O-acylterpineol derivatives which were expected to be hydrolyzed into nontoxic metabolites by esterase in the living epidermis, were synthesized from 4-terpineol (4-TER) enantiomers and straight chain fatty acids. Their promoting activities on the SR-flurbiprofen and its enantiomers were tested across full-thickness rabbit skin, as well as to correlate under in vitro and in vivo conditions. The permeation studies indicated that both d-4-O-acylterpineol and l-4-O-acylterpineol had significant enhancing effects, interestingly, d-4-O-aclyterpineol had higher enhancing effects than l-4-O-aclyterpineol with the exception of d-4-methyl-1-(1-methylethyl)-3-cyclohexen-1-yl octadec-9-enoate (d-4-T-dC18). The mechanism of 4-O-acylterpineol facilitating the drug penetration across the skin was confirmed by Attenuated total reflection-Fourier transformed infrared spectroscopy (ATR-FTIR) and molecular simulation. The mechanism of penetration enhancers promoting drug release was explored by the in vitro release experiment. Finally, a relative safety skin irritation of enhancers was also investigated by in vivo histological evaluation. The present research suggested that d-4-O-aclyterpineol and l-4-O-aclyterpineol could significantly promote the penetration of SR-flurbiprofen and its enantiomers both in vitro and in vivo, with the superiorities of high flux and low dermal toxicity.In order to devise more effective penetration enhancers, 4- The transdermal drug delivery system (TDDS) had received much attention in the pharmaceutical industry during recent years with superiorities of extended therapeutic action, decreased side effects, convenient use and better patient compliance , which is still recommended to patients with arthritis and gout, is one of the non-steroidal anti-inflammatory analgesics drugs  racemate and its enantiomers more effectively through the skin.Compared with other kinds of enhancers, 4-terpineol (4-TER), a natural cyclic terpene with a double bond, was more effective for delivering drugs through the skin.  was calculated by deconvolution to verify the feasibility of the penetration enhancer in vitro penetration test, a good IVIVC is always desirable in the drug development and optimization of formulation ; S-FP and R-FP were obtained from Toronto Research Chemicals (Canada); d-4-TER and l-4-TER were purchased from China National Pharmaceutical Group Co., Ltd. ; n-butyric acid, n-hexanoic acid, n-heptanoic acid, n-tetradeconic acid, oleic acid and dichloro sulfoxide were supplied by Jindong Tianzheng chemical reagent Co., Ltd. ; methanol and acetonitrile of chromatographic grade were purchased from the fisher Co, Ltd. ; other chemical reagents were conformed to the standard grade.DURO-TAK 87-235A, pressure sensitive adhesive (PSA), was obtained from Henkel Co., Ltd. ; isopropyl paraben was purchased from Beijing Xingjin Chemical Plant ; d-4-O-acylterpineol and l-4-O-acylterpineol are presented as follows : 0.046 mol fatty acid was dissolved in 0.098 mol dichloro sulfoxide, the reaction mixture was kept for 3 h at 60 °C, then added 0.032 mol 4-TER, and kept at 75 °C for 3 h. The product was extracted three times with ethyl acetate, and dried with anhydrous MgSO4, then the residue was purified by column chromatography on silica gel.The reaction sequences for the preparation of O-acylterpineol were confirmed by NMR  and HPLC-MS . The 1H NMR and MS data are as follows:d-4-methyl-1-(1-methylethyl)-3-cyclohexen-1-yl hexanoate (d-T-C6). ESI-MS m/z: 253.4 [M + H]+; 1H NMR(CDCl3), δ: 0.91 , 1.02–1.07 , 1.08–1.09 , 1.2 6–1.36 , 1.56 , 1.60–1.61 , 1.63–1.66 , 3.56–3.60 , 4.16 . Yield: 79.5%.l-4-methyl-1-(1-methylethyl)-3-cyclohexen-1-yl hexanoate (l-T-C6). ESI-MS m/z: 253.4 [M + H]+; 1H NMR(CDCl3), δ: 0.91 , 1.03–1.06 , 1.08–1.10 , 1.2 5–1.36 , 1.54 , 1.59–1.61 , 1.63–1.65 , 3.56–3.59 , 4.14 . Yield: 82.9%.d-4-methyl-1-(1-methylethyl)-3-cyclohexen-1-yl heptanoate (d-T-C7). ESI-MS m/z: 267.4 [M + H]+; 1H NMR(CDCl3), δ: 0.91 , 1.01–1.06 , 1.07–1.09 , 1.2 4–1.36 , 1.51 , 1.58–1.61 , 1.62–1.65 , 3.57–3.60 , 4.13 . Yield: 81.3%.l-4-methyl-1-(1-methylethyl)-3-cyclohexen-1-yl heptanoate (l-T-C7). ESI-MS m/z: 267.4 [M + H]+; 1H NMR(CDCl3), δ: 0.91 , 1.01–1.04 , 1.07–1.10 , 1.2 3–1.35 , 1.54 , 1.60–1.62 , 1.63–1.65 , 3.57–3.60 , 4.15 . Yield: 78.4%.d-4-methyl-1-(1-methylethyl)-3-cyclohexen-1-yl tetradecanoate (d-T-C14). ESI-MS m/z: 365.6 [M + H]+; 1H NMR(CDCl3), δ: 0.88 , 1.02–1.05 , 1.06–1.07 , 1.2 2–1.27 , 1.56 , 1.58–1.59 , 1.61–1.63 , 3.55–3.59 , 4.10 . Yield: 84.6%.l-4-methyl-1-(1-methylethyl)-3-cyclohexen-1-yl tetradecanoate (l-T-C14). ESI-MS m/z: 365.6 [M + H]+; 1H NMR(CDCl3), δ: 0.90 , 1.02–1.04 , 1.05–1.07 , 1.2 4–1.29 , 1.54 , 1.58–1.59 , 1.60–1.63 , 3.56–3.59 , 4.13 . Yield: 82.4%.d-4-methyl-1-(1-methylethyl)-3-cyclohexen-1-yl octadec-9-enoate (d-T-dC18). ESI-MS m/z: 421.7 [M + H]+; 1H NMR(CDCl3), δ: 0.91 , 1.02–1.06 , 1.07–1.09 , 1.25–1.27 , 1.58 , 1.59–1.62 , 1.63–1.65 , 1.96–2.02 , 2.25–2.27 , 3.57–3.60 , 4.11 , 5.48 . Yield: 86.7%.l-4-methyl-1-(1-methylethyl)-3-cyclohexen-1-yl octadec-9-enoate (l-T-dC18). ESI-MS m/z: 421.7 [M + H]+; 1H NMR(CDCl3), δ: 0.89 , 1.02–1.06 , 1.07–1.10 , 1.24–1.27 , 1.58, 1.59–1.61 , 1.63––1.66 , 1.96–2.02 , 2.26–2.28 , 3.56–3.59 , 4.13 , 5.48 . Yield: 85.2%.The structures of the 4-SR-FP or its single enantiomer (0.01 g) were sealed to 10 mL of pure water at 25 °C in glass vials and waved for 48 h in constant temperature air bath oscillator until fully dissolved. Finally, the contents were passed through the 0.45 μm microporous membrane and centrifuged at 10,000 rpm for 5 min, then aliquots for the supernatant saturated solution were diluted and analyzed by HPLC. The experiments were performed in triplicate.The excess n-octanol were mixed and shaken for 24 h at 25 °C, then the equal of water saturated n-octanol solution and n-octanol saturated aqueous solution through liquid-separation were obtained. The SR-FP or its single enantiomer (0.001 g) were added in the saturated solution, which were mixed to the ratio of 1:4, then shook for 24–48 h followed by stand for 24 h. (The shakings were in constant temperature air bath oscillator at 25 °C). Afterwards, the HPLC method was utilized to detect the concentration of the substance in the oil phase and the water phase.The same volume of water and Male, Japanese big-eared rabbits weighting 2.5–3.0 kg were purchased from the animal experimental center of North China University of Science and Technology. The experimental process was in accordance with the ‘Ethical Guide for Laboratory Animal Investigation’ and was approved by the Animal Experimental Center Committee of North China University of Science and Technology. All efforts were made to minimize animal suffering and to limit the number of animals used.The hair of back skin was removed by clipper and electrical shaver carefully when the rabbits were anesthetized with ether. Full thickness of back skin was excised after the rabbit was sacrificed by injecting air into the ear veins, and the subcutaneous fat tissue was carefully removed using the surgical scissor and scalpel. The integrity of the skin was confirmed by microscopic observation method. The treated rabbit skin was cut into a size of 1.5 cm × 1.5 cm and stored at –80 °C until use. Before use, it needed to be immersed in physiological saline for 20 min to thaw.® 9744, 3M, USA) by using lab coating unit . The coated laminate was dried at 50 °C for 15 min. After the solvent had been removed, it was covered with backing laminate .Drug-in-adhesive patch (drug loading of 5% by adhesive) was prepared by using solvent evaporation method. An appropriate amount of FP was dissolved in a suitable amount of ethyl acetate, which was later added to the adhesive and mixed thoroughly with a mechanical stirrer. The resulting solution was coated onto release liner , the receptor solution of 2.0 mL was withdrawn and equal volume PBS was added to maintain a constant volume of receptor solution.Modified two-chamber diffusion cells with an effective diffusion area of 0.95 cmQ) versus time was plotted.The cumulative amount of each drug permeating per unit area  was calculated from the slope of linear region of the plot. The lag-time was determined by extrapolating the linear portion of the curve to the abscissa.The steady-state flux (24hQ with enhancer to 24hQ without enhancer.Enhancement ratio (ER) was used to evaluate the activity of the enhancers, which is the ratio of A certain amount of FP was dissolved in a mixed solution of sterile water and Tween-80 at 2.0 mg/mL. The fresh intravenous solution was prepared just before intravenous administration.Six male rabbits weighing 2.5 kg were selected for IV. A capacity of 4.8 mg/kg FP (0.2 g FP was dissolved in a mixed solution of pure water and Tween80) was administered to the one ear vein of rabbit, the other ear vein of rabbit was phlebotomized about 0.5 mL at 5, 10, 15, 30 min and 1, 2, 3, 4, 6, 8, 10, 12 h. The blood samples were centrifuged at 4000 rpm for 10 min and kept at –80 °C.SR-FP; (2) enhancer group A: prepared SR-FP patch with 5% d-4-TER; (3) enhancer group B: prepared SR-FP patch with 5% d-4-T-dC18; (4) enhancer group C: prepared SR-FP patch with 5% l-4-T-dC18; (5) enhancer group D: prepared S-FP patch with 5% d-4-T-dC18; (6) enhancer group E: prepared R-FP patch with 5% d-4-T-dC18. In order to prevent the patches fallen off, gauze and medical tape were used to fix the patches. The blood from ear vein of rabbit was collected 0.5 mL at 0.5, 1, 2, 4, 6, 8, 12, 16, 20, 24, 26, 28, 30, 32, 36 and 48 h, and the patch was removed after 24 h, then the residual drug was wiped with physiological saline. The sample was processed by the same method as IV. All animal studies were carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and were also approved by the Life Science Research Center of North China University of Science and Technology.Thirty-six male rabbits weighing 2.5 kg were divided into six groups, and the back skin was intact without erythema. For patch administration studies, patches without or with enhancers (120 mg/kg) were stuck to the shaved area of the rabbits. They were administered as follows: (1) control group: only the After being thawed and equilibrated to room temperature, an aliquot (100 μL) of plasma sample was spiked with isopropyl paraben (10 μL) of 40.0 μg/mL and 400 μL methanol. The samples were vortexed for 5 min and followed by centrifugation at 16,000 rpm for 10 min at 25 °C. After centrifugation, the supernatant was transferred to a clean tube and evaporated to dryness at 40 °C under a gentle stream of nitrogen. The dried samples were reconstituted with 100 μL mobile phase, vortexed and centrifuged before analysis.