirrer. The samples were mixed thoroughly by vortexing. They were then put into a boiling water bath (100 °C) and allowed to saponify for 25 min under constant stirring. The tubes were cooled in a cold-water bath for around 10 min, after which 2.5 mL water and 2.5 mL ethanol were added. Tocols were extracted by adding 10 mL n-hexane:ethylacetate . The tubes were placed in a tumbler mixer to shake for 10 min. After shaking, the tubes were let to stand until the organic and non-organic phases separated. The upper organic layer was quantitatively transferred into a 100-mL separation funnel using a pasteur pipette. The extraction was repeated twice. The organic extract in the separation funnel was washed with 1 mL of 10% sodium chloride (NaCl) solution. The lower layer was removed from each washing. The organic extract was transferred into a 100-mL round-bottom flask, and the solvent was evaporated using the rotary evaporator, with the water bath at around 60 °C and the pressure around 300 mbar. The lipid residue was resolubilised in n-hexane and quantitatively transferred into a 5-mL volumetric flask. The volumetric flask was filled with n-hexane. An aliquot of the sample (around 1.5 mL) was filtered into an HPLC vial using a 45-μm PTFE syringe filter. The vials were stored at −20 °C until HPLC analysis. For normal phase HPLC Agilent 1200 , the following parameters were used: eluent n-hexane: 1,4-dioxane, 97:3 (v/v), flow rate 2 mL/min, injection volume 50 μL, temperature of column 30 °C, temperature of autosampler tray 4 °C, fluorescence detection (FLD) with excitation wavelength λex 292 nm and emission wavelength λem 325 nm, isocratic separation within 25 min.The tocopherols analysis was done following the method in . For eacThe method in  was adap® BEH C18   in UHPLC 1290 Infinity II Series . The composition of the eluent and the gradient program were A: Methanol:formic acid (90:2); B: Milli-Q water:formic acid (88:2): from 0% to 20% A (0–5 min), at 20% A (5–6 min), from 20% to 50% A (6–12.3 min), at 50% A (12.3–13.2 min), from 50% to 100% A (13.2–15.3 min), at 100% A (15.3–16.9 min), from 100% to 0% A (16.9–19.1 min). The RP-UHPLC parameters were as follows: flow rate 0.4 mL/min, injection volume 1 μL, column and autosampler at ambient temperature, DAD detection at 254, 280, and 325 nm. The samples were run using the ultra-high-performance liquid chromatography column (UHPLC-Column) ACQUITY UPLC0 = absorbance  at 0 min, At = absorbance (DPPH + sample) at 5 min and Ap = absorbance  at 5 min.For the antioxidant assays, the total phenolic acid extracts  were tesThe 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) Antioxidant Capacity Assay (KF01002) and the Oxygen Radical Absorbance Capacity (ORAC) Assay (KF01004) kits were from Bioquochem , and the ABTS and ORAC antioxidant assays were performed using the total phenolic acid extracts following the manufacturer’s protocols. Calculations for %inhibition, Trolox equivalence antioxidant capacity (TEAC), and vitamin C equivalence antioxidant capacity (CEAC) were included in the manufacturer’s protocols.n-hexane. The samples were vortexed for 2 min, sonicated for 10 min, then centrifuged at 4000× g at room temperature for 5 min. The supernatant was transferred to a new 50-mL Falcon tube, and the hexane was gently evaporated completely with nitrogen stream at room temperature. The residue was resuspended in 500 µL of n-hexane. To each replicate, 125 µL of the resuspended sample was added to 500 µL n-hexane and 300 µL 2M KOH in methanol, and the mixture was vortexed for 2 min. The samples were let to stand until the layers separate, and the upper layer was pipetted off and transferred to a gas chromatography (GC) vial for further gas chromatography-flame ionisation detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS) analyses using the ThermoFisher Trace 1310 Series GC . The GC standard used was Supelco 37 Component FAME Mix (Supelco).The fatty acid characterisation was performed only on whole kabog millet samples. To obtain the fatty acids from whole kabog millet, 5.0 g of ground flour was extracted with 20 mL p < 0.05) (https://astatsa.com/OneWay_Anova_with_TukeyHSD/). Differences among means in Tables were evaluated by Tukey’s HSD tests (p < 0.05) using the free statistical software R project, v. 3.6.3 (downloaded from https://cran.r-project.org/mirrors.html).All experiments were carried out in triplicates unless otherwise stated and data were reported as mean ± standard deviation. The data in Figures were evaluated using a one-way ANOVA  had comparable total starch with whole kabog millet sample 2 (47.4 ± 2.0 g/100 g sample). The same trend was noted in the dehulled kabog millet samples . The totFor the % amylose , the whoFor the dietary fibre, the whole kabog millet sample 1 (14.6 ± 0.6%) had comparable value with whole kabog millet sample 2 (13.8 ± 0.9%). Likewise, the dehulled kabog millet samples had similar values  as shown in The ash content of the whole kabog millet sample 1 (4.2 ± 0.0%) was significantly higher than the whole kabog millet sample 2 (4.1 ± 0.1%). On the other hand, the ash content of dehulled kabog millet sample 1 (1.0 ± 0.0%) was significantly lower than the dehulled kabog millet sample 2 (1.1 ± 0.0%). Overall, the whole kabog millet samples had significantly higher ash content than the dehulled kabog millet samples, all the rice samples, and the reference millet .In terms of total protein, the whole kabog millet sample 1 (12.0 ± 0.0 g/100 g sample) had comparable value as the whole kabog millet sample 2 (12.1 ± 0.0 g/100 g sample), while the dehulled kabog millet sample 1 (12.5 ± 0.0 g/100 g sample) had significantly lower content than the dehulled kabog millet sample 2 (12.9 ± 0.1 g/100 g sample) . Overallp < 0.05). Likewise, the Thr content of the dehulled kabog millet sample 1 (2.96 ± 0.21 g/100 g protein) was not significantly different with the dehulled kabog millet sample 2 (3.06 ± 0.16 g/100 g protein) (p < 0.05) (p < 0.05). The Lys, Ile, and Val content were comparable between whole kabog millet samples, the dehulled samples, and the reference millet, but significantly lower than in the rice samples (p < 0.05). The Leu content was comparable between whole kabog millet samples, the dehulled samples, and the reference millet, but significantly higher than in the rice samples (p < 0.05). The Phe content was not significantly different between whole kabog millet samples, the dehulled samples, and the reference millet. However, the Phe content of the dehulled kabog millet sample 2 (3.99 ± 0.13 g/100 g protein) was significantly lower than that of red rice (4.61 ± 0.07 g/100 g protein) and black rice (4.51 ± 0.53 g/100 g protein) (p < 0.05). In terms of Met, there was no significant difference among all the samples. Quinoa, however, still has the highest