Annona squamosa L.) is a popular tropical fruit and its peel is a municipal waste. An ultrasound-assisted extraction method was developed for the recovery of natural antioxidants from sugar apple peel. Central composite design was used to optimize solvent concentration (13.2%–46.8%), ultrasonic time (33.2–66.8 min), and temperature (43.2–76.8 °C) for the recovery of natural antioxidants from sugar apple peel. The second-order polynomial models demonstrated a good fit of the quadratic models with the experimental results in respect to total phenolic content , FRAP , and TEAC  values. The optimal extraction conditions were 20:1 (mL/g) of solvent-to-solid ratio, 32.68% acetone, and 67.23 °C for 42.54 min under ultrasonic irradiation. Under these conditions, the maximal yield of total phenolic content was 26.81 (mg GA/g FW). The experimental results obtained under optimal conditions agreed well with the predicted results. The application of ultrasound markedly decreased extraction time and improved the extraction efficiency, compared with the conventional methods.Sugar apple ( Fruit wastes are one of the main sources of municipal wastes. Due to the high consumption and industrial processing of fruit edible parts, fruits residues  are generated in large quantities in large cities and become a severe environmental issue. However, if the phytochemicals of fruit residues were extracted effectively by applying efficient extraction technologies, their value could be added ,2. RecenAnnona squamosal L. (Annonaceae) is an important plant and widely distributed in tropical America and Asia [Annona squamosal bears edible fruits called sugar apple. It is a popular tropical fruit, typically globular or heart-shaped, 5–10 cm in diameter, with many round protuberances. The pulp is sweetly aromatic with a custard-like flavor, and is widely used to prepare juices, jellies, and compotes. The processing of the fruits will leave behind a substantial amount of non-edible parts. In previous studies, extremely high phenolic content and antioxidant capacities were found in sugar apple peel, indicating its potential to be utilized as a resource of natural antioxidants [and Asia . It has and Asia . Annona oxidants ,8. So faoxidants ,12,13. Ioxidants . Ultrasooxidants ,16.The extraction efficiency and antioxidant capacity of natural antioxidants could be markedly influenced by many factors, such as solvent type and concentration, solvent-to-solid, as well as extraction temperature and time . TherefoIn the preliminary experiment of our study, the effects of solvent type, solvent-to-solid ratio, acetone concentration, ultrasonic time, and temperature on the extraction of natural antioxidants from sugar apple peel were studied. Under the same extraction conditions , the responses followed a similar trend among all the solvents tested a.p < 0.05). At the solvent-to-solid ratio of 20:1, a plateau in the mass transfer was reached. There were, indeed, no significant differences for solvent-to-solid ratios between 30:1, 40:1, 50:1, and 20:1 (p > 0.05). In consideration of the solvent consumption and extraction efficiency, solvent-solid ratio of 20:1 (mL/g) was selected. The effects of extraction temperature and time were shown in In the literature, several frequently-used solvents were reported, such as ethanol, methanol, and acetone ,19,20,21The results of 20 runs using CCD design were shown in p < 0.0001) with F-values of 22.23, 42.05, and 27.36, respectively (R2) is a measure of degree of fit [R2 should be at least 0.80 [R2 values of the second-order polynomial models for TPC, FRAP, and TEAC were 0.9524, 0.9743, and 0.9610, respectively, which implied that 95.24%, 97.43%, and 96.10% of the variations could be explained by the fitted quadratic models, respectively. Furthermore, the absence of any lack of fit (p > 0.05) also strengthened the reliability of all models. The models were used for the construction of three dimensional response surface plots to predict the relationship between the responses and independent variables.The analysis of variance (ANOVA) of the quadratic regression models for TPC, FRAP, and TEAC showed that the models were significant  and temperature (X3), and the cross product effect between acetone concentration and temperature , were positive and significant at p < 0.01, while the quadratic effect of acetone concentration (X12) were negative and significant at p < 0.001. It was evident that the linear effects of time  and temperature (X3) showed significantly positive linear effects and negative quadratic effects, respectively (p < 0.001). Ultrasonic time showed significantly negative linear effect on the response (p < 0.05). The cross product effect between acetone concentration (X1) and time (X2) was positive and significant at p < 0.05, whereas the cross product effect between time (X2) and temperature (X3) was negative and significant at p < 0.01. The 3D response surfaces plot for the effects of independent variables  on FRAP values were given in In term of FRAP, acetone concentration (X2), cross product terms of time (X2) and temperature (X3), all the terms had significant effects on the antioxidant capacities (p < 0.01). The 3D response surfaces plot X1, X2, X3). Seen from In aspect of TEAC, as shown in The optimum extraction conditions obtained by RSM was used to validate the predictive model of extraction for TPC, FRAP, and TEAC values from sugar apple peel. Seen from The compounds 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,4,6-tri(2-pyridyl)-S-triazine (TPTZ), 2,2′-azinobis(3-ethylbenothiazoline-6-sulfonic acid) diammonium salt (ABTS), Folin–Ciocalteu’s phenol reagent and gallic acid were purchased from Sigma–Aldrich . Acetone, ethanol, methanol, acetic acid, hydrochloric acid, potassium persulphate, iron(III) chloride hexahydrate, iron(II) sulfate heptahydrate, sodium acetate, and sodium carbonate were of analytical grade and obtained from Tianjin Chemical Factory . Deionized water was used throughout the experiment.Sugar apples were collected from markets in Guangzhou, China. Samples were cleaned with deionized water and the peel was separated. Immediately, the separated peel was ground into fine particles with a special grinder.i.e., acoustic power of 0.38 W/cm2), heating power of 800 W, equipped with a digital timer and a temperature controller. In RSM test, sample particles (0.5 g) were sonicated in the acetone (10 mL) for different time at set temperature. After sonication, the sample was centrifuged at 9600 g for 20 min, and then the supernatant was collected and diluted for analysis. The reaction process happened in a 15 mL centrifuge tube with screw cap and conical bottom, which was fixed in a plastic rack placing in the ultrasonic device.The ultrasound-assisted extraction process was performed in a ultrasonic device  with an electric power of 600 W  at 37 °C for 24 h.The FRAP assay was performed according to the procedure described by Benzie and Strain  with min•+ stock solution was prepared from 7 mmol/L ABTS and 2.45 mmol/L potassium persulfate in a volume ratio of 1:1, and then incubated in the dark at room temperature for 16 h and used within two days. A 100 μL of the tested sample was mixed with 3.8 mL ABTS•+ working solution and the absorbance was detected at 734 nm after incubation at room temperature for 6 min. The percent of inhibition of absorbance at 734 nm was calculated and the results were expressed as µmol Trolox/g FW of fruit peel.The TEAC assay was performed based on the protocol established previously  with minTotal phenolic contents were determined with Folin–Ciocalteu method . BrieflyX1), ultrasonic time (X2), and ultrasonic temperature (X3) were chosen for independent processing variables. The range and center point values of three independent variables presented in Y is the predicted response, β0 is an intercept, βi, βii, and βij are the coefficients of the linear, quadratic, and interaction terms, respectively. iX and jX are coded independent variables.Optimization of extraction conditions for natural antioxidants from sugar apple peel were carried out using response surface methodology (RSM). A three-variable, five level central composite design (CCD) was applied to determine the best combination of extraction variables. Acetone concentration (R2), and the Fisher test value  generated from the analysis of variance (ANOVA) by the software. The difference was considered significant at p < 0.05.The regression and graphical analysis of the experimental data were performed by Design Expert version 7.1.3 software . The suitability of the model was checked by evaluating the lack of fit, coefficient of determination  of solvent-to-solid ratio, 32.68% acetone and 67.23 °C for 42.54 min under ultrasonic irradiation. The application of ultrasound markedly decreased extraction time, and improved the extraction efficiency compared with the conventional methods.An ultrasound-assisted extraction method has been developed for the recovery of natural antioxidants from sugar apple peel. Response surface methodology was used to optimize the experimental variables such as solvent concentration, ultrasonic time, and temperature. The


Glycine max var) sprouts. Three influencing factors: liquid-solid ratio, period of ultrasonic assisted extraction and extraction temperature were investigated in the ultrasonic aqueous extraction. Then Response Surface Methodology (RSM) was applied to optimize the extraction process focused on DPPH radical-scavenging capacity of the antioxidants with respect to the above influencing factors. The best combination of each significant factor was determined by RSM design and optimum pretreatment conditions for maximum radical-scavenging capacity were established to be liquid-solid ratio of 29.19:1, extraction time of 32.13 min, and extraction temperature of 30 °C. Under these conditions, 67.60% of DPPH radical-scavenging capacity was observed experimentally, similar to the theoretical prediction of 66.36%.Response surface methodology (RSM) using a central composite design (CCD) was employed to optimize the conditions for extraction of antioxidants from black soybean ( Glycine max var) is the black seed of the soybean (Glycine max (L.) merr), also known as the black bean. It is mainly cultivated in the provinces of Shanxi and Hebei for both food and medicinal purposes. The Supplement to Compendium of Materia Medica states that black beans can be beneficial to sperm and bone marrow production, muscle strength, hair growth, and the immune system. Modern scientific research shows that black beans have hypolipidemic and antioxidant properties and can be used to beautify the skin [Black soybean  is a kind of optimization method which serves as an accurate, effective, and simple tool for optimizing the experimental process ,14, and The influences of extraction time, liquid-solid ratio and extraction temperature on antioxidant activities of extraction were shown in It can be seen from The effect of three independent variables, namely, extraction temperature (A), extraction time (B), liquid-solid ratio (C) on DPPH radical scavenging activity (Y) was investigated