® software  based on noncompartmental model, including the maximal plasma drug concentration (Cmax), the time to reach Cmax (Tmax) which were obtained from the observed data, the area under the time–concentration curve from time 0 to time t (AUC0–t) of distribution which was calculated using the linear trapezoidal rule, the mean residence time (MRT), the plasma clearance (Cl) and the apparent volume(V). Enhancement factor (fE) was calculated from the following equation:Pharmacokinetic parameters were calculated using WinNonlinFor IVIVC studies, the Wagner-Nelson method was often used for single-chamber model calculations, the IAO-Riegelman method was often used for two-compartment model calculations, and the Deconvolution method was used for non-compartment models.in vivo could be expressed by the following volume integral equation:The absorption concentration of the drug C represented the output function, i.e., the concentration function of the patch administration; W represented the weight function, i.e., the concentration function for IV, expressed as the AUC of IV during this time interval; R represented the input function, i.e., the average absorption rate during this time interval.The formula can be interpreted as:R:So, for each measurement point ……in vivo (vivo%) was obtained. The in vitro cumulative percentage (vitro%) was used as the independent variable, and vivo% was used as the dependent variable, correlation analysis was performed in GraphPad Prism 7.0.The data was calculated in Microsoft Excel and finally cumulative permeability 2. The patch was sandwiched in the double-layered round mesh disk and dipped into a dissolution cup with receptor fluid  of 900 mL (n = 6). Samples were withdrawn (5.0 mL) at the scheduled time point of 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h, and the same volume of phosphate buffer solution was replenished.In the drug release study, the drug dissolution apparatus  was set at 32 ± 0.5 °C, whose effective contact area with the patch was 16 cmin vitro analysis of SR-FP, thermo (USA) C18 column  maintained at 40 °C was used to elute SR-FP, the mobile phase was used methanol and 1% aqueous glacial acetic acid  and the wavelength was set at 247 nm. The HPLC conditions of S-FP and R-FP were as follows: The column was CHIRALPAK®IA  of DAOCEL . The mobile phase consisted of acetonitrile and trifluoroacetic acid , the wavelength was set at 254 nm. For in vivo analysis of S-FP and R-FP, the mobile phase consisted of acetonitrile and trifluoroacetic acid , and other conditions were the same to in vitro analysis.The concentrations of drugs in the receptor medium or rabbit plasma were determined using a validated HPLC method. The HPLC system was equipped with a Shimadzu HPLC apparatus, including an LC-20AT pump, a SIL20A syringe, an SPD-20A detector, and a CTO-20A column oven. The propylparaben was used as an internal standard, and the flow rate of chromatographic analysis was set 1.0 mL/min. For Molecular simulation was used to predict the intermolecular interaction among the stratum corneum ceramide and enhancers. The structures of molecular were constructed by ChemDraw 16.0. By calculation with COMPASSII force field, the lowest energy conformations of compound and mixture were found in Materials Studio software 7.0 with Forcite and Blend. The mixing frequency was set at 1000, and all the process were calculated in Fine. Then docking calculations were performed with the Dreding force field, the corresponding energy was calculated  and randomly divided into seven groups (n = 3). The marked areas were applied with 5% (w/v) enhancer solution of 20 μL. They were administered as follows: (1) control group only the absolute ethanol; (2) group A with d-4-TER; (3) group B with l-4-TER; (4) group C with d-4-T-C14; (5) group D with l-4-T-C14; (6) group E with d-4-T- dC18; and (7) group F with l-4-T-dC18. After the enhancer was infiltrated of 30 min, the skin of the penetrated area was peeled off. Then the residual solution was blotted up and dried of 15 min. Finally, the treated skin was placed on the surface of zinc selenide (ZnSe) with an incidence angle of 45° and detected by ATR-FTIR spectroscopy . The stratum corneum was scanned 200 times in the detection wavelength range of 1000–4000 cm−1 at a resolution of 2 cm−1. Infrared absorption peaks were analyzed using Oringe 7.0.The shaved back was divided into 21 areas (each area in 1 cm2). The marked areas were applied with 5% (w/v) enhancer solution of 20 μL. They were administered as follows: (1) control group (without enhancer); (2) group A with d-4-TER; (3) group B with l-4-TER; (4) group C with d-4-T-dC18 and (5) group D with l-4-T-dC18. After the enhancer was infiltrated for 1 h, the surface-relieving penetration enhancer was removed by alcohol cotton. Rabbits were sacrificed by injecting air into the ear veins, then the shaved skin was cut and fixed by 4% paraformaldehyde for one day. The skin was embedded in paraffin for 30 min under vacuum at 60 °C, subjected to paraffin vertical coronal sections and stained with HE. These samples were then observed under Olympus microscope and compared with the control sample that was free of exposure to drugs.The back hair of rabbits was removed with the same method above, then the shaved back was divided into five areas (each area of 1 cmp < .05. Correlation coefficients were examined for significance (p < .05) using Student’s t-test of the SPSS program.Statistical analysis was carried out using analysis of variance (ANOVA). Data was considered significant when d-4-TER level (w/w), the cumulative transports of SR-FP were 122.93 ± 8.02 μg/cm2, 168.14 ± 19.95 μg/cm2 and 266.01 ± 58.81 μg/cm2, respectively. The cumulative transports of SR-FP were 145.16 ± 13.64 μg/cm2 and 222.09 ± 19.19 μg/cm2 for 3% and 5% l-4-TER level, respectively. At 7% level of chiral 4-TER, the enhancer was precipitated from the patch for its high concentration. It can be seen that at 5% concentration, the cumulative permeation of SR-FP was higher than the other levels (p < .05), so the 5% level was chosen to perform the latter experiment. The concentration of 4-O-acylterpineol derivatives during the subsequent experiments were at the same molar concentration with 4-TER which was selected for SR-FP.When cumulative amounts permeating as a function of the enhancer concentration were compared at 24 h, the fluctuation was apparent. At 0%, 3% and 5% SR-FP and its enantiomers  with or without chiral 4-O-acylterpineol were listed in in vitro experiment indicated that all the enhancers had significantly higher enhancing effects on the permeation of SR-FP and its enantiomers than the control group (p < .05). By comparison, l-4-T-dC18 had the most significant enhancing effect, and the attained Q24h was 547.20 ± 35.94 μg/cm2, increasing the SR-FP in Q24h by 4.45-fold. It was also found that esterification resulted in greater lipophilicity due to the masking of the hydroxyl group of chiral 4-TER, so the effect of ester promoting penetration is stronger than that of the equimolar amount of alcohol . Miyazaki et al. has reported different permeation rates of propranolol enantiomers through rat skin . The solubilities of SR-FP, R-FP and S-FP were 89.11 ± 5.48, 49.82 ± 1.31 and 46.39 ± 4.30 μg/mL, respectively; The logP values of SR-FP, R-FP and S-FP were 2.28 ± 0.04, 1.83 ± 0.09 and 1.86 ± 0.05, respectively. The larger logP and solubility value of SR-FP may imply higher speed of permeant penetration across the SC (lipophilic layer) and the VE (hydrophilic layer) , which could be attributed to two possible reasons to explain this phenomenon. First, the elimination rate of S-FP was slower than that of R-FP (p < .05), which was available in the pharmacokinetic results of IV in vivo. This also has been confirmed in S-FP could be detected after administration of individual R-FP to rabbits, but inversion from S-FP to R-FP had not been observed, just as shown in S-FP and R-FP during in vitro permeation experiments. These results demonstrated that the penetration process of the SC could not provide any enantiomer selectivity, which also has been illustrated by molecular simulation results, presented in In observed  (p < .05in vivo skin penetration study are similar to that of in vitro. d-4-T-dC18 and l-4-T-dC18 had significantly greater Cmax and AUC0-t of FP than the control group (p < .05). Notably, the promoting ability of l-4-TER-dC18 was highest among all enhancers (p < .05). l-4-T-dC18 provided the highest AUC0-t and the maximum plasma concentration of R-FP (110.62 ± 21.62 h·μg/mL and 4.43 ± 1.03 μg/mL). Identically, l-4-T-dC18 provided the highest AUC0-t and the maximum plasma concentration of S-FP (209.04 ± 54.76 h·μg/mL and 8.27 ± 2.49 μg/mL). Despite the in vitro and in vivo permeability parameters were different attributing to the complex environment in vivo, these enhancers displayed a similar ability on the transdermal of FP, which further confirmed the results of the in vitro penetration. In contrast, there was no significant change in Tmax and MRT between the enhancer containing group and the control group (p > .05). The V and Cl values of the enhancer containing groups were significantly reduced compared with the control groups (p < .05), which is conducive to maintaining the prototype component of the drug in vivo, and prolonging the action time.The pharmacokinetic parameters were calculated in F) was calculated by the following equation:Absolute bioavailability , which indicated that in vitro isolated study was reliable to predict FP in vivo dynamics absorption.The correlation coefficient indicated excellent correlation, the exceptional IVIVC relationship indicated that the results of In vitro release experiment was carried out to explore the role of chemical enhancers in SR-FP release from patches. As is presented in supporting information Figure S3, due to the small error in the in vitro release experiment, the error line in the figure is not obvious. The cumulative percentages of SR-FP release from patches within 24 h for the control group, d-4-TER group and l-4-TER group were 70.15 ± 0.75%, 66.58 ± 1.57% and 69.63 ± 1.06%, respectively. There was no significant difference in the release amount among the above three groups (p > .05), however, the cumulative release percentage of l-4-T-C7 and l-4-T-dC18 were 78.29 ± 0.8% and 91.09 ± 1.65%, respectively, which were significantly higher than that of l-4-T-C7 and l-4-TER. It was noteworthy to point out that the effects of 4-O-acylterpineol on SR-FP release were consistent with the in vitro transdermal experiment and in vivo pharmacokinetic study. Usually, it is regarded that the transdermal delivery of drug in the patch includes two processes: release and penetration. The present result suggests that the promoting effect of enhancers depends not only on the promoting ability through the skin but also on the release speed from the patch.O-acylterpineol