content of Lys, Met, and Phe  in the rice and millet samples were compared with quinoa, a pseudocereal known for its protein quality . In term < 0.05) . In gene and Phe .The total carotenoid content of the whole kabog millet sample 1 (16.8 ± 0.4 µg/g) was not significantly different with the whole kabog millet sample 2 (15.7 ± 0.6 µg/g). The same comparable values were noted between dehulled kabog millet sample 1 (19.4 ± 0.3 µg/g) and dehulled kabog millet sample 2 (19.3 ± 0.5 µg/g). The whole kabog millet samples and the dehulled kabog millet samples had significantly higher total carotenoid content than the rice samples and the reference millet with the dehulled kabog millet samples having significantly higher total carotenoids than the rest of the samples .p < 0.05). The reference millet had significantly lower lutein content (4.7 ± 0.2 µg/g) than the kabog millet samples (p < 0.05). The rice samples contained lower amounts of lutein than the kabog millet samples. Zeaxanthin was present in the reference millet (1.7 ± 0.1 µg/g), in both whole kabog millet samples 1 and 2 , and in both dehulled kabog millet samples 1 and 2 . The reference millet had lower lutein and zeaxanthin content than the kabog millet samples. In this study, zeaxanthin was not detected in the white, brown, and red rice samples. Black rice had zeaxanthin and β-cryptoxanthin, but the amount was low (≈0.1 µg/g for both carotenoids). The kabog millet samples also contained β-carotene, which were lower than black rice (1.4 ± 0.0 µg/g sample) (p < 0.05).In terms of individual carotenoids, the whole kabog millet samples (11.6 ± 0.3 µg/g and 12.9 ± 0.3 µg/g) had significantly lower amounts of lutein than the dehulled kabog millet samples (14.5 ± 0.2 µg/g and 15.1 ± 0.2 µg/g) . The γ-tocopherol values ranged from 29.8 ± 1.4 µg/g for the reference millet to 83.9 ± 7.2 µg/g for the whole kabog 2. On the other hand, the δ-tocopherol contents ranged from 5.6 ± 0.2 µg/g for the dehulled kabog 2 to 13.9 ± 2.3 µg/g for the whole kabog 2. The reference millet contained 7.4 ± 0.7 µg/g δ-tocopherol. The δ-tocopherol form was not detected in rice. However, γ-tocotrienol was the most abundant form in rice, with values ranging from 10.8 ± 0.1 µg/g (white rice) to 80.1 ± 9.8 µg/g (black rice). The tocopherol, γ-tocotrienol, was not detected in the whole and dehulled kabog millet and the reference millet.Among the tocopherols, γ-tocopherol and δ-tocopherol were present in significant amounts in the kabog millet and reference millet samples (p < 0.05)  .p-coumaric and ferulic acids. In whole kabog millet 1, the amounts of p-coumaric and ferulic acids were 731 ± 28 and 660 ± 21 µg/g sample, respectively, while the amounts of p-coumaric and ferulic acids from sample 2 were 760 ± 13 and 699 ± 9 µg/g sample, respectively. Dehulled kabog millet 1 and 2 contained significantly higher amounts of p-coumaric acid  than the reference millet (13 ± 1 µg/g) and white rice (undetected) (p < 0.05). The same trend was observed for cinnamic acid, with the whole kabog millet samples 1 and 2 having  significantly higher amounts than the reference millet (6 ± 0 µg/g) and white rice (undetected) (p < 0.05). On the other hand, black rice had the highest amount of protocatechuic and vanillic acids . White rice contained 90 ± 2 µg/g ferulic acid and 4 ± 0 µg/g sinapic acid  A. The whpic acid B.p < 0.05). White rice had the lowest %RSA value (3.0 ± 0.2 %)  .p < 0.05). The reference millet and the dehulled kabog millet samples had significantly lower %inhibition than red, black, and brown rice samples (p < 0.05). The values from the rest of the samples were significantly higher than that of white rice.In the ABTS assay shown in p < 0.05). Meanwhile, the reference millet and the dehulled kabog millet samples 1 and 2 had comparable CEAC values  (p < 0.05). These values were significantly lower than those of red, black, and brown rice. With regard to the TEAC values from the ORAC antioxidant assay, the whole kabog millet samples 1 and 2 had similar values (1456.4 ± 14.0 and 1432.3 ± 56.8), which were comparable to those of black rice (1439.2 ± 61.9) and red rice (1312.9 ± 35.3) (p < 0.05). Brown rice (1104.2 ± 52.4), the reference millet (1217.9 ± 44.0), and the dehulled kabog millet samples 1 and 2  had significantly the same TEAC values (p < 0.05). White rice had the lowest CEAC and TEAC among all the samples analysed.In terms of CEAC values, from the ABTS antioxidant assay as shown in The fatty acid methyl esters methyl palmitate, methyl stearate, methyl oleate, and methyl linoleate were detected from the whole kabog millet samples 1 and 2. Methyl tricosanoate was also present in small amounts . Among tThe total starch values obtained from both whole and dehulled kabog millet samples in our study are comparable with published data for millets. In millets, starch comprises 56% to 65% of the total seed weight, although in proso millet, starch content can be as high as 80% of the total seed weight . This maThe % amylose is important in predicting starch physicochemical properties, such as pasting, gelatinization, and retrogradation properties, and even digestibility . The % aIn terms of total dietary fibre content, the whole kabog millet samples have the highest amounts, around three times more than those of brown, red, and black rice, and that of the reference millet, while the dehulled kabog millet samples have comparable values to that of the reference millet . This caIn the present study, the total protein content of the reference millet, the whole kabog millet, and the dehulled kabog millet (≈11% to ≈13%) indicates that they can be good sources of proteins compared to rice and its different types. Rice is not a good source of protein, having only around 7% for white rice and brown rice , with brCarotenoids cannot be synthesised by animals, and hence, are normally obtained through the diet. In terms of carotenoid content, both whole and dehulled kabog millet have the highest lutein and zeaxanthin amounts. β-carotene is also present in the kabog millet samples but is not comparable to black rice, which has the highest β-carotene. In comparison, carrot, a commonly consumed vegetable, is rich in β-carotene and contains lutein, but lacks β-cryptoxanthin and zeaxanthin, whilst red pepper is rich in β-carotene and contains lutein, β-cryptoxanthin, and zeaxanthin . The resIn a previous study , HPLC anp-coumaric and ferulic acids were observed in whole kabog millet. Ferulic acid is found bound to arabinoxylans and other polysaccharides or proteins within the cell walls of the aleurone layer [p-coumaric acid occurs widely in the cell walls of graminaceous plants. The pericarp fractions in barley, wheat, oat, and corn appear to be the fractions richest in p-coumaric acid [p-coumaric acid is found in the outer layers of the