using a three-factor CCD-RSM experimental and the results are shown in 2 − 0.01378B2 − 0.03845C2. Analysis of variance for response surface quadratic model was shown in After data fitting to the experimental findings, one gets the following regression equation: Y = 13.3375 + 0.23845A + 1.06255B + 2.22142C − 0.04950AB + 0.01833BC − 0.09750AC − 0.02269A2 are significant model terms. Values greater than 0.1000 indicate the model terms are not significant. There are many insignificant model terms (not counting those required to support hierarchy), so model reduction may improve the model.As shown in The analyses of the data were done using the Design-Expert 7.1 software. Response surfaces and contour plots were shown in The response surfaces and contour plots of 1 = (A−45)/15, X2 = (B−40)/10, X3 = (C−30)/10. The regression equation transfer into: Y = − 405.5275 − 100.62975X1 − 17.1745X2 −103.725X3 − 5.10525X12 −1.378X22− 3.845X32 − 7.425X1X2 + 1.833X2X3 − 14.625X1X3.To get the optimized value of the extraction conditions, the three independent variables, namely, extraction temperature (A), extraction time (B), liquid-solid ratio (C) were transformed as follows: XThe optimal conditions analyzed using Design Expert 7.1 were as follows: 30 °C of extraction temperature, 32.13 min of extraction time, and 29.19:1 of liquid-solid ratio. Under optimal conditions, the theoretical value of DPPH radical scavenging rate of black soybean sprout extract was 66.36%, and the measured actual scavenging rate was 67.60%, very close to the theoretical prediction. The results show that response surface methodology is a useful mathematical tool in finding the optimum extraction conditions for antioxidants from black soybean sprouts.Glycine max var) ; 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•), AR ; Vitamin C, AR .The following reagents and materials were obtained for this study: soybeans  was used in this investigation. Five grams of soybean seeds were soaked in distilled water (200 mL) for 16 h and then placed in a germinator . Germination occurred at 25 °C and 99% relative humidity in darkness with water sprayed every 60 min [Black soybean , peroxyl radical-trapping capacity (PRTC), Trolox equivalent antioxidant capacity (TEAC) and DPPH radical scavenging capacity (DPPH•). DPPH• is a stable organic free radical which can be used to give a global measure of antioxidant activity by spectrophotometry; therefore, antioxidant activity of extracts were measured using DPPH• scavenging method . Ethanoli.e., extraction time, liquid-solid ratio and extraction temperature to be used in design of experiments. It involved extraction times of 20, 30, 40, 50, 60 min at fixed liquid-solid ratio of 30:1 (30 mL of deionized water to 1 g powder) and extraction temperature of 30 °C. Then, liquid-solid ratio was evaluated at different ratios from 10:1 to 50:1 at a fixed extraction time of 30 min and extraction temperature of 30 °C. Extraction temperature was evaluated from 20 to 75 °C at a fixed extraction time of 30 min and liquid-solid ratio of 30:1.A screening study was conducted to determine appropriate ranges of independent variables, Response surface methodology was employed to establish the optimum conditions for extracting antioxidants from black soybean sprout. The effect of three independent variables, namely, extraction time (20–40 min), liquid-solid ratio (20:1–40:1), extraction temperature 30–60 °C) on DPPH radical scavenging activity was investigated using a three-factor CCD-RSM experimental runs to determine the optimal parameters of the extraction process ,28,29,30–60 °C onThe analyses of the data were done using the Design-Expert 7.1. .In this study, RSM was used to investigate the main and interaction effects of important independent variables for extraction of antioxidants from black soybean sprouts on the basis of single-factor experiments. The optimum conditions for antioxidant extraction from black soybean sprouts were established. Research shows that the optimized experimental process increases the antioxidant activity of antioxidants from black soybean sprouts. The manufacturing process optimized using RSM needs lower energy, shorter time and offers simplified manipulation. The extract was green, healthly, safe and thus shows potential as an additive in cosmetic products. In addition, the successful extraction of antioxidants from black soybean sprout provides a basis for the development and utilization of black soybean resources.


Cinnamomum camphora leaves. Response surface methodology of Box–Behnken design was employed to optimize the HUCSE process, and the optimum operation conditions attained with an extraction yield of 7.95 ± 0.27 mg/g were ethanol concentration 76%, homogenate/ultrasonic time 25 min, solvent-to-solid ratio 22 mL/g, and ultrasonic power 240 W. A second-order kinetic mathematical methodology was performed to depict the behaviors of HUCSE and heat reflux extraction method. The results suggested that the developed HUCSE is an efficient and green method for the extraction of C. camphora flavonoids or other plant natural products, where the obvious higher parameters of extraction capacity at saturation, second-order extraction rate constant, and original extraction rate were obtained when compared to the heat reflux method. The antioxidant activity assays in vitro showed that the C. camphora flavonoids possessed strong antioxidant activity and are promising to be applied as a natural alternative antioxidant.A free-of-dust pollution extraction method combined-homogenate and ultrasonic cavitation system, namely, homogenate-combined ultrasonic cavitation synergistic extraction (HUCSE), was proposed for the efficient extraction of flavonoids from  Natural products have gained great interest as natural alternatives to substitute the traditional synthetic compounds for health purposes as their remarkable health-promoting activity and consumer preference for the cleaning labels , 2. SpecCinnamomum camphora is an evergreen tree, which is widely distributed in Southern China as a Chinese folk medicine for the cure of many diseases [C. camphora is even extensively cultivated in Jiangxi province as a landscape tree species and an economic crop for the production of essential oil and camphor [C. camphora are mainly on essential oils used for their antifungal and insecticidal activities [C. camphora leaves with remarkable antioxidant activity and antibacterial activity [C. camphora leaves at regular intervals facilitates C. camphora to grow vigorously [diseases . C. camp camphor . Many retivities –15, and activity –18. Moregorously , so it iC. camphora flavonoids is much necessary.Generally, plant materials are ground to powders using mechanical disintegrators prior to the subsequent extraction process for sufficiently obtaining the target compounds. Among the physical smashing ways, homogenate is an effective technique as it possesses many inherent merits , which is conducive to the dissolution of plant bioactive compounds into extraction solvent , 21. ExtC. camphora leaves. An RSM-based Box–Behnken design (BBD) was applied to offer the optimal combination of ethanol concentration, homogenate/ultrasonic time, solvent-to-solid ratio, and ultrasonic power with which a maximum yield of C. camphora flavonoids can be attained by HUCSE and their antioxidant capability can be evaluated in vitro.The response surface method (RSM) is an efficient statistical optimization methodology which just needs a few numbers of experimental trials to investigate multiple variables and their interactive effects . RSM is C), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and 2,4,6-tripyridyl-s-triazine (TPTZ) were obtained from Aladdin Reagent Co. . Other analytically pure reagents were obtained from Beijing Chemical Reagents Co. . Deionized water used in the experiments was purified by a Milli-Q water system . C. camphor leaves were collected in November 2017 from Jiangxi Normal University campus  and authenticated by Prof. Ronggen Deng . Fresh leaves were dried at room temperature (20 ± 0.8°C) for 7 days, stored in a nylon bag, and then placed in a dry and ventilated place before the subsequent experiments.Chromatographically pure rutin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), vitamin C  and 2 mL of CH3COONa solution (1 mol/L) were added, followed by adjustment to the scale with 60% ethanol aqueous solution. The mixtures were placed for 20 min in atmospheric temperature and then detected at 400 nm against the mixture without coloration as a reference by a UV-Vis spectrophotometer . The yields of the flavonoids were conveyed as milligram of rutin equivalents per gram of dry weight of C. camphora leaves based on the standard calibration curve. The calibration curve  ranged from 0 to 0.05 mg/ml (R2 = 0.9963). All experiments were conducted three times, and the results were conveyed as mean ± SD.The quantification of g et al.  with sliC. camphora leaves were weighed and transferred into the HUCSE system accompanied with the addition of a definite volume of ethanol solution followed by treatment under the presetting conditions for flavonoids extraction. After each treatment, the suspension was centrifuged at 8,000 × g for 20 min to collect the crude extract solutions and then stored in a refrigerator prior to the analysis of flavonoids content by UV-Vis spectrophotometry. Each experiment trial was performed in triplicate for accuracy.Dried A, %), homogenate/ultrasonic time , solvent-to-solid ratio , and ultrasonic power . The scope and level of each factor as shown in BBD, a member of RSM, is characterized with a spherical and revolving design. It comprises a central point, and the intermediate points circumscribed on the sphere at the cube edges . Herein,β0 is presented as the constant; βi, βii, and βij are defined as the coefficients of linear, quadratic, and cross-product, respectively. Xi and Xj are given as the independent factors at different levels.The Design-Expert  software was employed for performing the model establishment, experimental design, analysis of data, and graph plotting. A quadratic equation was fitted to investigate the correlation between the determined values and the four independent variables as follows:C. camphora leaves powders (60 meshes of particle size) was weighted and placed into a glass flask, and then a definite volume of ethanol aqueous solution was added before subjecting to extraction by HRE. The other extraction conditions applied in HRE were the optimal conditions based on the results of the optimizing process for HUCSE. After the extraction, the mixture was transferred to certification and then analyzed by UV–Vis spectrophotometry as mentioned above.The conventional heat reflux method (HRE) was performed as the reference method to make a comparison with the proposed HUCSE . HRE wasC. camphora leaves by HUCSE and HRE were investigated on the basis of the second-order kinetic model. The model parameters h, k, Ys, and Yt are defined as the original extraction rate (mg/g·min), second-order extraction rate constant (mg/g·mim), extraction capacity (mg/g) at saturation, and the extract yield (mg/g) at any moment t (min), respectively. The dissolution rate of flavonoids contained in the solid transferring to solvent can be represented asThe second-order kinetic model offers an adequate 1 acceptable illustration with respect to the solid-liquid extraction procedures and has t = 0 to t and the corresponding Yt = 0 to Yt, the second-order model equation  can be denoted by equation  can be calculated experimentally by the intercept and slope of the t/Yt versus t plot.The three second-order kinetic model parameters /g extracts.According to the study of Benzie and Strain , the ant3Fe(CN)6 solution were mixed and incubated at 50°C for 20 min and then 2.5 mL 10% of trichloroacetic acid was added, followed by centrifugation at 10,000 × g for 10 min. Aliquots of 2.5 mL of the upper layer were collected and then blended with 2.5 mL of deionized water and 0.5 mL 0.1% of FeCl3 and then were detected at 707 nm. Positive control groups were carried out as mentioned above by replacing sample solution by VC, BHT, and BHA.The assay for the evaluation of reducing power was performed based on Oyaizu's study . 