promoted the release process of FP mainly by increasing the molecular mobility of PSA rather than decreasing the interaction between FP and matrix. The results of in vitro release indicated that hydrogen bond between FP and PSA was not formed due to neither l-4-TER nor d-4-TER could improve the release percentage of FP compared to the control group. Therefore, drug release from patches might mainly be dominated by PSA molecular mobility. So, it is postulated that higher molecular mobility of PSA would introduce a higher formation frequency of free volume in the PSA, thereby enhancing the FP release from PSA according to the free volume theory  were concerned, which formed hydrogen bond interactions with the polar head of the CER-NP assemblies, the experimentally determined ER increased in line with absolute values of docked energy. As the amount of FP which can penetrate through the skin increases, the enhancer binds more tightly with the CER, and the energy of hydrogen bonding increased to establish a positive correlation between ER and docked energy. It can be deduced that enhancers are combination with SC by hydrogen bond, the higher the docked energy between compounds and SC, the stronger promoting abilities that can be obtained in patch for permeant dissociate from the polar domain of CERs. To prove this hypothesis requires further investigation.As far as the other enhancers  and amide II   which was formed by the uncoiling of the α-helical structure (1662–1645 cm−1) for various systems .The histological evaluation was employed to gain insights into the physicochemical changes of the skin and roughly evaluate the skin irritation of enhancers. Histological studies of the optimized enhancer group  and normal enhancer group  were performed and compared with the control group (supporting information O-acylterpineol were used as enhancers, there was marginally focal disruption of the epidermis with thinning, which might be related to the conformational changes of the lipid bilayer. Meanwhile, skin injury also observed with chiral 4-T-dC18 showed slight dermal edema. Although the chiral 4-O-acylterpineol induced a mild impairment of the barrier function of the skin, it did not alter the viability of the skin, meanwhile, the impairment was reversible. The reduced skin barrier is reflected in the increased permeability of the skin and therefore increased flux (Prasad et al., O-acylterpineol. Meanwhile, the FP patch containing chiral 4-O-acylterpineol was safe for application on the skin, and the marginally pathological changes have no effect on skin integrity, which was in line with the expectation of chiral 4-T-dC18 derived from the terpene natural compound.The control group exhibited an intact morphology throughout the skin of epidermal keratinocytes. When chiral 4-l-4-T-dC18 was shown to be the most promising enhancer for increasing the transdermal amount of SR-FP and its enantiomers, with the superiorities of high flux and low skin irritation. The IVIVC analysis revealed that there was a good correlation for permeants between the penetration amounts from patches in vitro and the AUC0-∞in vivo when employing 4-O-acylterpineol derivatives as penetration enhancers. Moreover, the mechanistic insights studies suggested that chiral 4-O-acylterpineol could make lipids and keratin looser or more permeable, and bind more tightly with the CER-NP to decrease the affinity between FP enantiomers and the lipids in the SC. In addition, this investigation proposes a new idea for designing chiral penetration enhancers, and provides a reference for improving the transdermal delivery of chiral drugs through rationally selection of chemical enhancers.From the results of present investigations,


Cholesterol oxidase biosensors have been used to determine the level of cholesterol in different serum and food samples. Due to a wide range of industrial and clinical applications of microbial cholesterol oxidase, isolation and identification of a new microbial source (s) of cholesterol oxidase are very important.Streptomyces sp. strain NEAE-94 is a promising source of cholesterol oxidase. It was identified based on cultural, morphological and physiological characteristics; in addition to the 16S rRNA sequence. The sequencing product had been deposited in the GenBank database under the accession number KC354803. Cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 in shake flasks was optimized using surface response methodology. The different process parameters were first screened using a Plackett-Burman design and the parameters with significant effects on the production of cholesterol oxidase were identified. Out of the 15 factors screened, agitation speed, cholesterol and yeast extract concentrations had the most significant positive effects on the production of cholesterol oxidase. The optimal levels of these variables and the effects of their mutual interactions on cholesterol oxidase production were determined using Box-Behnken design. Cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 was 11.03, 27.31 U/mL after Plackett-Burman Design and Box-Behnken design; respectively, with a fold of increase of 6.06 times compared to the production before applying the Plackett-Burman design (4.51 U/mL).The local isolate 2HPO4 1, FeSO4.7H2O 0.01, MgSO4.7H2O 0.5), pH 7, inoculum size 4% (v/v), temperature 37°C, agitation speed of 150 rpm, medium volume 50 mL and incubation time 5 days.Maximum cholesterol oxidase activity was obtained at the following fermentation conditions: g/L (cholesterol 4, yeast extract 5, NaCl 0.5, K Streptomyces natalensis is required for the biosynthesis of the polyene macrolide pimaricin  which is used as a mould inhibitor in the food industry [Cholesterol oxidase EC 1.1.3.6) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyzes cholesterol oxidation to cholestenone (4-cholesten-3-one) and hydrogen peroxide .1.3.6 is, 3. It pindustry .Bordetella sp. cholesterol oxidase is used as a promising therapy for lung cancer treatment (both in vitro and in vivo) and led to irreversible cell apoptosis even after addition of cholesterol [Rhodococcus equi infects young horses as well as human immuno-compromised patients [Rhodococcus equi for the host cells [Anthonomus grandis”, which decreases the cotton yields [Pectinophora gossypiella), tobacco budworm (Heliothis virescens) and corn earworm (Helicoverpa zea) [The lesterol . Rhodocopatients . Cholestst cells . Purifien yields . When chn yields . Cholestrpa zea) .Streptomyces natalensis [Streptomyces cavourensis [Mycobacterium tuberculosis [Chromobacterium sp. DS1 [Bacillus pumilus W1 and Serratia marcescens W8 [The intracellular or extracellular cholesterol oxidase produced by a variety of microorganisms, including talensis  Streptomourensis , Mycobacrculosis , Chromob sp. DS1 , Bacilluscens W8 . In casescens W8 . Non-patMany scientists have sought to increase the yield of cholesterol oxidase because of low production by several microorganisms. The production of cholesterol oxidase is greatly influenced by the soluble component of the medium and culture conditions, including temperature, incubation time, pH, inoculum size and the rate of agitation . ResponsStreptomyces anulatus strain NEAE-94 using response surface methodology.The objectives of this study were to identify robust cholesterol-assimilating actinomycetes strain with high cholesterol oxidase activity, and to optimize the fermentation conditions for enhanced production of cholesterol oxidase by Streptomyces sp. strain NEAE-94, has been tested for its cholesterol oxidase activity with a plate-based method, the formation of the pink areas around the colonies indicated the presence of cholesterol oxidase activity . Streptomyces sp. strain NEAE-94 showed positive antimicrobial activities against Staphylococcus aureus, E. coli, Bacillus subtilis and Pseudomonas aeruginosa, but no activities were shown against Candida albicans, Rhizoctonia solani, Aspergillus niger, Fusarium oxysporum, Alternaria solani, Sacchromyces cerevisiae, Bipolaris oryzae or Klebsiella pneumonia. Streptomyces sp. strain NEAE-94 did not produce melanoid pigments in tyrosine agar, peptone-yeast extract iron agar or tryptone-yeast extract broth.The physiological properties of ase Fig. c, cellulStreptomyces sp. strain NEAE-94 was observed by scanning electron micrograph after incubation on medium of starch nitrate agar medium at 30°C for 14 days. Microscopic observation of Streptomyces sp. strain NEAE-94 showed rectiflexibiles spores chains ; 99.59% to that of Streptomyces parvus strain 3151, query cover 94% (GenBank accession no. EF063462.1); 99.38% to that of Streptomyces flavofuscus strain NRRL B-2594, query cover 94% (GenBank accession no. EF178690.1) and 99.19% to that of Streptomyces fimicarius strain BWL-H1, query cover 95% (GenBank accession no. MG197994.1).The obtained 16S rRNA sequence of Streptomyces sp. strain NEAE-94 and its closest phylogenetic neighbors of the genus Streptomyces which showed significant similarities are shown in Table Streptomyces anulatus) has been deposited in the Culture Collection Ain Shams University (CCASU). The culture collection accession number CCASU 20202 is assigned to the deposited strain.The whole morphological and physiological properties of Streptomyces anulatus strain NEAE-94 during cholesterol oxidase production in shake flask in submerged fermentation is shown in Fig. 4)2SO4 4; FeSO4.7H2O 0.01; MgSO4.7H2O 0.5; NaCl 0.5; K2HPO4 1 and pH 7; inoculum size was 4% (v/v) and incubated for 5 days at 37°C using an agitation speed of 150 rpm. In the run no. 4, the lowest production of cholesterol oxidase was obtained (0.87 U/mL) using 50 mL medium/250 mL conical flask consists of (g/L): Glucose 5; starch 10; cholesterol 1; yeast extract 1; peptone 1; (NH4)2SO4 4; FeSO4.7H2O 0.05; MgSO4.7H2O 0.1; NaCl 1; K2HPO4 1 and pH 9; inoculum size was 4% (v/v) and incubated for 7 days at 37°C using an agitation speed of 100 rpm.The design matrix used for screening of the significant factors for production of cholesterol oxidase and the appropriate responses are shown in Table 2HPO4 positively affect cholesterol oxidase production, whereas the remaining factors named incubation time, glucose, inoculum size, MgSO4, NaCl and FeSO4 negatively affect cholesterol oxidase production. The data revealed that, starch (G), peptone (K) and ammonium sulphate (L) with higher P-values , lower effects  and lower contribution %  are insignificant factors. The Pareto chart shows absolute effects values and illustrates the significance order of the factors that influence cholesterol oxidase production. The Pareto chart shows a reference line, any absolute effect value extending past this reference line is highly essential  and the adjusted determination coefficient (Adj. R2 = 0.9978) are very