grain. Milling (or decortication/dehulling) leads to a loss of up to 80% of the total phenolics present in whole grains [p-coumaric and ferulic acids in dehulled kabog millet compared to the whole kabog millet samples. Phenolic acids are one of the major bioactives found in brown, red, and black rice grains [p-coumaric are present in red rice, and protocatechuic and vanillic acids are present in black rice. Protocatechuic, vanillic, syringic and ferulic acids have antioxidant activity in the soluble-conjugated fraction, while protocatechuic and ferulic acid are in the insoluble-bound fraction from rice.For phenolic acids, the highest amounts of ne layer ,36, whicne layer . Similarric acid . In mille grains , which ee grains ,40. In aPhenolic acids have known antioxidant activities. In addition to that, pigmented rice varieties (red and purple) rich in phenolic acids and anthocyanins were found to have lower starch digestibility than white rice . The intThe antioxidant activities of the total phenolic extracts from rice and the millet samples were analysed through DPPH, ABTS, and ORAC antioxidant assays. The DPPH %RSA values are comparable between the whole kabog millet and the red and black rice samples. This makes the phenolic acids found in the whole kabog millet, red rice, and black rice as effective free-radical scavengers. The ability of antioxidant compounds to reduce the ABTS radical cation to its nonradical form was also measure and their activities were compared to Vitamin C (CEAC). The high ABTS %inhibition of whole kabog millet can be correlated to its high total phenolic acid content. In the case of red rice, which has lower total phenolic acid content than brown and black rice, the relatively high ABTS %inhibition cannot be attributed to phenolic acids alone. In a study analysing the antioxidant activity of phenolic acid extracts of millets, it was found that the contents of phenolics alone may not sufficiently explain the observed antioxidant activity of plant phenolic extracts . Other cCereal lipids are a good source of unsaturated fatty acids. Unsaturated fatty acids can offer several health benefits such as reducing cardiovascular risk, improve cognitive functions and lower GI ,46. In aPanicum. Cereals that contain gluten include wheat, barley, emmer, spelt, einkorn, khorasan, triticale, and rye. Oats contain avenins, which are also considered under the term “gluten” [One of the special traits of kabog millet is that it is gluten-free, due to its being grouped in the genus “gluten” . Cereals“gluten” . A gluteDigitaria exiliis) and Iburu or black fonio (Digitaria iburua) have been used and studied [Some of the food products from gluten-free cereals and pseudocereals that are currently gaining ground are sourdough bread and gluten-free beers. For sourdough bread, African cereals called Acha or white fonio . In addition, except for the kabog millet and the dawa millet, millets are not common cereals in the Philippines. Even then, the cultivation of these two native millets is concentrated in specific areas only, and these millets are not widely known in the Philippines. Another limitation is the use of quinoa data from previous literature  to compaBased on the results of this study, and considering the limitations, kabog millet, particularly the whole grain samples, were found to possess good nutritional quality in terms of total dietary fiber and total protein content, essential amino acid profiles, phenolic acid, tocopherol, and carotenoid content, fatty acid profile, and antioxidant activity by DPPH, ABTS, and ORAC assays. It can be promoted as a healthier cereal substitute to white rice in the Philippines and potentially, in other rice-consuming countries in the world. It is hoped that increased consumption of kabog millet will lead to the conservation of its genetic pool for future generations, and the rich cultural heritage of the kabog millet-growing regions in the Philippines. It will also provide livelihood to small-scale farmers and their families. More consumers will appreciate the global diversity of cereal crops and will have access to nutritional and sustainable food choices by incorporating kabog millet into their diet.


This work provides detailed insight into perplexing redox tailoring enzymology that sets the stage for the (chemo)enzymatic production and bioengineering of bioactive spiroketal-containing polyketides.The structural complexity and bioactivity of natural products often depend on enzymatic redox tailoring steps. This is exemplified by the generation of the bisbenzannulated -spiroketal pharmacophore in the bacterial rubromycin family of aromatic polyketides, which exhibit a wide array of bioactivities such as the inhibition of HIV reverse transcriptase or DNA helicase. Here we elucidate the complex flavoenzyme-driven formation of the rubromycin pharmacophore that is markedly distinct from conventional (bio)synthetic strategies for spiroketal formation. Accordingly, a polycyclic aromatic precursor undergoes extensive enzymatic oxidative rearrangement catalyzed by two flavoprotein monooxygenases and a flavoprotein oxidase that ultimately results in a drastic distortion of the carbon skeleton. The one-pot in vitro reconstitution of the key enzymatic steps as well as the comprehensive characterization of reactive intermediates allow to unravel the intricate underlying reactions, during which four carbon-carbon bonds are broken and two CO Rubromycin family of natural products belongs to aromatic polyketides with diverse bioactivities, but details of their biosynthesis are limited. Here, the authors report the complete in vitro reconstitution of enzymatic formation of the spiroketal moiety of rubromycin polyketides, driven by flavin-dependent enzymes, and characterize reaction intermediates. The intensely colored rubromycins comprise various griseorhodins ), hyaluromycin, purpuromycin, heliquinomycin, as well as the eponymous rubromycins )12. Often, these compounds act as potent enzyme inhibitors and 2 was consequently suggested as a lead structure for drug development13. The hallmark structural feature of the rubromycins is a bisbenzannulated -spiroketal pharmacophore that disrupts the planarity of the polycyclic, aromatic carbon backbone , as well as the rubromycin family belong to a growing group of biosynthetically related aromatic type II polyketide natural products with extended “pentangular” architecture that are produced by numerous actinobacterial speciesrhodin A ), hyalurrhodin A ), hyalurgrh) A biosynthetic gene cluster of Streptomyces sp. JP95 isolated from the marine tunicate Aplidium lenticulum, which was expressed in the heterologous producer S. albus sp. J107410. Initial steps resemble typical type II polyketide pathways involving a minimal polyketide synthase (PKS) that likely utilizes an