1 mL ofANOVA was used to analyze the statistical significance of all data. BBD was performed by Design-Expert 8.0 software , and the actual responses were the average values of each experiment trial in triplicate. The other experiments were conducted in three replicates, and the results are presented as the mean ± SD. OriginPro 2017 software was employed to fit the extraction kinetic curves for HUCSE and HRE according to the second-order kinetic model.C. camphora leaves (1 g dry weight) were extracted by HUCSE at an ultrasonic power of 250 W for 20 min with various proportions of ethanol (0–100%), and the solvent-to-solid ratio was 20 mL/g. As shown in C. camphora flavonoids showed an increasing tendency. However, an upward trend was observed in the extraction yield when the ethanol concentration was over 75%. So, 75–100% of ethanol concentration was applied for the following experiments as an apparent change of extraction yield was observed in this range.Ethanol is a generally used extractant owing to its remarkable penetration capacity into the plant materials, relatively low boiling temperature (easily recycled) and cost, safety, and nontoxicity . The proTo obtain the appropriate homogenate/ultrasonic time, a series of experiments were conducted at different HUCSE times, and the results are presented in The solvent-to-solid ratio is a crucial factor in the extraction procedure, excessive volumes of the solvent could give rise to the tedious extraction process and needless waste, and minute volumes of solvent may result in the incomplete separation. Hence, different solvent-to-solid ratios from 12 to 28 mL/g with 4 mL/g intervals were studied to analyze their effects. As presented in To analyze the effect of ultrasonic power on the extraction yield of flavonoids, tests were conducted at different ultrasonic powers  with 75% of ethanol concentration, 20 min of homogenate/ultrasonic time, and 20 mL/g of solvent-to-solid ratio, respectively. As shown in C. camphora flavonoids extraction and to determine optimal extraction conditions. The extraction yield was defined as the response. The design matrix, numbered, and actual forms at three levels  of four independent factors; the determined extraction yields by HUCSE process; and the predicted values generated from BBD are given in A), homogenate/ultrasonic time (B), solvent-to-solid ratio (C), and ultrasonic power (D) were studied as shown in BBD was used to build the combined influences of four factors on the HUCSE process for R2 = 0.9903), the adjusted coefficient of determination (adjusted R2 = 0.9754), and the coefficient of variation (C.V = 1.26%), which revealed that more than 99% of the obtained actual values can be interpreted by the selected model, and the model accuracy and generic availability are capable. The predicted R2 of 0.9516 was corresponded well with the adjusted R2. The adequacy precision estimated the signal-to-noise ratio, and a ratio higher than 4 is generally desirable. The adequacy precision of 38.78 revealed that the developed model could be employed to handle the design space.The model suitability was verified by the coefficient of determination  and a very low P-value (<0.0001) as shown in The significance of all the coefficients was examined by  P-value . The devC. camphora flavonoids suggested that the interactive influences of HUCSE process was extremely significant affected by the ethanol concentration, homogenate/ultrasonic time, and solvent-to-solid ratio and ultrasonic power, in both linear and quadratic manners. The interaction of solvent-to-solid ratio versus ultrasonic power, homogenate/ultrasonic time versus ultrasonic power, and ethanol concentration versus solvent-to-solid ratio or ultrasonic power showed extremely significant, highly significant, and significant influences on the extraction process, respectively. Nevertheless, the interaction effects between homogenate/ultrasonic time to ethanol concentration and solvent-to-solid ratio were statistically insignificant. By using multiple regression analysis on the determined values, the empirical relationship between the extraction yield and four factors in natural values was represented using a polynomial equation:Statistical analysis regarding the regression model for the extraction of The regression polynomial equation can be delineated by both the three-dimensional response surface and the two-dimensional contour graphs generated from BBD. These graphs offered a way to visualize the connection between response values and detailed experimental levels of four factors and the interactive effects between two test factors on the extraction yield in HUCSE process. The contour plot shapes can reflect the statistical significance of mutual interactions between the factors, with which a circular contour graph indicates that the interactive effect between the factors are insignificant, while an elliptical contour graph suggests that the interactive effect between the factors are significant . The conn=3), which was in good accordance with the predicted response by the model equation and further confirmed that the developed response model was capable for the HUCSE process optimization.As for the BBD optimization process, which needs less experiment trials to be performed for a three-level factorial than other experiment designs to illustrate the significant factors, the possible interactive effects between the variables studied on the extraction yield of target compounds to attain the optimal operation conditions and the satisfied response . By supeC. camphora flavonoids extraction. The variations of flavonoids extraction yield with changing the treatment time in the procedures of HUCSE and HRE were depicted as presented in Extraction kinetic modeling was employed to make a comparison of the inherent behaviors of concern on heating and mass transfer during HUCSE and HRE procedures for Ys (extraction capacity at saturation) and k (second-order extraction rate constant)  denoted that the second-order kinetic mathematical model was adequate for fitting the HUCSE processes of flavonoids. The parameter Ys was applied to determine the extraction efficiency of HUCSE compared to that of HRE; it was observed that HUCSE (Ys = 14.95 mg/g) was more efficient for the extraction of flavonoids than traditional HRE (Ys = 13.05 mg/g). Meanwhile, the proposed HUCSE possessed apparently higher original extraction rate (h) and the second-order extraction rate constant (k) for flavonoids separation in comparison to HRE. These results all declared that HUCSE is an efficient technique for the extraction of C. camphor flavonoids. It can be concluded that the synergistic effects of the homogenate and ultrasonic cavitation are the main reason that the proposed HUCSE method can dramatically shorten the extraction time and improve the extraction efficiency. A similar result was also observed in our previous study [The parameters of onstant)  were obtonstant)  of t/Ytus study .C. camphor flavonoids on scavenging DPPH radicals. Compared to the positive control sets of VC, BHT, and BHA, C. camphor flavonoids revealed relatively low DPPH radical-savaging activity at low concentrations, notably with a further increase of concentration, flavonoids showed approximately similar DPPH radical-savaging activity as Vc. FRAP test was performed in the present study to evaluate the antioxidant ability of C. camphor flavonoids. As presented in C. camphor flavonoids showed great potential as a natural antioxidant to substitute the traditional synthetic antioxidants based on the above results.Scavenging activity on DPPH-free radicals is an important indicator on the investigation of antioxidant activity of bioactive compounds .  FigureC. camphora flavonoids. BBD was employed to optimize the HUCSE process; the optimal operation conditions were attained and validated, and the results showed that the improved HUCSE method is adequate for the extraction of flavonoids. The second-order kinetic model was performed to investigate the extraction kinetics of HUCSE and HRE of flavonoids, and the kinetic parameters were obtained and analyzed. HUCSE, compared with traditional HRE, enjoyed the higher efficiency for flavonoids extraction and could be extended to isolate other bioactive compounds from plant materials. Additionally, C. camphora flavonoids had remarkable antioxidant ability compared to the commercial antioxidants.In this study, HUCSE technique was developed with the merit of no-dust pollution for the efficient extraction of