high. Cholesterol, agitation speed with a P-value of < 0.0001 was determined to be the most significant factors, followed by the concentration of yeast extract, incubation time (0.0001) then glucose (0.0002)  implies that the model terms are significant. The F-value of the model (573.21) means that it is significant. The first order polynomial equation representing the production of cholesterol oxidase in relation to the independent factors was obtained by neglecting the insignificant factors:The values of the determination coefficient (R2) Table . The P-v2HPO4, NaCl, MgSO4 and FeSO4; respectively.Where Y is the production of cholesterol oxidase and A,B,C,D,E,F,H,K,M,N,O,P are temperature, the time of incubation, size of inoculum, speed of agitation, pH, concentration of glucose, concentration of cholesterol, concentration of yeast extract, concentration of K4.7H2O 0.01; MgSO4.7H2O 0.5; NaCl 0.5; K2HPO4 1; yeast extract 4; cholesterol 3 and pH 7. The production medium was inoculated with inoculum size of 4% (v/v) and incubated at a temperature of 37°C in a shaker incubator at 150 rpm for 5 days. The production of cholesterol oxidase using the previous medium was 11.03 U/mL, which was increased 2.45 times compared to the enzyme activity obtained before application of Plackett-Burman design (4.51 U/mL).For assessment of Plackett-Burman design precision, the following production medium was used for cholesterol oxidase production (g/L): FeSOStreptomyces anulatus strain NEAE-94 and to study the interaction effects among these factors. In the current study, 15 experiments with various combinations of agitation speed, cholesterol concentration and yeast extract concentration were performed and the experimental and predicted cholesterol oxidase production and residuals for the 15 trials are provided in Table Box–Behnken design was used to obtain the optimal levels of the most significant factors influencing cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 (5.64 U/mL) was achieved in the 13th run when the agitation speed was 100 rpm, cholesterol concentration was 4 g/L and yeast extract concentration was 3 g/L. The maximum value of cholesterol oxidase production was achieved in the 12th run with value of 27.31 U/mL, when agitation speed was 150 rpm, cholesterol concentration was 4 g/L and yeast extract concentration was 5 g/L.Based on the variations in the agitation speed, cholesterol concentration and yeast extract concentration, the results showed variations in the cholesterol oxidase production. Cholesterol oxidase production ranged from 5.64–27.31 U/mL. The lowest cholesterol oxidase production by F-test (113.82) , mean value (15.16), adequate precision value (31.469), PRESS value (40.03) and standard deviation value (0.82) (Table Table 2) Table . The curP and F values listed in Table P-values and F-values that the linear coefficients of cholesterol concentration, interaction between the agitation speed and cholesterol concentration; agitation speed and yeast extract concentration; cholesterol concentration and yeast extract concentration and quadratic effects of agitation speed, cholesterol concentration and yeast extract concentration are significant as it is evident from the F-values of 119.05, 7.80, 62.24, 7.80, 321.59, 79.45, 526.73; respectively, and P-values of 0.0001, 0.0383, 0.0005, 0.0383, < 0.0001, 0.0003, < 0.0001; respectively. On the other hand, P-values of the linear coefficients of agitation speed (X1) and yeast extract concentration (X3) indicate that they had nonsignificant effects on cholesterol oxidase production by the strain under study.The significance of each coefficient was defined in terms of both Streptomyces anulatus strain NEAE-94, with a non-significant lack of fit  and a very low P-value< 0.0001 was shown in the fit summary results (Supplementary Table 2 of 0.9864 and 0.9427 and the lowest standard deviation (0.82) was reported in the summary statistics of the quadratic model.The quadratic model of Box–Behnken design used for cholesterol oxidase production by The optimum levels of agitation speed, cholesterol concentration and yeast extract concentration giving the maximum cholesterol oxidase production was evaluated by a second-order polynomial equation. Cholesterol oxidase production can be predicted by applying the following second-order regression equation in terms of the independent variables:1 is the coded value of agitation speed, X2 is the coded value of cholesterol concentration and X3 is the coded value of yeast extract concentration.Where Y is the cholesterol oxidase production, X1 - X3) and the optimum level of each factor required for the maximum cholesterol oxidase production, the 3D curves and its corresponding contour plots were generated by plotting the cholesterol oxidase production on the Z axis versus two factors are allowed to vary and the third variable is fixed at its zero level , cholesterol concentration (X2) while yeast extract was kept at the central point (5 g/L). The cholesterol oxidase activity increases gradually by increasing cholesterol concentration and agitation speed till reach its optimum, but further increase in both cholesterol concentration and agitation speed leads to decrease in cholesterol oxidase activity. By solving the Eq. (To understand the interaction among the three factors (Xtration X while ye1) and yeast extract concentration (X3) while cholesterol concentration (X2) was fixed at the central point (4 g/L). Figure 1) results in lower cholesterol oxidase production and with increasing the agitation speed, the cholesterol oxidase production increases beyond 150 rpm after which cholesterol oxidase production was reduced. Lower and higher concentrations of yeast extract (X3) results in lower cholesterol oxidase production, and the maximum cholesterol oxidase production, obviously obtained at the central level of the yeast extract concentration. By analysis of Fig. The three-dimensional surface and contour plots in Fig. tration X was fixe2) and the concentration of yeast extract (X3) by maintaining the agitation speed at the central point (150 rpm). With an increased concentrations of both cholesterol and yeast extract, cholesterol oxidase production by the selected strain (Streptomyces anulatus strain NEAE-94) was improved and the maximum cholesterol oxidase production was obtained at the middle levels of two factors, and further increase of cholesterol concentration or yeast extract concentration decreases cholesterol oxidase activity. By analysis of Fig. Figure Streptomyces anulatus strain NEAE-94 was 27.31 U/mL. The verification revealed a high degree of model accuracy (97.21%).Using the optimal levels of the process variables as obtained using Box–Behnken design, the experimental cholesterol oxidase production was verified and compared with the predicted value of cholesterol oxidase production (26.55 U/mL). The maximum experimental cholesterol oxidase production by Streptomyces sp. strain NEAE-94 mainly have the same characteristics as Streptomyces anulatus, Streptomyces flavofuscus, Streptomyces parvus and Streptomyces fimicarius in that it produced rectiflexibiles spore chains and did not produce melanin pigments. However, Streptomyces sp. strain NEAE-94 mainly differed from Streptomyces fimicarius in that it produces yellow aerial mycelium, yellow substrate mycelium, and yellow diffusible pigment on ISP medium 2. On the other hand, Streptomyces fimicarius lost the ability to produce a diffusible pigment and not produce distinctive substrate mycelium pigment. Also Streptomyces sp. strain NEAE-94 differed from Streptomyces fimicarius and Streptomyces parvus in the pattern of utilization of carbon sources .es Table . Moreove94 Table  indicateanulatus . Accordi2 = 0.9996) and the adjusted determination coefficient (Adj. R2 = 0.9978) are very high and suggests a strong model significance [P-value of the factor reveals that the factor is more essential for cholesterol oxidase production.The values of the determination coefficient  have been maintained at their highest levels (+ 1). Whereas, the factors with negative impact on cholesterol oxidase production  were fixed at their low (− 1) levels. On the other hand, the insignificant factors 2SO4) have been omitted in the subsequent experiments.Depending on the statistical analysis of Plackett–Burman design results, the factors with positive impact on production of cholesterol oxidase  [Micrococcus sp. (3.68 U/mL) [Streptomyces A (2.44 U/mL) [Brevibacterium sp. (1.483 U/mL) [Streptomyces lavendulae (2.21 U/mL) [Streptomyces sp. (6.2 U/mL) [Bacillus cereus (1.67 U/mL) [Streptomyces fradiae (0·03 U/mL) [The maximum cholesterol oxidase production obtained from this study 27.31 U/mL) by 24 U/mL) , Microco68 U/mL) , Strepto.31 U/mL 83 U/mL) , Strepto21 U/mL) , Strepto.2 U/mL) , Bacillu03 U/mL) .2 and adjusted R2 values were 0.9951 and 0.9864; respectively, implying that the model is appropriate to represent the real relationship between cholesterol oxidase production and the selected factors. The largest R2 value showed that experimental and expected cholesterol oxidase production values are in excellent agreement [2 value of 0.9427 shows that the model is sufficient to predict the value of the production of cholesterol oxidase in the range of factors used.The current Rgreement . PredictF-value, low standard deviation and high adequate precision indicating the validity and high degree of accuracy of the model prediction for the production of cholesterol oxidase by Streptomyces anulatus strain NEAE-94.The non- significant lack-of-fit, the high value of adjusted and predicted R-squared, low PRESS value, high Enterococcus hirae was achieved using 1 g/L cholesterol. Sojo et al. [Rhodococcus erythropolis and Streptomyces fradiae was achieved using 2 g/L cholesterol.Enhanced production of cholesterol oxidase was recorded with the use of different compounds as cholesterol, yeast extract ; malt exo et al.  and Yazdo et al.  reportedRhodococcus equi 2C and Rhodococcus equi no. 23 was recorded using 0.3% [Pseudonocardia compacta S-39 were ammonium nitrate, ammonium sulphate and sodium nitrate [Arthrobacter simplex were ammonium salts.There are different and opposite effects of nitrogen sources on the production of cholesterol oxidase in the literature. Voelker and Altaba  recordeding 0.3%  and 0.4–ing 0.3% ; respecting 0.3%  and Ahmaing 0.3%  reported nitrate . Liu et  nitrate  reportedAppropriate oxygen must be given in the fermentation media using shaken cultures to satisfy the organism’s growing demands and to produce the desired end product. The suitable agitation speed ensures that the dissolved oxygen in the medium is adequately supplied and can become vital for microbial biosynthesis of certain end products.4.7H2O 0.01, MgSO4.7H2O 0.5, NaCl 0.5, K2HPO4 1, pH 