acetyl-CoA starter unit and 12 malonyl-CoA extender units to generate a highly reactive acyl-carrier protein (ACP)-bound poly-β-ketone chain. Following enzyme-catalyzed regioselective ketoreduction, cyclization, aromatization and ACP elimination, further tailoring reactions modify the polyketide backbone and lead to the advanced and highly oxidized intermediate collinone (3) , which may serve as a direct precursor for spiroketalization10. This would necessitate an extensive oxidative backbone rearrangement as well as the elimination of two C1 units, which may be mediated by mechanistically versatile flavin-dependent enzymes22 that often facilitate redox tailoring reactions in natural product biosynthesis  via various reactive intermediates by the concerted action of the flavoprotein monooxygenases GrhO5 and GrhO6, as well as the flavoprotein oxidase GrhO1 that are encoded by the grh gene cluster. This process is primarily mediated by the multifunctional monooxygenase GrhO5 that oxidatively rearranges the carbon backbone and ultimately forms a -spiroketal and is assisted by GrhO1, before the ring-contracting GrhO6 generates the -spiroketal pharmacophore found in mature rubromycin polyketides  was previously reported to produce 3 as major shunt metabolite, which may thus represent the native substrate of GrhO510. To investigate this, 3 was isolated from the S. albus mutant, while GrhO5  was obtained from the heterologous producer Escherichia coli BL21 DE3 (see Online Methods section for details on gene cloning as well as production and purification of enzymes and compounds). GrhO5 is predicted to function as flavoprotein monooxygenase based on the amino acid sequence10 and is homologous to the NAD(P)H- and FAD-dependent class A flavoprotein monooxygenases with “glutathione reductase type” Rossmann fold21. Typically, these enzymes catalyze aromatic hydroxylation reactions via an electrophilic flavin-C4a-hydroperoxide oxygenating species, while some members instead act as Baeyer–Villiger monooxygenases (BVMOs) that employ a nucleophilic flavin-C4a-peroxide anion23. The purified enzyme showed an intense yellow coloration indicative of a bound flavin cofactor that was further determined as flavin adenine dinucleotide  indicated that 5 represents a reduced form of 3, which spontaneously reoxidized in the presence of O2, as shown by the color change and confirmed by RP-HPLC  citrate or DTT), which also afforded 5 . Notably, compared to the much faster GrhO5-dependent 5 formation, NADPH (±free FAD) only reduced 3 at very low rates  proved suitable and afforded a completely methylated and reduced derivative (6) of an enzymatically hydroxylated intermediate based on LC-HRMS and tandem MS (MS2) analysis , keto-enol tautomerization (that triggers water elimination at C18), and complete methylation of the free hydroxyl groups  prompts the first C–C bond cleavage of ring D via retro-aldol condensation to afford 7 (step III). The flavin-mediated hydroxylation most likely proceeds via an electrophilic flavin-C4a-hydroperoxide (FlC4aOOH) oxygen transferring agent, as discussed below.To confirm that m 5 Fig. . Based oo 5 Fig. , step I,7, compound 8 emerged in the HPLC chromatograms, followed shortly by shunt product 9 and on-pathway intermediate 10  of 3 with GrhO5 did not yield significant amounts of enzymatic product. Surprisingly, 11 was identified as the previously reported lenticulone featuring a -spiroketal10 and 10 accordingly as the respective ring A-reduced dihydrolenticulone based on LC-HRMS/MS2 analysis, UV-Vis spectra, NMR spectroscopy, and comparison with a standard acquired from S. albus sp. J1074 MP66 (ΔgrhO6)10 10 Fig. –19. In c7 first undergoes retro-aldol cleavage of ring C generating an α-keto aldehyde motif at C5–C6 . Although highly labile 8 could not be isolated for structural elucidation by NMR, isotope-labeling studies with 8 corroborate this proposal. Accordingly, up to two 18O atoms derived from H218O were observed in 8 by LC-HRMS/MS2 .To consolidate these results, we propose that –C6 Fig. , step IV8 allows the third C–C bond cleavage via facile decarboxylation. This is enabled by an appropriately positioned C15-ketone in ring B that acts as electron sink, thereby producing on-pathway intermediate 10 after keto-enol tautomerization, before ring A autooxidation yields 11 H . To further investigate this mechanistic proposal, both 9 and 11 were enzymatically produced in presence of either 18O2 or H218O. As expected, one 18O label from O2 was incorporated in the eastern molecule halves of 9 and 11 as a result of GrhO5’s oxygenase functionality  flavoenzyme family that typically catalyze (non-oxygenative) two-electron oxidation reactions rather than hydroxylations, although some exceptions exist24. Hence, we supplemented our in vitro GrhO5 assays with heterologously produced polyhistidine-tagged GrhO1 that features a covalently bound flavin  could not be converted by GrhO1  spectroscopy, revealing a racemic mixture. Presumably, enzymatically formed 11 is chiral at first but undergoes spontaneous racemization via rapid ring-opening and recylization. Consistent with that, the racemate could not be separated by chiral HPLC  with a mass of [4 + H2O]  identity (70% sequence coverage) with GrhO5. The ΔS. albus sp. J1074 KR7 (ΔgrhO7) for the production of 7,8-dideoxygriseorhodin C , while remaining largely stable under acidic conditions  is likely driven by the ring strain caused by the sp2-hybridized C6-ketone of 4 and proceeds via a transient ring-opened oxocarbenium that is quenched by water to form 12. Most likely, 4 represents the true GrhO6 product that rapidly converts to 12 when not further processed by downstream pathway enzymes , as quantified by UV-Vis spectroscopy 10, Actinoplanes ianthinogenes (native producer of purpuromycin26), and S. puniceus (native producer of 1 and griseorhodin C)25. RP-HPLC and LC-HRMS analysis clearly confirmed the presence of 10 (that likewise underwent spontaneous oxidation to 11) and 12 in various cell free lysates, while 5, unstable 8, and shunt product 9 were not observed. Also, 7 could not be directly detected as a result of the poor interaction with chromatographic stationary phases. However, complex compound mixtures including insoluble compounds prone to polymerization were previously reported (but not further characterized) in the ΔgrhO1 mutant strain S. albus J1074 KR510. Indeed, upon treatment of the cell free lysate of a culture of this strain with DMS, 6 could be obtained , 2 (of S. collinus)15, hyaluromycin 27, and of heliquinomycin (S. piniterrae)28 harbor grhO5, grhO1, and grhO6 homologs . Compared to that, respective candidate genes were absent in the biosynthetic gene clusters of the non-spiroketal compounds benastatin A, fredericamycin A (featuring a carbaspirocycle), pradimicin A, lysolipin, and