Catharanthus roseus leaves. The optimized method employed 60-mesh particles, 80% ethanol, a negative pressure of −0.075 MPa, a solid to liquid ratio of 1:20, 30 min of extraction and three extraction cycles. Under these optimized conditions, the extraction yields of VDL, CTR, VCR and VLB are 0.5783, 0.2843, 0.018 and 0.126 mg/g DW, respectively. These extraction yields are equivalent to those from the well-known ultrasonic extraction method and higher than the yields from maceration extraction and heating reflux extraction. Our results suggest that NPCE-RP-HPLC represents an excellent alternative for the extraction and quantification of vinca alkaloids for pilot- and industrial-scale applications. In the present study, an improved method termed negative-pressure cavitation extraction (NPCE) followed by reverse phase high-performance liquid chromatography (RP-HPLC) was developed for the extraction and quantification of vindoline (VDL), catharanthine (CTR), vincristine (VCR) and vinblastine (VLB) from  Catharanthus roseus (L.) G. Don (Madagascar periwinkle) is a terpenoid indole alkaloid (TIA)-producing plant that belongs to the Apocynaceae Family. This plant has historically been used to treat a wide assortment of diseases. Research shows that Catharanthus roseus (C. roseus) contains over 130 compounds, and many of which have cytotoxicity [C. roseus and it has become a model species for the study of secondary metabolism in plants. The interest in this species arises from its therapeutic role as the source of the anticancerous alkaloids vinblastine (VLB) and vincristine (VCR) [toxicity . Few spene (VCR) . VLB andne (VCR) . Vincrisne (VCR) . The antne (VCR) ,8.C. roseus have reported the isolation of vinca alkaloids by centrifugal partition chromatography, supercritical CO2 extraction, ultrasound-assisted extraction and microwave assisted extraction [Because they exist in trace amounts (0.01–0.1 mg/g DW) in the plant, the extraction of VLB and VCR and their precursors from the plant has been an important method on the industrial scale. In attempts to improve the production of valuable alkaloids such as VDL, CTR, VCR and VLB, several studies on traction ,11,12,13C. roseus leaves. This study evaluated the effects of particle size, solvent concentration, as well as NCPE operational variables including negative pressure, solid to liquid ratio, extraction time and cycles on the extraction yields of the four main vinca alkaloids. This work may provide valuable data for both pilot- and industrial-scale applications.In recent years, a new extraction method called negative-pressure cavitation extraction (NPCE) has increased in popularity; NPCE is more efficient at constant lower temperatures and intensities than classical extraction technologies such as maceration extraction, heat reflux extraction and ultrasonic assisted extraction. Since it was first developed in 2009, the NPCE technique has been widely used in the extraction of a number of bioactive compounds from Chinese herbs ,18,19,20The particle size of the plant material and solvent concentration has a large impact on extraction yields. This is because plant cell walls are broken, and the accessibility to the bioactive compounds is augmented for the extraction solvent. As shown in To increase the extraction efficiency and decrease the extraction time, the extraction solvent concentration was optimized. It is well known that increasing the ethanol concentration induces a reduction in the dielectric constant of the extraction solution and improves the solubility of bioactive compounds. A range of ethanol concentrations  were compared. As can be seen from In our experiment, the extraction pressure was adjusted by regulating the airflow. The effect of different extraction pressures on the extraction yields of vinca alkaloids are shown in The effect of the solid to liquid ratio on the extraction yields of the four main vinca alkaloids was determined by a series of experiments with different solid to liquid ratios . As is shown in The proper extraction time is necessary for optimal extraction efficiency and ensuring a distribution equilibrium of bioactive compounds between the plant material and the extraction solvent. In the present study, extraction was carried out for 25, 30, 35, 40 or 45 min and the results are shown in The effect of the number of extraction cycles on the extraction yields was investigated using 1, 2, 3, 4 or 5 cycles. Generally, an increase in the number of extraction cycles increases extraction yield by allowing greater exposure of the material to fresh solvent and favoring the material-solvent equilibrium. As shown in After optimization, NPCE was shown to be an efficient method with high extraction yields . We determined that the optimal parameters to be employed are: 60-mesh particle size, 80% ethanol, a solid to liquid ratio of 1:20 (w/v) and a 30 min extraction for three cycles. NPCE has received considerable attention for extracting several bioactive compounds from Chinese Herbs; to the best of our knowledge, NPCE of vinca alkaloids has not been reported. As a result, it is necessary to compare the extraction efficiency of NPCE with other extraction methods for the extraction of vinca alkaloids. C. roseus leaves.Different methods  were performed using the optimized conditions, and the results are summarized in C. roseus leaves (80% water content) were collected from the Greenhouse of Northeast Forestry University, Heilongjiang, China, and identified by Professor Shao-Quan Nie from the Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin, China. The material was dried in the shade, powdered by a disintegrator  and then sieved through 20–100 mesh sieves . The powder was kept at 4 °C prior to use.Reference standards of VDL, CTR, VCR and VLB were purchased from Shanghai Anticancer Phytochemistry Co., Ltd. . Methanol  and acetonitrile  used for HPLC were HPLC reagent grade. Deionized water was filtered using a Milli-Q Ultrapure water purification system from Millipore . Other reagents were all commercially available analytical grade chemicals.The apparatus (CN 2597047) used in the NPCE experiment was provided by the Engineering Research Centre of Forestry Bio-preparation, Ministry of Education, Northeast Forestry University, Harbin, China. The mode of action of NPCE is based on the continuous formation and collapse of tiny bubbles under the action of a vacuum, which was described in detail in our previous study ,22,23. C. roseus leaves were transferred into the extraction chamber with different concentrations of extraction solvent. Then, a vacuum pump was connected to the extraction pot through a condenser to generate negative pressure. The pressure was monitored by adjusting the airflow rate, which was supplied from the bottom of the device. The extraction process was performed under different conditions as described in the Results section. After each extraction, the extraction solutions were combined and dried on a rotary evaporator  at 45 °C. HPLC-grade methanol was added in a constant volume to prepare the samples for RP-HPLC analysis. The extraction efficiency of the compounds in a given sample was defined as follows:In the present study, 25 g of powdered C. roseus leaves were placed in a beaker with 500 mL of 80% aqueous ethanol solution. The beaker was placed at room temperature for 12 h, the filtered extraction solution was collected, and another 500 mL of 80% ethanol was added into the beaker for an additional 12 h to obtain optimal extraction efficiency. For maceration extraction (ME), as determined by the preliminary optimized experiments, 25 g of powdered of C. roseus leaves were added to a round-bottom flask with 500 mL of 80% ethanol. The round-bottom flask was then placed into a water bath and linked with a condenser at the joint of the flask. The extraction process was twice maintained at 80 °C for 3 h to obtain optimal extraction efficiency. For heat reflux extraction (HRE), after preliminary optimization experiments, 25 g of powdered C. roseus leaves were added to a triangular flask with 500 mL of 80% ethanol. The extraction was performed in ultrasonic bath at a frequency of 40 kHz with the maximum input power of 250 W at 45 °C for 30 min. Three replicates were performed to obtain the best extraction efficiency. Ultrasonic-assisted extraction (UAE) was run in an ultrasonic bath  using the conditions optimized for NPCE at normal pressure. In brief, 25 g of powdered of In this study, an RP-HPLC separation and analysis protocol is employed to detect the four alkaloids. UV absorbance is the method of choice for their detection, and 220 nm was the wavelength used. The criteria for the identification of the compounds were established based on comparisons of the retention time and spectrum of an unknown compound with HPLC library data of standards .18W column (4.6 mm × 250 mm). RP-HPLC analysis was carried out using a Jasco HPLC system , which included a PU-1580 pump, a 7725i manual injector and a UV-1575 detector connected to CkChrom chromatography data system software (Version 6.1). Separations were performed on a HiQ sil C3PO4). The mobile phase consisted of 620 mL solution A and 380 mL solution B. The flow rate was set at 2.0 mL·min−1 and the elution was monitored at 220 nm [The separation was carried out by an isocratic elution using solution A (methanol/acetonitrile = 4:1), and solution B .All experiments were conducted in triplicate，and the average values of extracted vinca alkaloids were used for statistical analysis. The one-way ANOVA test was used to calculate the significance of the differences in extraction efficiency of the target alkaloids. The results of RP-HPLC analysis are expressed as the means ± SD (C. roseus leaves and compared with other standard extraction methods. Under the optimal parameters, the extraction yields of VDL, CTR, VCR and VLB were 0.5783, 0.2843, 0.018 and 0.126 mg/g DW, respectively. These yields are comparable to those obtained by the well-known UAE method and significantly higher than those obtained by ME and HRE. The results indicated that NPCE offers a fast, environmentally friendly and straightforward route for vinca alkaloid production. Consequently, the NPCE-RP-HPLC described here represents an excellent alternative for the extraction and quantification of the four main C. roseus alkaloids in both pilot- and industrial-scale applications.NPCE was first applied to the extraction of VDL, CTR, VCR and VLB from


Alzheimer’s disease is a complex disorder encompassing multiple pathological features with associated genetic and molecular culprits. However, target-based therapeutic strategies have so far proved ineffective. The aim of this study is to develop a methodology harnessing the transcriptional changes associated with Alzheimer’s disease to develop a high content quantitative disease phenotype that can be used to repurpose existing drugs. Firstly, the Alzheimer’s disease gene expression landscape covering severe disease stage, early pathology progression, cognitive decline and animal models of the disease has been defined and used to select a set of 153 drugs tending to oppose disease-associated changes in the context of immortalised human cancer cell lines. The selected compounds have then been assayed in the more biologically relevant setting of iPSC-derived cortical neuron cultures. It is shown that 51 of the drugs drive expression changes consistently opposite to those seen in Alzheimer’s disease. It is hoped that the iPSC profiles will serve as a useful resource for drug repositioning within the context of neurodegenerative disease and potentially aid in generating novel multi-targeted therapeutic strategies. Further, biological state dynamics can be modelled through temporal patterns of expression6. In the therapeutic context, it has been established that disease-associated expression changes can distinguish between disease states and are consistent across independent data sets, thus facilitating the identification of robust biomarkers7. Disease-associated gene changes point to modulated pathways and affected cell types, thus providing valuable insights into mechanisms8. Interestingly, the quantitative nature of the transcriptional phenotype has allowed for a direct mapping of disease to potential therapeutic12. Here the obvious hypothesis is that drugs tending to reverse the expression changes seen in the disease state may act to reverse the biological changes associated with the disease itself. An important caveat here is that some expression changes associated with Alzheimer’s disease (AD) may in fact be compensatory and beneficial. Drug repurposing