7, inoculum size 4% (v/v), temperature 37°C, agitation speed 150 rpm, medium volume 50 mL and incubation time 5 days.In this study, the maximum cholesterol oxidase production (27.31 U/mL) is obtained using the following medium formula (g/L): yeast extract 5, cholesterol 4, FeSOStreptomyces cavourensis strain NEAE-42. The most important factors that significantly influenced cholesterol oxidase production were initial pH, cholesterol and (NH4)2SO4 concentrations. El-Naggar et al. [4)2SO4 8 g/L. However, Ahmad and Goswami [Rhodococcus sp. NCIM 2891 using the classical and statistical methods. They reported that the maximum cholesterol oxidase production (3.25 U/mL) was obtained in the statistically optimized medium under the optimal levels of the process factors that were 2.5 g/L (NH4)2HPO4, 9 g/L yeast extract and 3.5 g/L cholesterol in approximately 60 h of cultivation at 30°C and pH 7.0. On the other hand, Srivastava et al. [Streptomyces rimosus MTCC 10792. They reported that, out of the examined factors, yeast extract, dextrose, starch and ammonium carbonate were the most significant factors and the maximum cholesterol oxidase production was 5.41 U/mL in the optimized medium using the optimum concentrations of the four variables that were (g/100 mL medium): 0.05 ammonium carbonate, 0.1 starch, 0.8 dextrose and 0.99 yeast extract.El-Naggar et al.  used ther et al.  reporteda et al.  applied Streptomyces badius using Plackett-Burman design and optimized these variables using Box-Behnken design. They reported that, yeast extract, pH, Tween 20 and temperature were the most significant factors and the maximum cholesterol oxidase production by Streptomyces badius was 2.05 U/mL in the optimized medium using the optimum levels of the four variables that were determined to be: yeast extract, 0.45%; pH, 6.5; Tween 20, 0.05% and 30°C. Moreover, ElBaz et al. [Bacillus pumilus. The maximum cholesterol oxidase production (90 U/mL) was obtained after 6 days of fermentation at pH 8 with medium/flask ratio of 0.35 and the concentrations of cholesterol, NH4NO3, yeast extract and Tween 80 were 0.2, 0.3, 1 and 0.2%; respectively.Moradpour et al.  screen vz et al.  used a tBacillus cereus strain KAVK4. The highest production of cholesterol oxidase was achieved under flask conditions using the optimal levels of fructose (as a carbon source) in the production medium at a concentration of 2%, ammonium nitrate (as nitrogen source) at a concentration of 0.2% and magnesium sulphate as metal ion source at a concentration of 0.03%. The maximum production of cholesterol oxidase by Bacillus cereus strain KAVK4 of 1.67 U/mL was achieved at the optimum process variable values .Kuppusamy and Kumar  used traBrevibacterium sp. They found that, the optimal values of the three independent variables resulted in highest cholesterol oxidase production by Brevibacterium sp. (1.483 U/mL) were determined to be: 22.361 (min) treatment time, 0.2932% (v/v) Tween-80, 4.076 g/L cholesterol. In addition, El-Naggar et al. [Streptomyces aegyptia strain NEAE− 102. They found that, out of 15 variables screened by Plackett-Burman design experiments, pH, incubation time and cholesterol concentration were the most significant variables for cholesterol oxidase production. El-Naggar et al. [Yang and Zhang  used a tr et al.  used ther et al.  used a fStreptomyces lavendulae NCIM 2421. A peak of cholesterol oxidase activity of 1.8 U/mL was detected at 72 h. They found that, Streptomyces lavendulae NCIM 2421 is a constitutive producer of cholesterol oxidase where the addition of cholesterol to the medium did not enhance cholesterol oxidase activity. Whereas, Chauhan et al. [Streptomyces lavendulae NCIM 2499. They reported that the model predicted maximum cholesterol oxidase production (2.21 U/mL) could be achieved after 72 h of incubation using the medium of the following composition (g/L): sodium chloride 0.7, MgSO4 2, K2HPO4 0.6, soyabean meal 20, malt extract 20, and glycerol 10 mL/L. An initial pH of 7.5 supported the maximum production of cholesterol oxidase. Moreover, Srivastava et al. [Streptomyces rimosus was achieved using the optimal levels of process variables which found to be inoculum size , pH (7), incubation temperature (30°C), incubation time (48 h) and agitation speed (200 rpm). Fazaeli et al. [Escherichia coli when the induced culture production medium was incubated for 24 h at 15°C. Also, Niwas et al. [Streptomyces sp. at shake flask level. The results indicated that the maximum cholesterol oxidase production (6.2 U/mL) was achieved using 0.05%, w/v cholesterol, pH 7 and 35°C, 200 rpm.Varma and Nene  studied n et al.  used orta et al.  standardi et al.  achievedperature °C, incubStreptomyces anulatus strain NEAE-94 could be used as a promising, efficient source for cholesterol oxidase production. The maximum cholesterol oxidase production after statistical optimization of fermentation process variables was 27.31 U/mL with a fold of increase 6.06 compared with the cholesterol oxidase production before applying Plackett-Burman design (4.51 U/mL).Streptomyces sp. strain NEAE-94 was isolated from a soil sample collected from Baltim, Kafr el-Sheikh Governorate, in the north of Egypt. Streptomyces sp. strain NEAE-94 isolation has been made on the plates of starch-nitrate agar medium of the following constituents (g/L): FeSO4.7H2O 0.01; CaCO3 3; NaCl 0.5; MgSO4.7H2O 0.5; K2HPO4 1; KNO3 2; starch 20 and agar 20 in 1 L of the distilled water. The plates were incubated for 7 days at 30°C. For long time preservation, the isolate was stored as spore suspensions in 20% (v/v) glycerol. Streptomyces anulatus NEAE-94 is an antagonistic actinomycete exhibited a broad antimicrobial spectrum against several bacterial strains including multidrug-resistant Staphylococcus aureus, E. coli, Bacillus subtilis and Pseudomonas aeruginosa [ruginosa .Streptomyces sp. strain NEAE-94 for the production of cholesterol oxidase was investigated on agar plate medium containing cholesterol as the sole source of carbon according to the method of El-Naggar et al. [The efficiency of r et al. . The medr et al. .4)2SO4 7.5; cholesterol 2; K2HPO4 1; MgSO4.7H2O 0.5; FeSO4.7H2O 0.02; ZnSO4 0.002; CaSO4 0.002; MnSO4 0.008; NaCl 1; CaCl2 0.0002; peptone 4; yeast extract 6; starch 9; glucose 12; Tween 80 0.05) [Streptomyces sp. strain NEAE-94 grown on starch nitrate agar medium. The inoculated flasks were incubated at 30°C for 48 h in rotating shaker incubator at 200 rpm, and used as inoculum for subsequent experiments.Two hundred fifty millilitre Erlenmeyer flasks containing 50 mL of broth medium, comprised of g/L: (NH80 0.05)  were inog for 15 min.Fifty millilitre of the fermentation medium was inoculated with the prepared inoculum and incubated in rotating shaker incubator at 30–37°C and 150–200 rpm. After the incubation time, the cell free- culture supernatant containing the crude enzyme was obtained by centrifugation of the mycelium culture using refrigerated centrifuge at 4°C and 6000×2O2 /min at 37°C.The cholesterol oxidase activity was determined spectrophotometry by the method of El-Naggar et al. . One enzSpore mass color, substrate mycelium color (reverse side of the colony) and soluble diffusible pigments were investigated on ISP media as described by the method of Shirling and Gottlieb  method. Streptomyces sp. strain NEAE-94 was determined on slants of ISP media 1, 6 and 7; the production of brown or black soluble pigments was recorded after 7 days of incubation at 30°C that indicates positive production of melanin [Streptomyces sp. strain NEAE-94 was investigated on agar plate medium according to the method of El-Naggar et al. [The ability of the strain to utilize carbon sources was determined according to the method described by Shirling and Gottlieb  using 13 melanin . Degrada melanin , L-aspar melanin , sodium  melanin , chitosa melanin  and uric melanin  were tesr et al. .Streptomyces sp. under investigation was carried out via polymerase chain reaction (PCR) using universal primer designed to amplify the full length of the gene. The reverse primer was 1492r (5′-TACGGYTACCTTGTTACGACTT-3′) and the forward primer was 27f (5′-AGAGTTTGATCMTGCCTCAG-3′). The PCR reaction was conducted in a total volume of 100 μL that contains 0.5 μL Taq polymerase, 10 μL deoxyribonucleotide 5′-triphosphate (dNTP’s) (250 mM), 4 μL of both forward and reverse primers of 10 pmol, 1 μL template DNA 50 ng, 3.5 μL MgCl2 (25 Mm), 10 μL PCR buffer, up to 100 μL with water.According to the method described by Sambrook et al. , the totStreptomyces sp. strain NEAE-94 was aligned with the 16S rRNA sequences of the related species of Streptomyces by using BLAST program [The amplified DNA was sequenced and the resulting sequence of 16S rRNA was deposited under accession number KC354803 in the GenBank NCBI database. 16S rRNA gene sequence (1536 bp) of  program . The phy program  via the 4)2SO4 (4 and 8 g/L), FeSO4.7H2O (0.01 and 0.05 g/L), MgSO4.7H2O (0.1 and 0.5 g/L), NaCl (0.5 and 1 g/L), K2HPO4 (0.5 and 1 g/L), pH (7 and 9), inoculum size , incubation time (5 and 7 days), temperature (30 and 37°C) and agitation speed (100 and 150 rpm). As well, 4 dummy variables (D1-D4) were included.The Plackett-Burman statistical design  is an efPlackett–Burman design is based on the first order model:0 is the model intercept, Xi is the level of each independent variable and βi is the linear coefficient.Where, Y is cholesterol oxidase production, βP-values, F-values, effects and contribution % were selected for further optimization using Box–Behnken design [1, 100–200 rpm), cholesterol concentration  and yeast extract concentration . Each factor varies on three different levels , with three center points resulting in a total of 15 different experiments. BBD was used to estimate the optimal levels of these variables and to study the individual and interaction effects between the selected process variables affecting cholesterol oxidase production. Linear, quadratic and interaction effects of the three significant independent variables on cholesterol oxidase production were calculated to correlate the relationship between production of cholesterol oxidase (Y) and these factors and to predict their optimal levels using the following second order polynomial equation:Based on the results of Plackett-Burman experiments, three factors with the highest n design . These fi is the coded levels of independent variables, β0 is the regression coefficients, βij is the interaction coefficients, βi is the linear coefficient and βii is the quadratic coefficients”.In which “Y is the cholesterol oxidase production, X“The experimental designs and statistical analysis were performed using Design Expert software version 7 for Windows. The 3D surface plots were drawn using STATISTICA software version 8.”Additional file 1.