arixanthomycins  that produces key intermediate 10, which is subsequently transformed by GrhO6 into 4. Both GrhO5 and GrhO6 resemble characterized class A flavoprotein monooxygenases, e.g., GrhO5 is related to the aromatic hydroxylase RdmE  from aklavinone biosynthesis31 and to XanO4  catalyzing oxygenation and demethoxylation in xanthone biosynthesis32. Moreover, GrhO5 resembles the predicted aromatic hydroxylases GrhO8  and GrhO9  from 1 biosynthesis that presumably catalyze early tailoring steps en route to 310. On the other hand, GrhO6 is more closely related to aromatic hydroxylases such as OxyS  from oxytetracycline biosynthesis33 and verified BVMOs, e.g., MtmOIV  from mithramycin biosynthesis34  with various non-redox steps . While flavoenzyme functionalities such as quinone reductiont 9 Fig. . The fla 10 Fig. . Presuma10, an aromatic hydroxylation appears likely . This hydroxylation then enables the final fourth C–C bond cleavage via decarboxylation, followed by recyclization to the -spiroketal of 4  as well as biosynthetic strategies and precursor molecules. Conventional enzyme-dependent routes rely, e.g., on a dihydroxy-ketone motif allowing hemiketal formation and dehydrative cyclization to afford the -spiroketal of the pesticide avermectin41. Similarly, the proposed cascade-like -spiroketal formation in the generation of the polyether antibiotics such as lasalocid A and monensin requires a hydroxy-ketone-epoxide motif and proceeds via initial hemiketal formation followed by epoxide-dependent spiroketalization42. Moreover, a radical mechanism has been proposed in griseofulvin biosynthesis43. Compared to these straightforward pathways, precursor 3 lacks evident structural requisites and functionalities for spiroketal formation and instead the pentangular precursor undergoes an unanticipated series of oxidative backbone rearrangements. Other noteworthy examples of flavoenzyme-driven oxidative rearrangements in type II polyketide biosynthesis are found, e.g., in chartreusin44, xanthone32, and rishirilide45 biosynthesis. Interestingly, an alternative strategy to disrupt aromatic polyketide planarity is employed in enterocin biosynthesis47, where a single flavoprotein monooxygenase drastically rearranges a linear poly-β-ketone chain49 rather than the cyclized backbone. Aside from flavoenzymes, other types of multifunctional monooxygenases  have also been reported in natural product biosynthesis50.The complex flavin enzymology underlying the formation of the rubromycin pharmacophore thus provides an astonishing solution for the generation of stable chroman spiroketals in nature. These complex redox transformations of a polycyclic, aromatic backbone starkly contrast with synthetic approaches enzymatic formation of novel rubromycin analogs and hybrid compounds. Our work illustrates a counterintuitive principle for the generation of structurally complex polyketide scaffolds and spiroketal pharmacophores.grhO5, grhO1, and grhO6 genes were amplified from the cosmid pMP319 containing the grh gene cluster , the grhO1 gene between the NcoI and EcoRI sites of the expression vector pHis851 and the grhO6 gene between the EcoRI and PstI sites of the expression vector pETDuet-1 . The rubL gene was synthesized (codon optimized) and purchased already cloned between the NdeI and XhoI sites of the expression vector pET-16b (BioCat).The grhO5 plasmid was transformed into E. coli BL21 (DE3) (Thermo Fisher Scientific). LB-medium was inoculated with the transformed strain and supplemented with 100 µg/mL ampicillin at 37 °C overnight under shaking at 180 rpm. Then, 1 L of TB-medium with 100 µg/mL ampicillin was inoculated with 10 mL of the starter culture and grown to an OD600 of 0.5–0.6. The culture was induced with a final concentration of 250 µM IPTG and the cells were grown overnight at 18 °C under shaking (130 rpm). The cells were retrieved by centrifugation at 4000 × g for 20 min at 4 °C. Cell pellets were suspended in buffer A  with additional 1 mg/mL lysozyme and 1 mM phenylmethylsulfonyl fluoride and then stirred for 30 min at 4 °C. Then, the cells were lysed by sonication and the cell fragments removed by centrifugation at 18,000 × g for 30 min at 4 °C. The supernatant was filtered through a 0.45-µm syringe filter and then loaded onto a 5-mL MBPTrap HP Dextrin SepharoseTM column  connected to an FPLC system. Following a washing step with buffer A, bound mbp-tagged GrhO5 was eluted by a gradient ranging from 0% to 100% buffer B  over 5 column volumes. GrhO5 was concentrated using a 30-kDa MW cut-off MACROSEP spin column  and subsequently desalted with a 5-mL HiTrap desalting column . The identity and purity of recombinant GrhO5 was analyzed by SDS-PAGE.The recombinant pMal-c5x-His-grhO1 plasmid was transformed into E. coli Lemo21 (DE3). The production and purification was performed according to the procedure described above using kanamycin (50 µg/mL) and chloramphenicol (30 µg/mL) as antibiotics and 750 µM L-rhamnose for optimized expression levels. A 5-mL HisTrapTM FF crude column  was used for purification. Unbound protein was removed by washing with buffer A  and 0–6% buffer B  over five column volumes and bound his-tagged protein was then eluted with 100% buffer B. The protein was concentrated using a 10-kDa MW cut-off MACROSEP spin column  and then further purified by size exclusion chromatography with a HiLoad 16/600 Superdex 200 pg column  equilibrated with buffer A  and connected to a FPLC system. Finally, 10% (v/v) glycerol was added to the fractions containing GrhO1. The identity and purity of the eluted protein was confirmed by SDS-PAGE.The recombinant pHis8-grhO6 plasmid was transformed into E. coli BL21 (DE3) (Thermo Fisher Scientific). The production and purification was performed according to the procedure described above with ampicillin (100 µg/mL) and the respective buffers .The recombinant pETDuet-1-rubL (BioCat) was transformed into E. coli BL21 (DE3). The production and purification was performed according to the procedure described above with ampicillin (100 µg/mL) and the respective buffers .The recombinant pET-16b-S. albus grhO5 knock-out strain were inoculated into 50 mL of TSB medium supplemented with apramycin (50 µg/mL) and grown at 30 °C, 200 rpm for 24 h. The resulting culture was used to inoculate 3 L of TSB medium distributed to 1 L flasks with stainless-steel springs. Each flask contained 300 mL of culture supplemented with apramycin (50 µg/mL) and was incubated at 30 °C, 200 rpm for 4 days. The cells were harvested by centrifugation and the pellet was suspended in dH2O (300 mL). The pellet/water solution was acidified to pH 1 with 2 N HCl, EtOAc (500 mL) was added and the mixture then stirred overnight. The supernatant was also acidified to pH 1 with 2 N HCl and extracted with EtOAc (3 × 500 mL for 1 L culture). The