or repositioning has resulted in successful initiatives across several maladies19. Further, and of more specific interest to the present project, drugs with profiles showing significant anti-correlation to AD gene changes have been shown to be conspicuous for their reported neuroprotective activities12. In a recent development, disease-associated gene expression changes have begun to be inferred from genomic risk variant data with the Genotype-Tissue Expression repository20 and harnessed to predict repurposing candidates for major psychiatric conditions21. Although there is some intriguing psychotherapeutic association of the candidate drugs in this approach, the predicted transcriptional perturbation does not have an overlap with that seen in diseased brain tissue . In the absence of further validation of the predicted gene changes, one must fall back on data from patient samples.Global gene expression profiling can be thought of as a high content quantitative phenotypic measure characterising tissue22. The main aim of transcription-based drug discovery is not target discovery, but rather the discovery of drugs that have a disease-modulating effect based on their global transcriptional activity. A particularly attractive aspect of the approach is that it naturally lends itself to repositioning existing drugs thereby bypassing the hurdles that novel entities must overcome on the road to the clinic. AD has been extensively studied in relation to the expression changes following pathological and cognitive decline26. The wealth of data points to consistent and characteristic changes associated with the disease and thereby makes a repositioning strategy particularly attractive.There are no established disease-modifying drugs for the treatment of AD, there have been no new symptomatic treatments licensed for AD for >20 years and the pipeline of emerging therapies is very limited. Target-based drug research in AD has led to many insights into the disease and provided the research community with useful tool compounds. However, the promising results seen in the laboratory have so far failed to be carried over to the clinic and this has led to researchers casting around for novel, non-target-based approaches11. A more extensive drug set has been profiled on a variety of induced pluripotent stem cell (iPSC)-derived cells, including neural stem cells and differentiated cortical neurons. However, this data constituting the LINCS project27 is based on profiling a set of 1000 landmark genes and then using an optimised linear mapping to generate full profiles. This motivated the present initiative to define the full expression profiles of the CMAP candidate drugs in the more AD relevant cell type of iPSC-derived cortical neurons. The new phenotypes can then be compared to the CMAP profiles and more pertinently scored against the disease profiles to see whether they preserve or enhance their anti-correlation with AD. In this context, iPSC-derived cortical neurons have now been established as a model system for the study of neurological diseases especially the tracing of the effects of disease-related genetic variants31. This model provides for an efficient moderate throughput platform to assess the transcriptional effects of the candidate drugs in a more neurological context. It must be remembered, however, that AD is a complex pathology also involving multiple cell types, such as microglia and astrocytes. In this context, assaying drug perturbations within isolated iPSC cultures facilitates an important but limited insight into the disease.The application of gene expression profiling to drug repositioning is limited at present by the fact that full drug profiles are available only on a restricted set of immortalised human cell lines. This data is provided by the Broad Institute connectivity map project (CMAP)The motivation for the work presented here is to generate a neuronal-specific transcriptional database of compounds with a view to drug repositioning in AD and other neurodegenerative conditions. The initial compound set was assembled based on CMAP profiles that showed a tendency to reverse AD-associated expression changes observed across a variety of independent studies. The drug candidates were then profiled for their transcriptional effects on iPSC-derived human cortical neurons. The results indicate that at the global level there is a degree of correspondence between the CMAP and iPSC profiles. Furthermore, 51 of the drugs have profiles that drive transcription changes counter to those in AD. The consistently regulated genes correspond to those implicated in AD. It is hoped that the transcriptional data for these drugs will be of use to the wider community of researchers interested in neurodegenerative conditions and facilitate further repositioning efforts.32 was queried for series containing samples derived from postmortem AD patient brains for various stages of the disease. Similarly, murine AD model brain samples were also collected based on relevant query key words: 5xFAD, 3xTG, Alzheimer’s disease+mouse. Profiles were generated based on relative levels of non-disease and disease state sample averages, with the scaled fold level defined as c) and disease (d) samples. The statistical significance is measured by Student’s t test and those folds falling below the 95% confidence interval were dropped as were those with folds of <20%. The human disease versus control AD set comprises 21 profiles derived from 13 series  showing intra-profile consistency based on the regression scores for significant (Student’s t test p < 0.05) correlations, see Supplementary Table 43 represented by two profiles from two independent series and Clinical Dementia Rating (CDR)44 profiles from one series. Similarly, series corresponding to murine models of AD were gathered from 5xFAD and 3xTG mice resulting in seven profiles from three series  for the 5xFAD set and nine profiles from eight series  for the 3xTG set. Series corresponding to BRAAK stage progression  were generated with a linear mixed model analysis, by fitting the gene expression level across the samples in the series to a linear function of the BRAAK stage with categorical calls on cell type and gender as covariates. The resulting residual correlation Z score for gene expression against BRAAK stage constituted the BRAAK profile. Profiles corresponding to full BRAAK progression were not considered to be sufficiently different to the overt disease profiles derived from the same series, where disease assignment is also based on BRAAK staging. However, gene expression changes driving mild BRAAK pathology should capture early disease biology invisible in the overt profiles. In total, six profiles corresponding to mild BRAAK pathology, level 0 to level 3, formed the mild BRAAK AD set. Similar profiles were generated for psychiatric measures MMSE and CDR . In the case of the MMSE profile, the regression signs were reversed as MMSE scores decrease with disease progression, see Table The NCBI GEO databasementclass2pt{minimmentclass2pt{minimt test of p < 0.05. Owing to the categorical nature of the representative profiles, correlation with the iPSC profiles was based on an enrichment analysis. The enrichment score was generated based on a binomial probability sum with gene probabilities scaled according to their frequencies in SPIED53.Representative profiles for each set were based on genes showing consistent changes across the constituent profiles. In particular, the sense changes  for significantly regulated genes were summed over the profiles and only those genes retained that had an absolute regulation fraction of >20% and with a significant regulation statistic measured by Student’s www.broadinstitute.org/connectivity-map-cmap) 11. This consisted of probe sets for each sample ranked according to expression level relative to batch control. The data consist of 6100 samples covering 1260 drugs and 4 cell types. The relative probe expression ranks, defined as R in the rank of a given gene’s expression change (Rmax being the highest and Rmin being the lowest ranks), were averaged over replicates ignoring cell type and filtered based on significance using a one-sample Student’s t test. For genes with multiple probes, the probe with the largest significant change was mapped to the gene. This resulted in a unique profile for each drug in CMAP. The compound data can be queried through SPIED53.CMAP data were downloaded from the Broad connectivity map site . Each drug treatment at a concentration of 10 μM for 6 h was performed on 3 independent HyCCN cultures (average density 300 K/cm2) and RNA from each treated well extracted by direct cell lysis and recovery using the Absolutely RNA Microprep Kit . Each drug-treated plate also consisted of a vehicle-only control set of triplicate cultures. Integrity of total RNAs was determined using an Agilent Bioanalyser as per the manufacturer’s instructions and only samples with RNA integrity number >7 were progressed to transcriptome analysis. Transcriptome changes driven by exposure to the candidate drugs were determined using the Nugen Ovation V2 labelling system (https://www.nugen.com/products) followed by Human U133 Plus 2 GeneChips as per the manufacturer’s instructions .Human iPSC-derived cerebral cortical neurons  were cultured as per the manufacturer’s guidelines . Rapamycin, which has a well-defined transcriptional signature, served as a positive control. The expression changes were either measured as scaled folds filtered for significance with Student’s 54. Animal model data were included in this study because the expression changes seen in the model systems have established causes, i.e. the inserted mutations, 5xFAD or 3xTG in our case. Consequently, candidate drugs reversing these changes may have more focused mechanisms of action. Furthermore, the evidence for neuroprotection is to a large extent derived from experiments in animal models.To capture as much as possible of the transcriptional landscape of AD, different categories were defined based on overt disease versus healthy profiles, profiles following early pathological and cognitive measures, together with those from animal models, as described in ‘Materials and methods’. There is a good degree of overlap between the overt AD profiles and those following cognitive decline, see Table 19. The relatively small number of compounds that are put forward for rigorous bio-assaying to establish firmer evidence for a disease-modulating potential of course reflects the experimental resource required. The basis of the present project was to select candidates to populate a database of iPSC profiles for drugs biased towards their predicted anti-AD and wider neuroprotective activities. It was therefore reasoned that the thresholds for deeming a drug a repositioning candidate had to be relaxed to allow for over a hundred candidates to be taken forward. To this end, five AD-based profile sets that capture distinct aspects of the disease were separately queried against CMAP and three selection criteria were applied. In the first instance, data were gathered on the anti-correlation rank of