N-acetylaspartate network begins in neurons with N-acetylaspartate production catalyzed by aspartate N-acetyltransferase from acetyl-CoA and aspartate. Clinical studies reported a significant depletion in N-acetylaspartate brain level in type 1 diabetic patients. The main goal of this study was to establish the impact of either hyperglycemia or oxidative stress on the N-acetylaspartate network. For the in vitro part of the study, embryonic rat primary neurons were treated by using a nitric oxide generator for 24 h followed by 6 days of post-treatment culture, while the neural stem cells were cultured in media with 25–75 mM glucose. For the in vivo part, male adult Wistar rats were injected with streptozotocin  to induce hyperglycemia (diabetes model) and euthanized 2 or 8 weeks later. Finally, the biochemical profile, NAT8L protein/Nat8l mRNA levels and enzymatic activity were analyzed. Ongoing oxidative stress processes significantly affected energy metabolism and cholinergic neurotransmission. However, the applied factors did not affect the N-acetylaspartate network. This study shows that reduced N-acetylaspartate level in type 1 diabetes is not related to oxidative stress and that does not trigger N-acetylaspartate network fragility. To reveal why N-acetylaspartate is reduced in this pathology, other processes should be considered.The  N-acetylaspartate (NAA) is a brain amino acid mainly produced by neurons from acetyl-CoA and aspartate in the presence of aspartate N-acetyltransferase  ..N-acetylp < 0.05)   . Further < 0.05) a. This i < 0.05) . Meanwhictively) a. With tctively) a.2+ concentrations resulted in a severe reduction of the N-acetylaspartate (NAA) level in SN56 neuroblastoma cells (the cellular model of cholinergic neurons) . T. Tp < 0.ogenase) a,b. Receogenase) a. Howeveogenase) . In orde< 0.001)  22,23].,23.p < 0N-acetylaspartate network (NAA network) are closely connected with each other, we consider hyperglycemia as one of the potential risk factors for NAA network stability   a,b. The  < 0.01) b.Astrocytes are known to be cells, which prefer the LDH/lactate-dependent energy production pathway instead of the tricarboxylic acid cycle ,37. AddiHyperglycemia triggered neurons to overproduce pyruvate, which is catalyzed by pyruvate dehydrogenase complex, which in theory should accelerate tricarboxylic acid turnover. However, an increased NADH/NAD ratio is an activation factor for pyruvate dehydrogenase kinase, which by the phosphorylation of PDHC prevents its over-activation f,g 43]..43]. As N-acetylaspartate production is closely related to cholinergic neurotransmission . Fo. Fo63]. Embryonal brain cortices (E18) obtained from pregnant Wistar rats were dissected in Hank’s buffered salt solution (HBSS) supplemented by 8 mM HEPES (pH = 7.4), 100 U/mL penicillin, and streptomycin. Using different culture strategies, the cells were settled for either primary neuronal culture (PR) or neural stem cells culture (NSC) ,64.Isolated cells were seeded as a monolayer in poly-l-ornithine and laminin-coated dishes. In order to obtain mature neurons, the cells were cultured for 14 days in a neurobasal medium supplemented with B27 supplement, 2 mM Glutamax, 10 ng/mL nerve growth factor (NGF), 20 nM AraC, and 100 U/mL penicillin with streptomycin. Every third day of the culture, the media were changed in half of their volume. For further experiments, culture dishes were assigned randomly to different treatment experimental time points. Mature primary neurons were cultured in the same medium, although in order to study the impact of oxidative stress on the cells, the B27 supplement was changed to B27 minus antioxidant supplement. For 24 h after maturation, the primary neurons were cultured in the presence of 0–0.8 mM S-nitroso-N-acetylpenicillamine (SNAP). On the following day, the media were changed to fresh ones without SNAP and the cells were maintained in culture for an additional three to six days in vitro (DIV) .Dissected cortices were cultured as neurospheres in DMEM/F12–GlutaMAX media supplemented with B27 supplement, 10 ng/mL basic fibroblast growth factor, 20 ng/mL epidermal growth factor, and 100 U/mL penicillin with streptomycin. Neurospheres were passaged every third day of culture. Cells between 1 and 3 passages were considered as suitable for this study. Neural stem cell types were recognized by β-III-tubulin (neurons), glial fibrillary acidic protein (GFAP) (astrocytes), and CNPase  specific markers . For theg , each sample was immediately used for studies or kept at −80 °C until analyzed.Brain tissues (without cerebellum) or cells were homogenized in chilled: 4% HClO4 (for metabolic studies); 0.1 M HCl (for NAD assay); 0.2 M KOH (for NADH assay); or 0.32 M sucrose buffered by 5 mM HEPES (pH = 7.4) with 0.1 mM EDTA . After centrifugation at 13,000× Images were captured under 40× magnification using an inverted light microscope  .To analyze enzymatic activity in the cell lines, from each dish, two independent cell lysates were collected and reported as one average result. To analyze enzymatic activity in brain tissue, 3 tissue lysates per one brain were collected, and then, the enzymatic profile of each lysate was measured in two independent samples. Eventually, 3 average results per 1 brain were reported in this study. Protocols for enzymatic assays were described with details in our previous study .N-acetyltransferase  [Aconitase  , asparta.3.1.17) , citrate.3.1.17) , choline.3.1.17) , isocitr.3.1.17) , pyruvat.3.1.17) .2, 0.25 mM NADH, 1 U glucose-6-phosphate dehydrogenase , and 20 μg of cell homogenate protein. Enzymatic assay was initiated by the addition of 10 μL of 0.1 M ATP [Hexokinase  activity was determined using the NADPH/NADP conversion technique at λ = 340 nm and 37 °C. Then, 1 mL of reaction buffer contained 0.1 M Tris-HCl (pH = 7.4), 5 mM glucose, 0.2 mM MgCl.1 M ATP .Lactate dehydrogenase or LDH in media assay  activity was determined using the NADH/NAD conversion technique, at λ = 340 nm and 37 °C. Then, 1 mL of reaction buffer contained 0.1 M Tris-HCl (pH = 7.4), 0.2 mM NADH, and 20 μg of cell homogenate protein . Enzymatic assay was initiated by the addition of 10 μL of 0.1 M pyruvate .2, 10 mM dithiothreitol, 10 mM pyruvate, 2 mM thiamine pyrophosphate, 0.2 mM CoA, 2.5 mM oxaloacetate, 2 mM NAD, and 0.15 U citrate synthase (EC 4.1.3.7). Reaction was terminated by thermic shock . Finally, the produced citrate level was determined using NADH/NAD conversion technique at λ = 340 nm and 37 °C. The reaction buffer contained 0.1 M Tris-HCl (pH = 7.4), 0.1 mM NADH, 0.2 U malate dehydrogenase (EC 1.1.1.37), and 100 μL of achieved supernatant in a final volume of 0.7 mL. The assay was initiated by the addition of 10 μL of 0.1 U citrate lyase (EC 4.1.3.6). The blank sample was treated in the same way as the measured sample, but the blank sample was not reached in CoA [Pyruvate dehydrogenase complex  activity was determined using cycling method. In each lysate (100 μg of cell homogenate protein), the following reactions were performed: (1) citrate production and (2) citrate level measurement. The first reaction (1) was carried out in 250 μL for 30 min , the buffer contained 0.1 M Tris-HCl (pH = 8.3), 2 mM MgCld in CoA .g . Next, ice-cold samples were neutralized and immediately assayed.To analyze metabolic profiles, we collected two independent acidic supernatants per each cell culture dish or three independent acidic supernatants per one rat brain. After 15 min of deproteinization (at 4 °C), samples were centrifuged at 13,000× N-acetylaspartate, oxaloacetate, pyruvate, and TBARS assays were described with details in our previous study [Protocols for acetoacetate, acetyl-CoA, aspartate, β-hydroxybutyrate, lactate, us study .Protocols for ATP and ADP, AMP, adenosine , citrate6 cells or 0.1 g of brain tissues were vortexed or homogenized in a sterile tube with 0.5 mL (cells) or 1 mL (brain tissue) of RNA Extracol extraction buffer . The extraction was initiated by the addition of 250 μL chloroform (per 1 mL of RNA Extracol buffer). After vigorous shaking, each sample was incubated at 4 °C for 15 min and spun down . The upper aqueous phase was transferred to a new tube and refilled by isopropanol in a 1:2 ratio . The RNA precipitation was carried out overnight at −20 °C, and on the following day, each sample was centrifuged . The RNA pellet was washed firstly with 99.8% and then with 75% ethanol, finally air-dried, and reconstituted in nuclease-free water (15–20 μL) . The obtained samples were kept at −20 °C until analyzed. The quantity of isolated RNA was determined using the Qubit RNA HA assay kit according to the manufacturer’s instructions . The gene expression levels of Nat8l encoding the NAT8L enzyme and Chat encoding the ChAT enzyme were determined by real-time RT-qPCR performed in a Light Cycler 480 II  using a Path-IDTM Multiplex One-Step RT-PCR Kit  and Universal ProbeLibrary for the rat species, and gene-specific intron-spanning primers (Actb). Reverse transcription program: 48 °C–10 min and 95 °C–10 min. Amplification program: 95 °C–15 s, 60 °C–45 s for 45 cycles. Data were processed with Light Cycler 480 II software 2.0 [The 2 × 10 primers . The rea primers . The tarv/v) with two different hosts  . Images were captured under 60x magnification using an inverted fluorescence microscope  [For immunocytochemistry, cells were seeded on cover slips and cultured as usual. Next, the cells were fixed with 4% paraformaldehyde in sterile PBS  followed by 3 washes with PBS. The fixed cells were left in PBS at 4 °C until needed. Before immunostaining, the cells were permeabilized for 30 min in PBS buffer with 0.1% Triton X-100 and 5% bovine serum albumin. The overnight incubation of cover slips was carried out in 0.1% Triton/0.5% bovine serum albumin/PBS and two primary antibodies diluted to 1:200  . FinallyOlympus) .2PO4) at 4 °C. The obtained lysates were kept at −20 °C until analysis. Each sample (40 µg of protein/20 µL of 10 mM dithiothreitol/Laemmli buffer) was incubated for 1 h at 57 °C followed by the addition of 2 µL 0.5 M 2-chloroacetamide and incubation for an additional 1 h at room temperature. Next, the spun down samples  were loaded to ExpressPlusTM PAGE Gel 4–20% gradient BisTris-PAGE gels . Then, the BisTris-PAGE gel was run at 300 V for 17 min in MOPS/SDS running buffer  in a MINI-PROTEAN electrophoresis system with cooling . Next, proteins were transferred from the Bis Tris-PAGE gel to a PVDF membrane  using the iBlot® Dry Blotting System with P0 program  . The PVDF membrane was washed 2 × 10 min in TBTS buffer . Non-specific bindings were blocked with 5% BSA in TBST . Next, the PVDF membrane was incubated with specific primary antibodies  in 5% BSA/TBST buffer  (v/v) with 5% BSA/TBST buffer   [The brain tissue samples were lysed for 30 min in lysis buffer  . On the erature) . The PVDmL BCIP) .Protein was assayed as previously .p < 0.05 were considered statistically significant. We performed all statistical analyses using the Graph Pad Prism 4.0 statistical package .The results are presented as a means ± standard deviation of the mean (SD). The Kolmogorov–Smirnov normality test excluded normal data distribution. Therefore, the results were tested by either the Mann–Whitney test or Kruskal–Wallis followed by Dunn’s Multiple Comparison post-test, where values of N-acetylaspartate network is created by a reaction between acetyl-CoA and aspartate metabolites introduced by aspartate N-acetylaspartate (NAT8L), while cholinergic neurotransmission starts from the reaction between acetyl-CoA and choline catalyzed by choline acetyltransferase. Our data clearly show that type 1 diabetes-like conditions triggers disturbances in energy metabolism. Under these conditions, brain cells kept the N-acetylaspartate level and NAT8L activity stable, but cholinergic markers were downregulated. In our previous study, we showed that the progression of Alzheimer disease-associated cholinergic neurodegeneration accompanied the NAT8L inhibition. The same pattern was identified in type 1 diabetes patients. Thus, we conclude that these diseases share pathomechanisms that trigger N-acetylaspartate depletion by the inhibition of NAT8L gene expression or enzyme activity rather than by substrate shortages.The