combined extracts of the supernatant were purified by column chromatography . Fractions containing 3 (detected via thin layer chromatography (TLC)) were combined, dried, and further purified by semi-preparative HPLC. The extract of the pellet fraction was directly purified by semi-preparative HPLC. An average of 10 mg/L cell culture of 3 was obtained. The purity of the red solid 3 was confirmed by high-resolution liquid chromatography mass spectrometry (LC-HRMS) and 1D NMR analysis. The acquired NMR data are in accordance with previously published data15. TLC : Rf = 0.59; UV/Vis (CH3CN/H2O): λ 244, 340, 518 nm; HRMS (m/z): [M + H]+ calcd. for C27H18O12, 535.087; found, 535.088.Spores of the S. albus KR42 strain were inoculated into 50 mL of LB medium supplemented with apramycin (50 µg/mL) and grown at 30 °C, 200 rpm for 4 days. The cells were harvested by centrifugation and the supernatant was acidified to pH 3 with 2 N HCl and extracted with EtOAc (2 × 100 mL). The solvent of the combined extracts was removed under reduced pressure and the residue dissolved in CH3CN and EtOAc. After centrifugation for 10 min at 18,000 × g, samples were analyzed by HPLC using a NUCLEODUR 100-5 C18ec column  coupled with a UNIVERSAL RP guard column .Spores of the 3) and and 536 nm for lenticulone (11) using a Shimadzu Photometer . A mixture of 50 mM Tris pH 7.4, 1.5 mM NADPH or NADH and substrate  or 20, 50, 100 µM lenticulone (11), both dissolved in CH3CN) was prepared. The reaction was started by addition of 1 µM GrhO5 or GrhO6 (or free FAD as control) that allowed the measurement of the linear decrease in absorption. The turnover rates were calculated based on extinction coefficients of 4850 L × mol−1 × cm−1 at 556 nm (3) and 1700 L × mol−1 × cm−1 at 536 nm (11) according to Beer-Lambert law. The slope of the absorption was read out by the software .The kinetic parameters for GrhO5 and GrhO6 were determined photometrically at wavelengths of 556 for collinone (ν/ν) hydroxypropyl-beta-cyclodextrin (HPCD) was prepared. The reaction was started by addition of collinone ) before incubation for 0.5–8 min at 30 °C while shaking at 750 rpm. The reaction was terminated by addition of EtOAc/formic acid [9:1 (ν/ν)] and extracted by vigorous shaking. The mixture was centrifuged for 30 s and the layers then separated. The solvent was removed under reduced pressure and the compounds dissolved in CH3CN (20 µL) and EtOAc (20 µl). After centrifugation for 10 min at 18,000 × g, samples were analyzed by HPLC using a NUCLEODUR 100-5 C18ec column  coupled with a UNIVERSAL RP guard column . The assay was also performed using identical conditions with RubL instead of GrhO5.A mixture of 50 mM Tris pH 7.4, 1.5 mM NADH and NADPH, 10 µM GrhO5, and 1%  HPCD and collinone ) to solution B . After 1 min the reaction mixture was extracted [EtOAc/formic acid = 9:1 (ν/ν)] inside the anaerobic chamber. The mixture was centrifuged for 30 s and the layers then separated inside the anaerobic chamber. The next steps were performed under minimal exposure to O2. The solvent was removed under reduced pressure and the residue dissolved in CH3CN and EtOAc. After centrifugation for 10 min at 18,000 × g, compound 5 was purified by HPLC using a NUCLEODUR 100-5 C18ec column  coupled with a UNIVERSAL RP guard column . The yield of 5 was ~12 mg of orange solid. The product was analysed by 1H, 13C, 2D NMR, UV/Vis and ESI-HRMS. 1H NMR : δ 2.07–2.11 , 2.18–2.21 , 2.30 , 2.92–2.96 , 3.33–3.39 , 3.61 , 4.00 , 6.24 , 6.35 , 6.73 , 6.88 , 9.55 , 9.89 , 12.29 , 14.68 , 15.48 .; 13C NMR : δ 19.4, 26.1, 29.1, 56.5, 76.6, 80.1, 100.6, 102.8, 103.8, 104.2, 104.6, 107.5, 114.9, 115.7, 117.7, 137.3, 141.0, 147.4, 152.2, 154.0, 154.9, 160.4, 161.4, 162.7, 167.0, 196.0, 197.1; UV/Vis (CH3CN/H2O): λ 259, 338, 455, 481 nm; and HRMS (m/z): [M-H]− calcd. for C27H20O12, 535.088; found, 535.088.The enzymatic assay was performed under anaerobic conditions using the above described assay composition. The reaction was started in an anaerobic chamber by adding solution A  HPCD) was prepared. The reaction was started by addition of compound 9  CH3CN in assay) before incubation for 5 min at 30 °C while shaking at 750 rpm. The reaction was quenched by addition of EtOAc/formic acid [9:1 (ν/ν)] and extracted by vigorous shaking. The mixture was centrifuged for 30 s and the layers then separated. The solvents were removed under reduced pressure and the compounds dissolved in CH3CN (20 µl) and EtOAc (20 µl). After centrifugation for 10 min at 18,000×g, samples were analyzed by HPLC.A solution of 50 mM Tris pH 7.4, 1.5 mM NADH/NADPH, 10 µM of either GrhO5, GrhO1 or GrhO6, and 1% : δ2.04–2.08 , 2.15 , 2.61–2.65 , 3.96 , 6.33 , 6.55 , 6.57 , 10.71 , 12.13 , 13.01 ; 1H NMR : δ 2.10–2.16 , 2.73–2.78 , 3.94 , 5.24 , 6.13 , 6.39 , 6.55 , 8.10 , 10.82 , 12.92 ; 13C NMR  δ 18.6, 24.3, 33.7, 57.6, 76.9, 103.9, 103.9, 104.2, 110.7, 116.1, 118.4, 128.3, 128.3, 137.4, 141.7, 141.7, 147.3, 147.3, 151.2, 151.2, 161.5, 166.1, 166.1, 183.1, 190.0, 197.9; Most signals are unambiguously assigned, also due to HSQC and HMBC signals. Some signals of quaternary carbon atoms are extraordinarily strong because of the complete overlap of two carbons. The signals in italics show the carbon atoms with lacking HSQC and HMBC signals that are assigned due to chemical shifts observed for biosynthetically related molecules  strongly support the proposed structure : λ 232, 364, 503 nm; HRMS (m/z): [M + H]+ calcd. for C26H18O12, 523.087; found, 523.089.A large scale enzyme assay (200 mL distributed to Eppendorf tubes each containing 500 µL) was performed using the same composition of constituents as described for the standard GrhO5 assay. The reaction was quenched after 15 min and compound ν/ν) HPCD was prepared. The reaction was started by addition of lenticulone  CH3CN in assay) before incubation for 30 min at 30 °C while shaking at 750 rpm. The reaction was quenched by addition of EtOAc/formic acid [9:1 (ν/ν)] and extracted by vigorous shaking. The mixture was centrifuged for 30 s and the layers then separated. The solvents were removed under reduced pressure and the compounds dissolved in CH3CN (20 µl) and EtOAc (20 µl). After centrifugation for 10 min at 18,000 × g, samples were analyzed by HPLC.A solution of 50 mM Tris pH 7.4, 1.5 mM NADH and/or NADPH, 10 µM GrhO6, and 1% (10/11 were isolated as described above for 5 but under aerobic conditions (collected compound 10 subsequently autooxidized to 11). The yield of 11 was ~1.1 mg of red solid. The product was analysed by 1H, 13C NMR, UV/Vis, and ESI-HRMS. The acquired NMR data are in accordance with previously published data10. UV/Vis (CH3CN/H2O): λ 233, 354, 496 nm; HRMS (m/z): [M + H]+ calcd. for C26H16O12, 521.072; found, 521.070.The large scale enzyme assay (75 mL distributed to Eppendorf tubes each containing 