each compound, with compounds showing a high rank in either of the profiles considered as candidates, see Supplementary Table In general, transcription-based repositioning results in tens of candidates out of a total of just over a thousand drugs constituting CMAP53, see ‘Materials and methods’. The top 1000 genes in the iPSC rank profile consists of 959 upregulated and 41 downregulated genes and this served as a query in the SPIED search. It is perhaps worth pointing out here that the level of gene expression unique to a given cell type will tend to be elevated relative to a background consisting of a variety of tissue types. An analogy would be in the context of division of labour one is characterised by what one does not by what one does not do. The top SPIED hits show a high correlation with human brain-derived samples, validating the cell’s lineage, see Supplementary Table As a first step in establishing the phenotype of the model cell system, the overall iPSC transcriptional profile was queried against a database of publicly deposited gene expression profiles via SPIEDZ score threshold of |Z| > 3, see ‘Materials and methods’ for details, capture most of the compound-associated changes. The enrichment is that of the rank of a given iPSC compound score with itself in CMAP. The enrichment plot is shown in Fig. p = 5.1E−6.The extent to which an iPSC profile correlates with its CMAP equivalent can be assessed by querying the CMAP database with the iPSC profile and ranking the CMAP equivalent. The extensively studied perturbagen rapamycin served as a positive control and eight independent profiles were generated to assess the degree to which these profiles are consistent with each other and with the rapamycin profile in CMAP. The rapamycin profiles had consistently high overlaps among themselves, but less so with the CMAP profile, with only one returning rapamycin as a top hit, rank seven, in a CMAP query, see Supplementary Fig. Z scores are: 2.43 for ADC pairs and 0.77 for all other pairs. It is therefore of interest to see to what extent the ADC set regulate a common set of transcripts. In Fig. 56, with growing evidence that gene variation affecting mitochondrial function may play a role in AD58. The downregulated set appears to be less consistent. Nonetheless, the enrichment of immune-associated pathways points to a possible anti-inflammatory activity of the candidate drugs. Interestingly, the following drugs have been reported to have neuroprotective activity: fluocinonide59, kawain63, allantoin64, dipyridamole67, estriol68, levamisole69, mycophenolic acid70, neostigmine71, probenecid73, chlorpromazine74, and phenoxybenzamine75, and xamoterol has been reported to ameliorate neuroinflammation and pathology in 5xFAD mice76 and shown to enhance cognition in a Down syndrome mouse model77. The atypical antipsychotic risperidone prescribed to manage psychosis in AD has demonstrated neuroprotection in animal models of ischemia78. Furthermore, cholinesterase inhibition is a therapeutic strategy for AD79 and there are two such inhibitors in the candidate list with galantamine as an established AD therapeutic80, while neostigmine exhibits poor blood–brain barrier penetrance and is therefore not in clinical use for AD. There does not appear to be any gene expression signature distinguishing compounds with reported neuroprotective activities from the other ADC compounds. This is to be expected as not all compounds have been assayed for neuroprotection and biological activity is not expected to be solely encoded in the transcriptome.Further to assessing the extent to which compounds orchestrate similar expression changes in the cancer cell lines and differentiated cortical neurons, it is critical to test whether the drugs also act in an anti-AD manner in the neuronal context. To this end, the drug profiles were scored against five representative AD reprofiles derived from the AD sets defined above, see ‘Materials and methods’ for details. Table www.norpd.no), showing that salbutamol use reduced PD risk81. A middle ground between target-based and epidemiological approaches is a methodology based on the disease phenotype gleaned from gene expression changes observed in pathological states. Underlying this approach is the observation that disease states can effectively be represented by characteristic expression changes, in the sense that these changes are consistent and can function as high content quantitative biomarkers. One avenue available to drug repositioning is to use these transcriptional phenotypes together with the hypothesis that an anti-correlation in phenotypes is indicative of the therapeutic potential of the compound. Whereas the transcriptional landscape of neurodegeneration and AD in particular has been well characterised, the corresponding data for compounds are either limited to full profiles defined on non-neuronal proliferating cells or partial profiles on iPSC-derived neuronal cells. The basis of the present study is to go some way to fill this gap in the compound-associated transcriptome with an emphasis on drugs with an anti-AD potential.Neurodegenerative diseases present a therapeutic challenge due to the difficulty in establishing a clear protein or mechanistic culprit for classic target-based intervention. Another hurdle is a consequence of the temporal extent of disease progression and the probable need to treat before overt symptom onset. This is a particular problem in designing clinical trials. With this in mind, alternatives to target-based approaches are increasingly being pursued. One recent report compared Parkinson’s disease (PD) incidence and chronic therapeutic use data from the Norwegian Prescription Database (82. This approach would have the advantage of including non-neuronal factors contributing to AD pathology such as inflammation. However, practical considerations limit whole-animal approaches to smaller drug sets and will therefore form part of a subsequent endeavour based on a more limited set of drug candidates selected based on the iPSC data.In the context of defining the neurotherapeutic potential of candidate drugs, a further development would be to treat wild-type or mutant AD mice with the compounds and measure expression changes in the brain, along the lines of the DrugMatrix projectIn the present work, we have established an AD transcriptional profile landscape and shown this to have a high degree of internal consistency. This disease-associated transcriptional landscape served as the basis for selecting a series of candidate drugs from the CMAP database of cancer cell line profiles, which were then assayed for their transcriptional effect on iPSC-derived cortical neurons. The iPSC profiles show a degree of overlap with the corresponding CMAP profiles, with a highly significant overall comparison in terms of the ranks observed for iPSC queries of CMAP. Out of the 153 iPSC drug profiles, 51, termed the ADC set, showed a high degree of anti-correlation with transcriptional changes seen in AD. A pathway enrichment analysis performed on each of the ADC set showed that pathways related to mitochondrial function were commonly upregulated while commonly downregulated pathways represented immune-associated pathways. Interestingly, these pathological features are found in multiple neurodegenerative disorders, such as PD and Huntington’s disease, and it would be of interest to investigate whether these compounds may have wider therapeutic potential. Notably, 18 of the ADC drugs already have established neuroprotective ability in published studies. Whereas we expect that initial CMAP filtering against AD profiles has led to increased likelihood of discovering compounds that tend to reverse AD-associated expression changes in the context of iPSC cultures, this can only be rigorously assessed by generating iPSC profiles for a series of compounds randomly selected from the CMAP database, which is outside the scope of the present study. In conclusion, approaches to identifying a broader range of candidate therapies for AD are urgently needed. It is therefore expected that the iPSC database will serve as a useful platform for drug repositioning across multiple neuropathological disorders as well as AD.Supplementary Figure LegendsSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Figure 1Supplementary Figure 2Supplementary Table 6


Major biological processes rely on the spatial organization of cells in complex, highly orchestrated three-dimensional (3D)tissues. Until the recent decade, most of information on spatial neural representation primarily came from microscopic imagingof “2D” (5-50 μm) tissue using traditional immunohistochemical techniques. However, serially sectioned and imaged tissuesections for tissue visualization can lead to unique non-linear deformations, which dramatically hinders scientists’ insight intothe structural organization of intact organs. An emerging technique known as CLARITY renders large-scale biological tissuestransparent for 3D phenotype mapping and thereby, greatly facilitates structure-function relationships analyses. Since then,numerous modifications and improvements have been reported to push the boundaries of knowledge on tissue clearingtechniques in research on assembled biological systems. This review aims to outline our current knowledge on next-generationprotocols of fast free-of-acrylamide clearing tissue (FACT) and passive CLARITY (PACT). The most important question is whatmethod we should select for tissue clearing, FACT or PACT. This review also highlights how FACT differs from PACT onspanning multiple dimensions of the workflow. We systematically compared a number of factors including hydrogel formation,clearing solution, and clearing temperatures between free-acrylamide and acrylamide-based passive sodium dodecyl sulfate(SDS) tissue clearing and discussed negative effects of polyacrylamide on clearing, staining, and imaging in detail. Suchinformation may help to gain a perspective for interrogating neural circuits spatial interactions between molecules and cellsand provide guidance for developing novel tissue clearing strategies to probe deeply into intact organ.  