Trim28, an essential epigenetic regulator, suffer gradual testicular degeneration. Contrary to previous reports we detect Trim28 expression in spermatogonia, albeit at low levels. Further reduction through Trim28 heterozygosity increases the propensity of SSCs to differentiate at the cost of self-renewal.Spermatogenesis relies on exquisite stem cell homeostasis, the carefully balanced self-renewal and differentiation of spermatogonial stem cells (SSCs). Disturbing this equilibrium will likely manifest through sub- or infertility, a global health issue with often idiopathic presentation. In this respect, disease phenotypes caused by haploinsufficiency of otherwise vital developmental genes are of particular interest. Here, we show that mice heterozygous for   •TRIM28/KAP1 haploinsufficiency causes testicular degeneration and infertility•TRIM28/KAP1 is expressed in spermatogonia stem cell compartment•Stem cell homeostasis in the testis is dependent on proper TRIM28/KAP1 levels In this article, Tan et al. show haploinsufficiency of TRIM28/KAP1 causes premature degeneration of the seminiferous epithelium in young adult mice. The requirement of proper TRIM28 levels is cell-autonomous and specific to spermatogonial stem cells, previously shown to be devoid of TRIM28 expression. They instead reveal Trim28’s vital role in spermatogonia to maintain proper stem cell homeostasis. Trim28 (Trim28Het) male mice.Haploinsufficiency—a mechanism by which a loss-of-function mutation leaves only one functional copy of a gene and so leads to insufficient gene product —is a majIn the seminiferous tubules of the testis, millions of spermatozoa are produced on a daily basis through a process termed spermatogenesis . During Trim28 haploinsufficiency-induced infertility phenotype using conditional, constitutive, and inducible genetic approaches. We redefined the expression domain of TRIM28 in spermatogenesis and revealed a new, essential role of TRIM28 in the maintenance of the SSC compartment.In this study, we uncovered and characterized a Trim28 (Trim28Het) males in our colony  with their histology revealing numerous Sertoli cell-only tubules  mice. In these animals, one Trim28 allele is excised only in germ cells shortly before birth using the Mvh-cre deleter strain , induced through Amh-cre deletion  resulting in less pre-spermatogonia just before birth (E18.5)  E and 1F.Trim28-heterozygous mouse model (Trim28iHet) based on the Mvh-creERT2  4 weeks later , SYCP3 , and SOX9 ––at various ages. This analysis revealed four types of tubules commonly observable in both heterozygous mutant models (Trim28Het and Trim28cHet): (1) normal tubules that contain SALL4/SYCP3-positive cells (SG+/SC+), (2) spermatogonia-deficient tubules containing SYCP3-positive but not SALL4-positive cells (SG−/SC+), (3) spermatogonia- and spermatocyte-deficient tubules without SALL4/SYCP3-positive cells (SG−/SC−), and (4) Sertoli cell-only tubules containing only SOX9-positive cells (SE-only) (−/SC− or SE-only tubules and rarely observed SG−/SC+ tubules (4.25% ± 0.79%) , the proportion of SG−/SC+ tubules increased dramatically (31.08% ± 6.67%) at 8 weeks (with 64.26% ± 7.55% remaining SG+/SC+). This phenotype was exacerbated with the appearance of SG−/SC− (22.26% ± 7.21%) and SE-only (17.90% ± 1.03%) tubules, further reducing SG+/SC+ (40.78% ± 9.75%) tubules at 16 weeks   (We established that SE-only) A. Note t± 0.79%) . Convers16 weeks . Trim28H± 7.72%) . This is± 7.72%) E. We als6 weeks) B and S3.Trim28iHet model when analyzed 4 weeks after tamoxifen injection   defect B. ImportSG+/SC+) C and S3.Mvh-creERT2-inducible, germ cell-specific Trim28-knockout mouse model (Trim28iKO). Although in adult control (Trim28Ctr) animals SALL4-positive spermatogonia continued faint TRIM28 staining upon induction, adult Trim28iKO SALL4-positive spermatogonia lost all TRIM28 staining post-recombination (Trim28 was detectable in SSCs (ID4-eGFP bright cells) as well as progenitor undifferentiated spermatogonia (ID4-eGFP dim cells) at the transcript level  was correlated to the proportion of SALL4-positive tubules (32.39% ± 10.66%), indicating that the replenished germ cells were derived from SSCs as expected , subsequent rounds of spermatogenesis initiate from the SSC pool, which is sustained by self-renewal division . To deterecovery A–4C. Theexpected C and 4D.Trim28-heterozygous SSCs. According to the recent “Revised Asingle model” for SSC self-renewal, Asingle spermatogonia that express high levels of ID4 are the SSCs  directly correlates with their propensity toward a more differentiated state  or an Apaired cell pair (differentiation) (Trim28WT (73.27% ± 1.77%) with Trim28cHet (56.55% ± 4.48%) and Trim28Het (59.84% ± 3.88%) “Asingle” events, TRIM28-deficient SSCs showed a propensity to form Apaired spermatogonia and hence rather differentiate than self-renew (Trim28cHet compared with Trim28WT testes at P7 (91.91% ± 1.01% vs 95.49% ± 1.02%) and 2 weeks (52.82% ± 3.41% vs 62.73% ± 4.89%)  E. Comparlf-renew F, thereb± 4.89%) G. Hence,Trim28iKO model, we generated a germ cell-specific  Trim28 knockout mouse (Trim28cKO) using the Mvh-Cre deleter strain to study the progression of TRIM28-null germ cells into adulthood.Having characterized the phenotype in the heterozygotes, we proceeded to address the obvious question: What ensues from the complete knockout of TRIM28 in germ cells? Apart from our Trim28cKO testes had started losing half their SG+/SC+ tubules at 4 weeks (48.23% ± 21.84%) and almost all of the rest at 8 weeks (3.17% ± 2.44% SG+/SC+) (Trim28 in adult germ cells furthered progression (compared with Trim28iHet) of testis size reduction (0.16% ± 0.03% vs 0.23% ± 0.02% BW) and spermatogonia loss (31.81% ± 13.60% SG−/SC+ vs 17.61% ± 3.45% SG−/SC+)  resulted in accelerated degeneration in terms of testis size A as wellSG+/SC+) B. This dSG−/SC+) C and 5D.SG−/SC+) E, which Stra8-Cre deleter strain (Trim28Stra8KO)  and 8 weeks (89.98% ± 6.29% SG+/SC+) (Trim28Stra8KO testes still harbored various TRIM28-negative germ cell layers (including SALL4-positive spermatogonia) at 62 weeks (more than thrice the time Trim28Het and Trim28cHet took to become infertile) (Stra8-cre in late undifferentiated spermatogonia (in response to retinoic acid) just before transiting into differentiated spermatogonia   did not Stra8KO) A. This wSG+/SC+) B and S3.fertile) C. Althouatogonia . This woTrim28cHet mice, and propensity of TRIM28-deficient SSC to differentiate, clearly point to a previously unknown role of TRIM28 in SSC.Ablation of key developmental genes has been shown to be incompatible with life, but the haploinsufficient effects of such genes are relatively poorly understood. Our study on TRIM28 reveals that heterozygous mice are viable but exhibit a premature infertility phenotype in male animals. Characterization of this haploinsufficiency phenotype using different mouse models specifies the spatial  importance of TRIM28 function within spermatogonia and provides a new insight into the role of this developmentally essential regulator in spermatogenesis, namely the cell-autonomous homeostasis of the SSC compartment. This expands on a previous study that suggests, on the basis of restricted detection of TRIM28 in mid-pachytene spermatocytes to early elongating spermatids, a TRIM28 requirement for spermatogonia maintenance through a paracrine feedback mechanism from Sertoli cells, or meiotic or post-meiotic germ cells . ConversTrim28 deletion using the Stra8-Cre deleter strain yielded an initially unexpected milder testicular degeneration phenotype, which could be explained by the activation of Stra8-cre in response to retinoic acid in late undifferentiated spermatogonia   , possiblTrim28 floxed (Trim28f/f) mouse was reported previously . Derivation of eviously . Mvh-Cre or modified Davidson's fixative (mDF)  and embedded in paraffin. Testes of embryonic day 18.5 (E18.5) embryos were fixed in mDF for 5 h. Quantification of Sertoli cell-only tubules on hematoxylin and eosin (H&E)-stained slides involved three sections (>200 μm apart) from each testis. For immunofluorescence staining, heat-induced epitope retrieval was done in 10 mM sodium citrate buffer . Sections were blocked  for 1 h at room temperature (RT), incubated with primary antibodies at 37°C for 1 h, before secondary antibody incubation for 1 h at RT. Coverslip was mounted with Hydromount  containing 1 μg/mL DAPI (Sigma). Counting of tubules (and cells) was done manually and all tubules within each testis section were counted. See Spermatogonia isolation was performed on neonate testes (P6–P8) as described previously . Magnetiβ-actin and their relative expression analyzed using the 2−ΔΔct method. See RNA from spermatogonia was isolated using the Arcturus PicoPure RNA Isolation Kit (Thermo Fisher Scientific) following the manufacturer's instructions. Total RNA was converted using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). qPCR assays were designed with the Universal Probe Library Assay Design Center (Roche), and performed  on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Target genes were normalized to Busulfan (Sigma) was prepared by dissolving in DMSO (Sigma) and diluting with an equal volume of water. A single dose (30 mg/kg) was administered via i.p. injection. Percentage of regenerated seminiferous tubules  was quantified on H&E-stained testis sections.single cells were cells that stood alone or if cytoplasmic connections were present, cells that were more than one nuclear length apart. An Asingle event was counted when an Asingle cell was spotted or when two Asingle cells were found in close proximity (within the counting frame). Apaired cells were defined as two cells that were connected and within one nuclear length from each other. See P5 neonates were given 10 μL of Amersham Cell Proliferation Labeling Reagent  per gram of body weight via i.p. injection and testes were isolated 5 days later. The tunica albuginea of the testis was removed, and seminiferous tubules were mechanically separated and fixed in 4% paraformaldehyde (Sigma) for 5 h at 4°C. Fixed tubules were denatured with 3.5 M HCl for 2 min and blocked  for 4 h at RT. Tubules were incubated with primary antibodies overnight at 4°C followed by secondary antibodies for 1.5 h at RT. Coverslip was mounted using DAPI-containing Hydromount. At least 1 cm of tubule was analyzed. AData are represented as mean ± SD and were analyzed using Student's t test . N.S. denotes not significant (p > 0.05). At least three biological replicates were used for each experiment.J.H.L.T. conceived and performed the experiments, analyzed the data, and wrote the manuscript. H.W. performed the experiments and analyzed the data. A.M.M.v.P. contributed reagents, supervised analysis, and contributed to the manuscript. P.K. conceived and supervised the experiments. D.M.M. conceived and supervised the study, conceived and performed the experiments, analyzed the data, and wrote the manuscript.