1 mL) was performed using the same composition of constituents as described for the standard GrhO5 assay with additional 5 µM of GrhO1. The reaction was quenched after 10 min and ν/ν) HPCD) to solution B . After 1 min the reaction mixture was extracted [EtOAc/formic acid = 9:1 (ν/ν)], the layers separated and the mixture analyzed on a photometer inside the anaerobic chamber.The enzymatic assay was performed under anaerobic conditions. The reaction was started in an anaerobic chamber by adding solution A citrate and 1%  HPCD and 10% glycerol) was thoroughly removed by 10 cycles of treatment with vacuum and nitrogen gas. Solution B was added to solution A in an anaerobic chamber and the reaction was started by injection of about 97% 18O2 gas (Campro Scientific). The reaction was stirred at room temperature for 1 and 5 min and then extracted [EtOAc/formic acid = 9:1 (ν/ν)]. The solvent was removed under reduced pressure and the residue dissolved in CH3CN (20 µl) and EtOAc (20 µl) before LC-HRMS analysis. The assays with H218O were performed corresponding to typical GrhO5 assays with at least 55% of labeled water in the reaction mixture.The dioxygen in solution A  and solution B  with trifluoroacetic acid . EtOAc (1 L) was added and the solution was stirred overnight at room temperature. After centrifugation, the layers were separated and the solvent of the organic layer was removed under reduced pressure. In total, 1.4 g crude extract was obtained from 9 L of culture. To a suspension of crude extract (1.4 g) of S. albus KR5 and anhydrous K2CO3  in dry acetone (150 mL), dimethyl sulfate (DMS)  was added dropwise. The mixture was stirred at room temperature for 1 h and then heated under reflux for 20 h at 40 °C. Afterwards, the mixture was cooled to room temperature and diluted with H2O (300 mL). After stirring for additional 30 min, the mixture was extracted with EtOAc (500 mL). The layers were separated and the organic layer was washed with water. The solvent was removed under reduced pressure. The remaining extract was purified by column chromatography . Fractions containing 6 (detected via TLC) were combined, dried, and further purified by semi-preparative HPLC. After a second purification step, the isolated yield was 4 mg of a yellow solid. The purity of 6 was confirmed by LC-HRMS and NMR analysis. TLC : Rf = 0.42; 1H NMR : δ 2.16 , 2.83–2.85 , 2.90–2.91 , 3.79 , 3.79 , 3.81 , 3.87 , 3.88 , 3.91 , 3.97 , 6.40 , 7.01 , 7.06 ; 13C NMR : δ 19.3, 25.6, 31.1, 56.9, 57.1, 61.5, 61.5, 61.6, 62.0, 62.0, 63.1, 102.7, 103.2, 112.5, 115.6, 121.9, 124.8, 128.8, 130.4, 135.4, 135.8, 140.4, 144.4, 147.4, 151.0, 153.9, 153.9, 154.4, 156.2, 156.4, 158.5, 158.6, 180.7, 183.6; UV/Vis (CH3CN/H2O): λ 231, 265, 350, 396 nm; HRMS (m/z): [M + H]+ calcd. for C34H34O12, 635.212; found, 635.214. The methylation of 7 in the course of enzymatic assays with GrhO5 was performed analogously in smaller scale and analyzed by HPLC as described for the standard assays with GrhO5.S. albus KR7 spores (50 µL) were inoculated into 50 mL LB medium and grown at 30 °C, 200 rpm for 24 h. 5 mL of the starter culture was transferred into 10 flasks containing 300 mL LB medium and cultivation was continued at 30 °C, 200 rpm for 96 h. This procedure was repeated four times resulting in a total culture volume of 12 L. After cell harvesting by centrifugation  the supernatant was separated from the pellet and the pellet was resupended in H2O (4 × 500 mL). Pellet suspensions were acidified with HCl  in 500 mL portions. EtOAc (2 × 500 mL) was added and the mixture was stirred overnight. The combined organic layers were concentrated under reduced pressure and the residue subjected to a short silica column . Fractions containing compound 13 were combined. Solvents were removed under reduced pressure and further purified via semi-preparative HPLC, which yielded 120 mg of red solid from the pellet. The supernatant was acidified with conc. HCl (10 mL/L) and extracted with EtOAc (1 L culture with 2 × 500 mL). The combined organic layers were filtered and evaporated under reduced pressure yielding 2.60 g of crude extract from the supernatant. Further purification was performed by column chromatography  and provided 750 mg of crude product. The crude product was further purified via semi-preparative HPLC. Solvents were removed under reduced pressure yielding again 11.6 mg of red solid isolated from the supernatant. The overall yield was 131.6 mg of red solid compound 13. The product was analysed by 1H, 13C NMR, UV/Vis and ESI-HRMS. The acquired NMR data are in accordance with previously published data52. TLC : Rf = 0.26; UV/Vis (CH3CN/H2O): λ 233, 316, 354, 505 nm; HRMS (m/z): [M + H]+ calcd. for C25H18O11, 495.092; found, 495.092.13)  was dissolved in dichloromethane (100 mL) by vigorously stirring for 1 h. DMP solution   was added and the reaction mixture was stirred at room temperature for 24 h. The solvent was removed under reduced pressure at room temperature and the residue dissolved in CH3CN (100 µL). After centrifugation , 4 was isolated by semi-preparative HPLC. The collected compound 4 was directly frozen in liquid nitrogen and solvents were removed by subsequent lyophilization for at least 24 h yielding 15 mg of 4  as dark purple solid. The product was analysed by NMR, UV/Vis and ESI-HRMS. The acquired NMR data are in accordance with previously published data53. TLC : Rf = 0.40; UV/Vis (CH3CN/H2O) λ 232, 255, 317, 352, 508 nm; HRMS (m/z): [M + H]+ calcd. for C25H16O11 493.077, found 493.077.Dideoxygriseorhodin C (4)  CH3CN in assay) and 1% (ν/ν) HPCD in aqueous solution at different pH values  was prepared. The mixtures were incubated at different temperatures  for 1 h (750 rpm). The reactions were quenched by addition of EtOAc/formic acid [9:1 (ν/ν)] and extracted by vigorous shaking. The mixture was centrifuged for 30 s and the layers then separated. The solvents were removed under reduced pressure and the residues dissolved in CH3CN (40 µl). After centrifugation for 10 min at 18,000 × g, samples were analyzed by HPLC.A solution of 50 mM Tris pH 7.4, 7,8-dideoxy-6-oxo-griseorhodin C (A. ianthinogenes spores (60 µL) were inoculated into 60 mL GYM medium and grown at 30 °C and 200 rpm for 48 h. 5 mL each of the preculture were then transferred into 10 flasks equipped with a stainless-steel spring containing 200 mL GYM medium and culture was continued at 30 °C and 200 rpm for ~96 h. The metabolite production was monitored by HPLC. If sufficient amounts of compound 12 were detected, the culture was harvested by centrifugation . The separated supernatant was acidified with HCl (6 M to pH = 1) before dichloromethane (700 mL) was added. The mixture was stirred vigorously for 1 h followed by centrifugation . The solvent of the combined extracts was removed under reduced pressure and the