Biological systems are capable of forming complex neuronal network of feedforward, feedback and horizontal circuits , the mos2 transparent for deep microscopic imaging  are among the first to make fixed tissues as large as 2 cm imaging , compare imaging , dibenzy imaging , 3D imag imaging , 12, see imaging , ClearT imaging , clear u imaging , 16, sys imaging , and ul imaging . HoweverA cutting-edge technique (termed CLARITY) developed by Chung and Deisseroth , provideThree clear contributions have been made via these techniques: stabilizing tissue structures using hydrogel embedding , use of In this review, we summarize some important findings related to the FACT and the PACT for whole tissue imaging, with systematically comparing differences on recent published protocols between free-acrylamide method and PACT method for whole tissue imaging, including gel formation, clearing solution concentrations, clearing temperature, and negative effect of polyacrylamide. Before, it should be noted that this article discusses most of publishedresearch and review articles comparing or introducingacrylamide-based methods together or with other methodsof clearing. It means the newly developed method, the FACT, has not been compared with acrylamide-based clearing methods. Based on comprehensive comparisons between the two tissue clearing techniques, FACT and PACT, we propose that FACT method performs better in the whole clearing processing, which may give novelinsights into mapping the architecture of neural circuitsand developing new approaches for tissue visualization. CLARITY technique has gained considerable success owing to its potential for mapping detailed structural and molecular information of intact biological systems and is supported by extensive research data, the applications of hydrogel embedding and electrophoretic tissue clearing (ETC) are at the very heart of the CLARITY clearing procedures associated with tissue preservation and clearing efficiency, both of these two techniques have had broad impact on the rate of tissue clearing, and had been incorporated into the design, or redesign, continually. Although active transport organ-electrophoresis approach which capitalizes on the highly charged nature of ionic micelles, can accelerate the lipid extraction by orders of magnitude, the payoffs of ETC are inevitable tissue degradation during sample heating and complexity to implement. Extending their prior work, Chung et al.  developeNevertheless, the main limitation of the slow rate of clearing makes the protocol impractical for scaling up, not to mention intact biological body mapping. On the basis of the passive CLARITY method, the concept of the PACT was first scientifically described by Yang et al. , when thRecently, an exciting new wave of improvements avoids incomplete tissue hydrogel hybridization and fine has emerged from free-of-acrylamide SDS-based cytostructure destruction in this protocol. However, the tissue clearing (FASTClear), which greatly reduces most prominent disadvantages of FASTClear is being the whole clearing time. Most notably, the replacement limited by immunolabelling to the full thickness of tissue. of acrylamide hydrogel by formaldehyde largely Moreover, problems of tissue shrinkage and archival formalin-fixed tissues clearing still exist. To challenge the above imperfections in 3D tissue imaging, simultaneously with FASTClear, the FACT  was intrIncrease of our knowledge on gel formation that preserves proteins of cytoarchitecture but may conversely influence tissue expansion by clear solution, depth of antibody or light penetration in tissue, and speed of lipid removal is crucial. The hydrogel-embedding clearing protocols, including active CLARITY, passive CLARITY, PACT and so on, were developed based on polymerizing the fixed tissue into an acrylamide hydrogel prior to the process of lipid-removal. This hypothesis of hydrogel embedding provides a beautiful and simple account for how the use of hydrogel may provide physical framework for the cleared tissues and act as an impediment allowing for deep penetration of the antibodies into the tissue . It steInterestingly, a more careful examination of different polyacrylamide concentrations in modified CLARITY protocol, found that the retention of RNA and penetration of antibodies improved with reducing acrylamide hydrogel composition from 4 to 1% . FurtherSimultaneous with the FASTClear, the FACT was developed  for furtOne plausible explanation for PFA connection with peptides which is consistent with most of these data, is that aldehyde plays a crucial role in reaction with nitrogen or other atoms within proteins and peptides and therefore, creates a methylene bridge made of a -CH2- cross link . The maThe optimal methods for 3D tissue mapping dependon a set of processing factors that include selection ofappropriate ingredients, optimization of pH, suitable incubation temperatures, thickness of sample, and perhaps also effort-based manipulation proficiency. For lipid removal, the clear solution composition at certain pH and solution concentration, could contribute to thespeed of diffusion of detergent micelles and the rate oflipid solvation by detergent micelles in tissues . Each oEarly recordings revealed that the presence of free radicals which is associated with pH value of solution used in the tissue clearing process, closely affect the clearing speed , 57-59, Recent studies have also revealed that temperature is also one of the most sensitive factors during clearing incubation process to preserve the fluorescent signal. Exposure to high temperatures can accelerate delipidation of tissue; however, 50°C adopted in FASTClear method, resultiThe limitations of hydrogel crosslinking have substantial effects on the clearing process, including clearing speed, protein preservation, tissue character, which are thought to contribute to the index of clearing quality. The slow clearing speed using polyacrylamide has been brought under the spotlight. Theoretical basis of the FASTClear postulatPreservation of structural integrity is the most prominent factor of 3D tissue imaging, which is thought to provide an intuitive and detailed picture of how neural circuits and other connectomics work. Polyacrylamide was once considered to chemically incorporate native biomolecules into the hydrogel mesh, however, investigation of the necessity of hydrogel involvement has just started. Although, analysis based on daily measurement of tissue weight (served as an indicator of tissue and water absorption) and tissue size (served as an indicator of tissue size change) area revealed increased daily protein loss by removal of hydrogel from brain fixation comparing with the PACT and passive Clarity approaches. Intriguingly, the total protein losses showed no significant differences among hydrogel-based and FACT groups . This dFurthermore, the field has begun to appreciate that acrylamide-embedded tissues during SDS clearing undergo expansion and become more fragile because of loss of structural integrity , 36. ConTherefore, PFA-based tissue clearing protocols can achieve a rapid clearing without influencing total protein loss and tissue area under specific conditions, comparing with hydrogel-based protocols. However, given that some degree of protein loss is inevitable in all protocols, and given the potential of more or less deformation of samples during lipid removing process occur no matter it is in PACT or FACT approaches. More effort should be made to further preserve more tissue structure details upon tissue transparency.Avariety of unfavorable factors of polyacrylamide, such as specific antibody, non-specific staining, low antibody penetration, and being time consuming on staining, made it heavily implicated. According to hydrogel-based hypothesis, pores of polyacrylamide matrices assist lipid exchange and therefore enhance antibody penetration by changing hydrogel composition . This hyPolyacrylamide hydrogel application in imaging are also subjected to modifications such as alteration of depth of penetration and light scattering, to increase background during imaging. Tissues are opaque for conventional light microscopy, mainly owing to their lipids, induce light scattering , 19 whicAs mentioned above, hydrogel polymerization can be considered for the expansion and fragility of tissue during the imaging procedure. Deformation of tissue disrupts the evaluation of fine structures such as neuronal processes and microglia. In addition, lesions to tissue swelling is irreversible, a major adverse event for 3D tissue imaging quality, which RI cannot be corrected by using FocusClear. Abnormalities in tissue processing caused by polyacrylamide, heavily influence fluorescent signal intensity, antibody penetration depth as well as light scattering. In contrast, the absence of hydrogel in the FACT method circumvents these limitations and optimizes the preservation of antigenicity . MoreoveAided by merging and adjusting the FASTClear and PACT methods, Xu et al.  effectivWe have witnessed tremendous progress in refining clearing methods introduced for identifying complex cytoarchitecture and mapping neural circuitry, involved in simplified protocols, higher quality and speed of data generation, safety and economical materials and reagents application. Notably, the PACT and the FACT methods have recently attracted considerable attention as they could optimize the clearing time, temperature, and reagents and preserve the fluorochrome signal. Notably, the FACT technique which has been developed by merging and modifying the FASTClear and PACT methods, includingavoiding hydrogel perfusion and embedding, decreasingthe temperature to 37°C, adjusting the clear solution pH to 7.5, has been identified with reinforcing properties compared with PACT for mapping detailed structures suchas neuronal processes and branches. As FACT facilitates thewhole workflow of tissue clearing including clearing timesaving, fluorescent signals preservation, cytoarchitectural retention, confocal microscopy optimization, data collectionacceleration, the distribution of such a 3D tissue map provides a more intuitive brain tissue picture of how connections of large populations of cells and their networks and howphysiological and pathological conditions lead to changesin expression of corresponding proteins and molecules. The FACT protocol suggests optimized conditions of rapid high-resolution imaging for brain tissue, which has made a big step over the PACTprotocol. In the following, we highlight several frontiers which should be addressed by further investigations to deepen our understanding of the FACT.To test the validity and versatility of the FACT in 3D tissue imaging, a compelling set of experiments with tissue-type specificity and temporal precision recordings, remains warranted. Recently, a series of other tissue clearing methods has strongly established the visualization efficiency of various tissues including the spinal cord , whole eAlthough some articles have been recently done using FACT protocol , 69; butA potential problem of using scanning microscopes is that low scanning speeds make them impractical for imaging large fields of samples, resulting in light falloff, lens distortions, potential micro movements of the tissues inside the chamber . The molIt would be an effective and reliable tool tosimultaneously map subcellular molecular architecturebetween neighboring or distant cells of various organs, such as the brain and stomach, using multiple fluorophore signal in the same biological samples to examine how feedback loops and neuronal circuits works among central and peripheral system. Moreover, based on molecular-level and projection-based neural circuit tracing, it would be especially useful to comprehensively explorepathological and physiological conditions by comparing the functionality of protein and cell population. Theadvantages of the FACT protocol, such as maximal preservation of fluorophore signal hold promise for classifying and sub-classifying cytoarctitectures, and for molecular localization and projection in other projects.