Plectranthus spp. The cytotoxic diterpene 6,7-dehydroroyleanone (1) is the main component of the P. madagascariensis (Pers.) Benth. essential oil, while 7α-acetoxy-6β-hydroxyroyleanone (2) can be isolated from acetonic extracts of P. grandidentatus Gürke. The reactivity of the natural royleanones 1 and 2 was explored to obtain a small library of new P-gp inhibitors. Four new derivatives , 6,7-dehydro-12-O-methylroyleanone (21), 6,7-dehydro-12-O-benzoylroyleanone (22), and 7α-acetoxy-6β-hydroxy-12-O-benzoylroyleanone (23) were obtained as pure with overall modest to excellent yields (21–97%). P-gp inhibition potential of the derivatives 20–23 was evaluated in human non-small cell lung carcinoma NCI-H460 and its MDR counterpart NCI-H460/R with the P-gp overexpression, through MTT assay. Previously prepared diterpene 7α-acetoxy-6β-benzoyloxy-12-O-(4-chloro)benzoylroyleanone (4), has also been tested. The P-gp inhibiting effects of compounds 1–4 were also assessed through a Rhodamine 123 accumulation assay. Derivatives 4 and 23 have significant P-gp inhibitory potential. Regarding stability and P-gp inhibition potential, results suggest that the formation of benzoyl esters is a more convenient approach for future derivatives with enhanced effect on the cell viability decrease. Compound 4 presented higher anti-P-gp potential than the natural diterpenes 1, 2, and 3, with comparable inhibitory potential to Dexverapamil. Moreover, derivative 4 showed the ability to sensitize the resistant NCI-H460/R cells to doxorubicin.Cancer is among the leading causes of death worldwide. One of the most challenging obstacles in cancer treatment is multidrug resistance (MDR). Overexpression of P-glycoprotein (P-gp) is associated with MDR. The growing incidence of cancer and the development of MDR drive the search for novel and more effective anticancer drugs to overcome the MDR problem. Royleanones are natural bioactive compounds frequently found in  Although P-gp inhibitors showed high efficacy in vitro and in vivo studies, very few have successfully passed all phases of the clinical trials and none of them have been approved by the U.S. Food and Drug Administration (FDA) for clinical use in cancer treatment (Cancer is among the leading causes of death worldwide with an estimated 18.1 million new cancer cases and 9.6 million cancer deaths in 2018 . One of reatment . After treatment . TherefoPlectranthus (Lamiaceae) is used in traditional medicine in southern Africa and it is known as a source of bioactive natural products (1) (P. madagascariensis (Pers.) Benth essential oil (2)  , which hucts (1) , is the tial oil . Other e oil (2) , that caal agent  with a sal agent . Althougal agent , the two3) and 7α-acetoxy-6β-benzoyloxy-12-O-(4-chloro)benzoylroyleanone (4)  (2. The derivate 5 has shown selective modulation on Protein kinase delta isoform (PKC-δ). A key study reports that 5 strongly inhibited the proliferation of colon cancer cells by inducing a PKC-δ-dependent mitochondrial apoptotic pathway involving caspase-3 activation   were suce (4) (2 . Compounmpound 2 . Furthernone (5)  was alsotivation . Besides 2 and 5 . Herein 1 and 2  group. Royleanone 2 was also subjected to methylation reaction and introduction of Boc (tert-butyloxycarbonyl) group. The predicted structures and the isolated derivatives (6–23) are shown in 3.In this work, the reactivity of two royleanones was explored to prepare a small library of compounds of enhanced effect on the cell viability decrease potential and anti-P-gp activity. Several hemisynthetic reactions were performed on natural compounds 1 and 2 . Compoun6 to 19 (Scheme 1 and 2) tend to degrade after isolation. On the other hand, the introduction of Boc group (20), methylation (21), and benzoylation (22 and 23) reactions .Unfortunately, the obtained products have encountered stability issues: the derivatives eactions  were acc6 to 11, 6 to 11, they invariably decomposed during such steps. This degradation was also observed in the carbamoylation reactions , and thiocarbamoylation (19). Despite the presence of allyl (12) and m-tolyl (16) carbamates and silyl ether (18) in the TLC analysis of the reaction mixtures, the compounds decomposed before purification and no characterization could be done.The Mitsunobu products 6 to 11,  have disO-substituents, and we used the derivative 6,7-dehydro-12-(prop-2′-yn-1′-yloxy)-royleanone , using its characteristic UV spectrum as a fingerprint. This fact may indicate that compound 6 tends to decompose to a much more stable scaffold–the starting material (1).In the analytical HPLC analysis of compound 6 was subjected to LC-MS analysis. The ESI positive mass spectra indicated a mass of m/z 355 [M + Na]+. Although it was not possible to identify the moiety or cleavage pattern responsible for the result, some decomposition mechanisms based on the presence of the mentioned fragment were considered  of 42.0 kcal/mol and, upon the establishment of hydrogen bonds network with water molecules. Notwithstanding the high energy barrier, the process is energetically favorable with the intermediate 6B being 9.3 kcal/mol more stable than 6A. The 1,2-hydride migration in 6B delivers the aromatized molecule identified in HRMS.The decomposition of derivative l Theory  to elabol Theory . Differe6A in a conjugate addition fashion, was considered but determined highly unlikely due to an energy barrier of 55.6 kcal/mol. However, this energy barrier lowers significantly when in the presence of acid catalysis  and sensitivity index (SI)   in these 6 and 7 positions. This suggests that the substituents in position 6 (-OH) and 7 (-OAc) can also contribute to P-gp interaction. Further studies should be conducted to assess this hypothesis.The P-gp inhibition potential of all stable derivatives obtained, derivatives prepared , was alsdex (SI) . Based oI 26.90) . A recenard P-gp . On the 4 and 23 showed to be promising candidates for P-gp inhibition, nonetheless, due to the small amount of compound 23 available, we choose the royleanone 4 for further studies. Accordingly, the effect of compound 4 was investigated in NCI-H460, NCI-H460/R cell lines, and normal embryonal bronchial epithelial cells MRC-5. Royleanone 4 showed high toxicity in all cell lines tested, with IC50 of 1.9 ± 0.4 µM for NCI-H460, 2.2 ± 0.4 µM for NCI-H460/R, and 2.0 ± 0.3 µM for MRC-5 cell lines. In previous studies, 1, 2, and 3 in the same cell lines. Royleanone 2 is more efficient than 1 and 3, with IC50 of 2.7 µM for NCI-H460, 3.1 µM for NCI-H460/R, and 8.6 µM for MRC-5 cell lines  accumulation assay  can reverse doxorubicin resistance. Therefore, derivative 4 can be considered as a new P-gp inhibitor useful in combination with classic chemotherapeutics.The P-gp inhibiting effects of compounds on assay . The obterapamil . Dexveraerapamil . In our erapamil . It is werapamil . On the erapamil . Accordiorubicin . Results20 to 23 as pure with overall good yields.In this work, the reactivity of two natural royleanones was explored to obtain a small library of new P-gp modulators. Several hemisynthetic reactions were performed and successful results were obtained when performing methylation and benzoylation, and introduction of Boc group, affording compounds 20–23) was evaluated. Previously prepared diterpene 4, has also been tested. From the tested derivatives, compounds 4 and 23 have significant P-gp inhibitory potential.P-gp inhibition potential of the stable derivatives  Benth. and P. grandidentatus Gürke were cultivated in Parque Botânico da Tapada da Ajuda  from cuttings obtained from the Kirstenbosch National Botanical Garden . Voucher specimens were deposited in Herbarium João de Carvalho e Vasconcellos (ISA). The plant name has been checked with http://www.theplantlist.org  or 2 , corresponding alcohol (5 eq.), triphenylphosphine (5 eq.) and DIAD (5 eq.) in 4.5 ml dry THF, were irradiated with microwaves at 300 W and 60 °C for 45 min under argon atmosphere. Conditions for Carbamoylation: A mixture of 1 , DMAP (5 eq.) and excess of the corresponding isocyanate, in 0.5 ml dry CH2Cl2, were stirred at room temperature under inert conditions until consumption of the starting material as judged from TLC. Tosylation Conditions: A mixture of 1 , triethylamine (4.5 eq.), DMAP (0.3 eq.) and p-toluenosulfonyl chloride (3 eq.), in 0.5 ml dry CH2Cl2 were stirred until consumption of the starting material as judged from TLC. Introduction of the TBDPS group: A mixture of 1 , imidazole (2 eq.) and excess of tert-butyldiphenylchlorosilane in 1 ml dry CH2Cl2 was stirred at room temperature Thiocarbamoyl: 1  was added to a suspension of sodium hydride (1 eq.) in 0.4 ml THF, followed by sodium iodide (0.5 eq.) and dimethylthiocarbamoyl chloride (1.2 eq.). The mixture was left stirring at room temperature until consumption of the starting material. Introduction of Boc group: A mixture of 1 , DMAP (0.5 eq.) and Boc2O (2.2 eq.) in 0.5 ml of dry CH2Cl2 was left stirring at room temperature. Methylation: A mixture of 1  (1 eq.), methyl iodide (8.4 eq.) and silver oxide (8.4 eq.), in 0.5 ml of dry CH2Cl2 was left stirring at room temperature. Benzoylation: A mixture of 1  or 2 , pyridine (12 eq.) and benzoyl chloride (12 eq.), in 2 ml dry CH2Cl2 was left stirring at room temperature until complete consumption of the starting material as judged by TLC.The Mitsunobu reactions were carried out with microwave irradiation, according to a previous report : 1 , equipped with a Zorbax® XDB-C18  column, from Merck and ChemStation Software. The sample was injected in acetone, 10 mg/ml. Each injection was analyzed with a gradient elution mixture composed of solution A (methanol), solution B (acetonitrile), and solution C (0.3% trichloroacetic acid in water) was used as follows: 0 min, 15% A, 5% B, and 80% C; 20 min, 70% A, 30% B and 0% C; 25 min, 70% A, 30% B and 0% C; and 28 min, 15% A, 5% B and 80% C. The flow rate was set at 1 ml/min and 20 μL of the sample were injected.LC-MS/MS analysis was performed using a Zorbax Eclipse XBD-C18, 4.6 × 250 mm (5 μm) and the mobile phase consisted of 0.5% formic acid in Milli-Q water (eluent A) and acetonitrile + 0.5% formic acid (eluent B). A flow rate of 0.3 ml/min was used, with the following gradient program: 0–30 min from 70 to 5% A, 30–45 min at 5% A, 45–65 min 70% A.2 for NCI-H460 and 16,000 cells/cm2 for NCI-H460/R.Non-small cell lung carcinoma cell line NCI-H460 was purchased from the American Type Culture Collection, Rockville, MD. NCI-H460/R cells were selected originally from NCI-H460 cells and cultured in a medium containing 100 nM doxorubicin . Cell li2 tissue flasks were trypsinized, seeded into flatbottomed 96-well tissue culture plates , and incubated overnight in 100 µL of appropriate medium. After 24 h, the cells were treated with compounds 1 to 4 (1–25 µM) and incubated for 72 h in complete medium. The combined effects of 4 simultaneously applied with doxorubicin were also studied. In simultaneous treatments, three concentrations of 4  were combined with five concentrations of doxorubicin . After 72 h, 100 µL of MTT solution (1 mg/ml) was added to each well, and plates were incubated at 37°C for 4 h. Formazan product was dissolved in 200 ml dimethyl sulfoxide. The absorbance of the obtained dye was measured at 540 nm using an automatic microplate reader . Half maximal inhibitory concentration  was defined as the concentration of the drug that inhibited cell growth by 50% and calculated by non-linear regression analysis using GraphPad Prism6 software.MTT assay is based on the reduction of 3--2,5-diphenyl-2H-tetrazolium bromide into formazan dye by active mitochondria of living cells. Cells grown in 25 cm1 to 4. NCI-H460 and NCIH460/R cells were grown to 80% confluence in 75 cm2 flasks, trypsinized, and resuspended in 10 ml centrifuge tubes in a Rhodamine 123-containing medium. The cells were treated with diterpenes and Dexverapamil (5 and 20 μM) and incubated at 37°C in 5% CO2 for 30 min. At the end of the incubation period, the cells were pelleted by centrifugation, washed with PBS, and placed in cold PBS. The samples were kept on ice in dark until the analysis of the CyFlow Space Partec flow-cytometer . The fluorescence of Rhodamine 123 was assessed on the FL1 channel. A minimum of 20,000 events was assayed for each sample and the obtained results were analyzed using Summit Dako Software.Rhodamine 123 accumulation was analyzed by flow cytometry utilizing the ability of Rhodamine 123 to emit fluorescence. The intensity of the fluorescence is proportional to Rhodamine 123 accumulation. Studies were carried out with Dexverapamil and compounds All calculations were performed using the Gaussian 16 software package , withoutTransition state optimizations were performed with the Synchronous Transit-Guided Quasi-Newton Method (STQN) developed by The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The manuscript was written through the contributions of all authors.Support for this work was provided by FCT through UIDP/04567/2020, UIDB/04567/2020, and Ph.D. grant SFRH/BD/137671/2018. CSC-IT center for Science Ltd., Finland is acknowledged for the computational resources’ allocation. The Academy of Finland is acknowledged for the financial support to N.R.C (Decisions No. 326487 and 326486).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.