residue dissolved in CH3CN. After centrifugation for 10 min at 18,000 × g, samples were analyzed and compound 12 isolated by HPLC using a NUCLEODUR 100-5 C18ec column  coupled with a UNIVERSAL RP guard column . The purification via semi-preparative HPLC yielded 31 mg of dark purple solid. The product was analysed by 1H, 13C and 2D NMR, UV/Vis and ESI-HRMS. 1H NMR : δ 2.15 , 2.34 , 2.73–2.75 , 3.91 , 6.30 , 6.62 , 6.64 , 9.30 , 10.75 , 12.22 ; 13C NMR : δ 18.6, 24.4, 34.1, 56.9, 103.9, 104.1, 109.8, 116.2, 128.2, 137.4, 141.7, 147.3, 151.0, 158.2, 166.2, 194.2; The signals of 16 carbon atoms are unambiguously assigned, also due to HSQC and HMBC signals. The remaining nine carbon atoms are not detectable possibly due to interaction with metals that is often observed for such compounds. However, IR spectroscopy and DFT calculations  strongly support this structure; UV/Vis (CH3CN/H2O): λ 235, 321, 374, 508 nm; HRMS (m/z): [M + H]+ calcd. for C25H18O12, 511.087; found, 511.087.6 (90% B) and compound 9 (60% B) at a flow rate of 3–5 mL/min at 30 °C and absorbance was measured at 254 nm as well as 350 nm, 488 nm, and 500 nm. For analysis of the flavin cofactors, a NUCLEODUR Sphinx RP  with a UNIVERSAL RP guard column  was used. The mobile phases were water with 0.1% TFA (solution A) and acetonitrile with 0.1% TFA (solution B). The following gradient was used: 2% B for 0–1 min, 2–100% B from 1 to 15 min, 100% B from 15 to 20 min and 2% B from 20 to 21 min with a flow rate of 1 mL/min at 30 °C. Absorbance was measured at wavelengths of 210 nm, 254 nm, 260 nm, and 280 nm.Compounds from enzymatic assays were analyzed or prepared by RP-HPLC on a 1100 series chromatographic system (Agilent Technologies) with a VP NUCLEODUR 100-5 C18ec column  and a UNIVERSAL RP guard column . The mobile phases were water with 0.1% trifluoroacetic acid  and acetonitrile with 0.1% TFA (solution B). A gradient was run with 2% B from 0 to 1 min, 2–100% B from 1 to 15 min, 100–2% B from 20 to 21 min and 2% B from 21 to 24 min. As second purification step for NMR samples, a Kinetex® Biphenyl 100 Å column  was used with a UNIVERSAL RP guard column . The mobile phases were water with 0.1% TFA or 0.1% formic acid  and acetonitrile with 0.1% TFA or 0.1% FA (solution B). Isocratic methods were used for compound 11) were analyzed by chiral HPLC on a 1100 series chromatographic system (Agilent Technologies) using a CHIRALPAK® AS-H . The mobile phase was 100% ethanol. The flow rate was 0.25 mL/min at 30 °C and absorbance was measured at 240 nm as well as 350 nm, and 495 nm. Additional tested columns: CHIRALPAK® AD-H , CHIRALCEL® OD-H  and MultoHigh Chiral-AM-HR .Dissolved and filtered samples of lenticulone  coupled with a UNIVERSAL RP guard column . The gradient was as follows: 0–1.5 min, 10% B; 1.5-22.5 min, 100% B; 22.5–30 min, 100% B; 30–30.1 min, 10% B; and 30.1–37.5 min, 10% B. Mobile phase A and mobile phase B consisted of water with 0.1% formic acid and acetonitrile with 0.1% formic acid, respectively. Reference mass correction was performed with constant infusion of purine and HP-0921 with 10 µL/min through the secondary nebulizer needle. Mass spectral analysis was performed in full-scan mode and in MS² mode. MS-Source parameters were as follows: Gas temperature, 250 °C; drying gas flow, 12 L/min; nebulizer pressure, 30 psi; capillary voltage, 3000 V, fragmentor voltage, 150 V; skimmer voltage, 65 V; octopole voltage, 750 V. Collision energy was set to 36 V, yielding overall best fragmentation efficacy. Additional measurements of intermediates of enzyme assays were conducted with a Waters ACQUITY I-class UPLC in combination with a Waters HSS T3 (C18) column  coupled to a Waters Acquity photo diode array detector (Waters) and a Waters Synapt G2-Si HDMS electrospray ionization (ESI)/quadrupole time-of-flight (Q-TOF) system. The following software was used for MS and HPLC data analysis: Agilent MassHunter Workstation Acquisition B.04.00, Agilent OpenLAB ChemStation Edition Rev. C.01.07, Agilent MassHunter Workstation Qualitative Analysis B.08.01, Agilent OpenLAB ChemStation Edition Rev. C.01.07, and Waters MassLynx V4.1.The spectra were recorded on UV-1800PC (inside the anaerobic chamber) and UV-1650PC spectrophotometers (Shimadzu Corp.) with a 10-mm QS quartz cuvette .Silica gel chromatography was performed using silica gel from Macherey-Nagel . The solvents for the mobile phase were chosen as described in the according sections.® Xtra SIL G/UV254 (Macherey-Nagel). The compounds were detected with UV light (254 nm and 366 nm).TLC was performed using pre-coated TLC-sheets ALUGRAM4) and lenticulone (11) were recorded in a 0.2-mm path length cuvette at 20 °C with a Jasco J-810 spectropolarimeter  equipped with a Peltier temperature controller PTC-423S (Jasco). The samples were dissolved in methanol, final concentrations: 0.6 mg/mL of compound 4 and 0.2 mg/mL of compound 11. Instrumental parameters: spectral range, 180–700 nm, bandwidth, 1 nm, scanning speed, 500 nm/min. The CD spectra shown in Supplementary Figs. CD spectroscopy of 7,8-dideoxy-6-oxo-griseorhodin C (4 and 12 were recorded on a Tensor 27 FTIR spectrometer (Bruker) equipped with an attenuated total reflection (ATR) cell (Pike instruments) at a resolution of 4 cm−1. To facilitate the assignment of characteristic vibrational modes, 11 conformers of 4 and 27 conformers of 12 were modeled using the conformer search algorithm in SPARTAN . To identify the most abundant conformers for each compound, geometry optimization, energy calculation, and frequency calculation on all unique conformers were performed at the DFT level (B3LYP/6-31 G(d)) in Gaussian 16 Rev. C.01 . A conformer was identified with 99% abundance for 4 and a conformer with 97% abundance for compound 12  that were used for comparison to the corresponding experimental spectra. IR bands were constructed with Lorentzian band shapes around each calculated intensity with a band width of 6 cm−1. Calculated frequencies were uniformly scaled by 0.97. Characteristic bands were assigned through visualization of vibrational modes in GaussView 6 (Gaussian Inc.).Solid state IR spectra of compounds 1H- and 13C-NMR spectra of compounds 9 and 11 were recorded with an Avance III HD 400 instrument (Bruker) . All other 1H- and 13C-NMR spectra were recorded with a FT-NMR DMX 600 instrument (Bruker) . DMSO, CDCl3 and 1,4-dioxane-d8 were used as solvents. Chemical shifts refer to the δ-scale and are reported in ppm with a solvent resonance as an internal standard . Data was analyzed and processed using the Bruker TopSpin 4.0.7 software.Chemical structures for figures were drawn with PerkinElmer ChemDraw 19.0. UV and MS traces of enzymatic assays were plotted using GraphPaD Prism 8.3.0.Further information on research design is available in the Supplementary InformationReporting Summary