For illustration purposes, the previous travel time probability density (BTTPD) approach is extended to assess the 3-d deep groundwater susceptibility to non-point source contamination within a sequence stratigraphic framework observed in the Kings River fluvial fan (KRFF) aquifer. The BTTPD, which represents complete age distributions underlying a single groundwater sample in a regional-scale aquifer, is used as a quantitative, transient measure of aquifer susceptibility. The resultant 3-d imaging of susceptibility using the simulated BTTPDs in KRFF reveals the strong influence of regional-scale heterogeneity on susceptibility. The regional-scale incised-valley fill deposits increase the susceptibility of aquifers by enhancing rapid downward solute movement and displaying relatively narrow and young age distributions. In contrast, the regional-scale sequence-boundary paleosols within the open-fan deposits “protect” deep aquifers by slowing downward solute movement and displaying a relatively broad and old age distribution. Further comparison of the simulated susceptibility index maps to known contaminant distributions shows that these maps are generally consistent with the high concentration and quick evolution of 1,2-dibromo-3-chloropropane (DBCP) in groundwater around the incised-valley fill since the 1970s’. This application demonstrates that the BTTPDs can be used as quantitative and transient measures of deep aquifer susceptibility to non-point source contamination.Groundwater susceptibility to non-point source contamination is typically quantified by stable indexes, while groundwater quality evolution  can be a long-term process that may last for decades and exhibit strong temporal variations. This study proposes a three-dimensional (3- The ultimate goal in most groundwater protection studies is to ascertain groundwater vulnerability, which refers to the likelihood that an aquifer will become contaminated as a function of the contaminant type, source type and distribution, and the system hydrogeology ,2. CurreDepth to water table, Recharge, Aquifer, Soil, Topography, Impact of the vadose zone, and hydraulic Conductivity. The DRASTIC index motivated many other indicators to map aquifer vulnerability in the last three decades. For example, the SINTACS index modified the DRASTIC index by adding options for the weight rating of each parameter embedded in DRASTIC [Concentration, Overlying layers, and Precipitation [First, the subjective rating (or index) method assigns a numerical score or rating to each (subjective) attribute affecting groundwater vulnerability, without the computational burden of statistically analyzing or numerical modeling of contaminant transport dynamics . This ap DRASTIC . The aqu DRASTIC . The vul DRASTIC . The COPpitation ,23. The pitation .Second, the statistical method uses descriptive statistics or complex regression to analyze contaminant concentrations and spatial distribution, or link observed contamination and environmental factors ,26. For Third, the process-based method simulates the process of groundwater flow and contaminant transport at the study site, and hence the advantage of this method is the detailed information on the spatiotemporal dynamics of contaminant migration and comprehensive information of groundwater vulnerability. Examples of the process-based method can be found in the recent review of Katyal et al. .As a specific process-based method, the backward travel time probability density (BTTPD) approach was proposed by Fogg et al.  to addreAlthough tremendous efforts have been made to assess groundwater vulnerability, there are two historical challenges. The first challenge is that efforts are needed to assess deep aquifer vulnerability for the following two reasons. First, the indices produced by the rating and statistical methods may only reflect potential of contaminant loading to the water table and do not represent vulnerability of deeper aquifers, especially in highly heterogeneous settings . The worThe second challenge is that previous studies usually quantified the groundwater vulnerability as a static value, while groundwater quality evolution is a long-term process. A transient index may describe better and provide more information about the vulnerability of real-world aquifers.d), deep aquifers. To check the feasibility of the transient, 3-d index map, we will apply it to assess the vulnerability of Kings River fluvial fan (KRFF) aquifer, located southeast of Fresno, California, USA  [vulnerability, by adjusting the initial/boundary conditions in flow/transport models and/or adding reactive terms in transport dynamics. Clearly, susceptibility analysis, or the intrinsic capability for contaminants to intrude on an aquifer system, is a prerequisite for reliable vulnerability analysis.Different from previous vulnerability assessment studies, here we address the less restrictive ristics) . Thus, cFogg et al.  applied X at time t originates from location Y at time s (s is a time before t), Θ is the effective porosity, D is the local hydrodynamic dispersion tensor. Note that here the dispersion tensor has the same value but different meanings in the backward and forward ADEs. In the forward-in-time ADE, D accounts for the uncertainty in concentration caused by molecular diffusion and differential advections; whereas in the backward-in-time ADE, D accounts for the uncertainty in the initial location and travel time of the particle as it moves backward [The BTTPD obeys the Chapman-Kolmogorov backward differential equation , which hbackward . The abobackward ,37,41. Award ADE ,38,43, wd continuous imaging of aquifer susceptibility instead of Fogg et al.’s [d susceptibility image can provide whole-system mapping of aquifer susceptibility. It may also help us to distinguish the responses of different subsurface materials, including aquifers and aquitards, to contaminations. Aquitard susceptibility is considered in this study since aquitard materials can form significant sources/sinks of contaminants found in aquifers. Second, the susceptibility of a single aquifer system is treated as a time-dependent transient variable, not the static index calculated by Fogg et al. [d susceptibility maps to evaluate the influence of subsurface heterogeneity on aquifer susceptibility. This evaluation has not been done by Fogg et al. [Three logic extensions of Fogg et al.’s  approachet al.’s  discreteg et al. . A transg et al.  due to td susceptibility map for a million-node model in a PC. We circumvent this problem by distinguishing particles based on their releasing locations during one simulation of particle tracking. In other words, particles with labels are simultaneously released from each node and are then back tracked to obtain the BTTPDs  during one simulation. Second, if the node is close to the model boundaries, many particles can exit the model through the lateral or bottom boundaries. The groundwater age refers to the elapsed time since the water enters into the groundwater system through the water table. Therefore, the age is unknown for particles exiting boundaries other than the top boundary  in backward particle tracking experiments. In this study, we adjust the ages for these particles based on the depths at which they exit the model as was also proposed by Weissmann et al. [The particle-tracking algorithm used by Fogg et al.  needed tn et al. . Note thThe backward-in-time method is much more computationally efficient than forward methods, i.e., see . The sold advective flow field and dispersion, each particle history produces a different travel time, therefore generating full distributions of groundwater age within the modeled groundwater “sample”. As will be shown later in this paper, such estimates of complete age distributions are useful and necessary not only for estimating susceptibility, but also for analyzing long-term evolution of groundwater quality as well as the transient susceptibility. The entire age distribution within single water sample generated by the particle tracking method distinguishes this study from other age simulation methods, such as Goode [In addition, because each particle takes on a different path due to the 3-as Goode , where o2 area of the Kings River fluvial fan aquifer  . ChannelThe third structure type is a coarse-grained incised-valley fill (IVF) deposit identified across the study area ,57. ThisHydrofacies within each of the stratigraphic sequences were simulated using transition probability geostatistics, as described by Weissmann and Fogg  and WeisDefinition of sequence stratigraphy is important for fluvial fan characterizations . WeissmaK) distributions, we apply the U. S. Geological Survey software MODFLOW [d, steady-state flow field. The conductivities assigned to each hydrofacies were 1.0 × 10−2, 1.0 × 10−3, 1.0 × 10−5, 1.0 × 10−6, 1.3 × 10−7 m/s for gravel, sand, muddy sand, mud, and paleosol, respectively [K of overlying beds for each cell at the water table [Using the geostatistical realizations to generate stochastic hydraulic conductivity  and the effective porosity (0.33). The single effective porosity was used for all hydrofacies (assigned with different K), since the spatial variation of porosity is relatively smaller than that for K, and the impact of the spatial variation of porosity on solute transport is secondary compared with the impact of K on transport [X × Y × Z) m. This assumption is based on (1) the conclusion of LaBolle and Fogg [The modeled flow velocities were incorporated into the particle tracking algorithm to simulate the groundwater BTTPD. The same transport parameters in Weissmann et al.  were useransport . The totand Fogg , who fouand Fogg , who foud susceptibilities are shown by the following five slices across the model domains: three horizontal slices at 5, 20, and 40 m below and parallel to the water table, respectively; one vertical slice parallel to the general groundwater flow direction and through (inside and below) the IVF; and another vertical slice parallel to the general groundwater flow direction and outside the IVF (X = 7100 m), where the high-K IVF truncates most parts of the uppermost paleosol. The vertical slice outside the IVF (X = 3600 m) contains all of the three layers of paleosols, with the uppermost paleosol lying close to the water table.The simulated 3- the IVF a. The thK hydrofacies since young water can also be found in some low-K zones or aquitards. Most of the discrete regions representing old waters, however, generally correspond to the location of aquitards. At a few small, separate high-K zones surrounded by low-K materials we observe very old water. Second, the IVF appears to significantly influence groundwater age and susceptibility. Inside or underlying the IVF, the system is dominated by relatively young water along the whole valley, independent of the corresponding hydrofacies type. This is especially true for saturated zones 20 and 40 m below the water table, where the shape of valley can be seen clearly from the correspondent susceptibility maps. Outside of the valley, the areas with relatively young water are not well connected horizontally, but intermingled with areas representing very old water.The susceptibilities along the three horizons  provide The susceptibility along the two vertical slices basically shows different behaviors from each other . In the To further explore the influence of heterogeneity on aquifer susceptibility, we compared the complete BTTPD along two vertical columns inside of the two vertical slices  through the IVF, the simulated BTTPD has three main characteristics  outside the IVF , which is different from the classical index  method [Although the influence of local heterogeneity on solute transport has been well-known for decades ,63,64, t) method .The regional-scale IVF significantly increases the susceptibility of aquifers by enhancing rapid downward solute movement. This is because of the ~80% of sandy-materials deposited in this valley. These high permeable materials incise through the uppermost paleosol layer, and pull in higher recharge than other areas in the model domain ,65. TherFor areas inside and below the IVF, the mean groundwater age may somewhat reflect the aquifer susceptibility, because the mean is fairly close to the mode and also the age distributions are relatively narrow. In addition, the relatively uniform susceptibility within/below the IVF at a same depth is caused by relatively homogeneity of the incised valley fill deposits and lack of extensive aquitard units within it. For aquifers inside and below the IVF, groundwater age usually increases with depth gradually. Hence, the regional-scale IVF has a dominant effect on aquifer susceptibility, overwhelming the influence of local-scale heterogeneity discussed above.2 of DBCP contaminated groundwater in the California Central Valley in 1986 [The measured concentration of pesticides is used here to test the susceptibility maps, and speculate on the historic role of the IVF on contaminant transport in KRFF aquifer system. 1,2-dibromo-3-chloropropane (DBCP) was a widely used nematicide during the 1950s through the 1970s, with the most frequent use between 1960 and 1979 [ in 1986 ). It was in 1986 ,67, muchThe regional-scale boundary paleosols within the open-fan deposits affect the BTTPD in two ways: (1) the relatively low permeability of paleosols retards the vertical transport of particles; and (2) the paleosols change the age distribution at a well by bringing more particles from deep zones, where the water age is generally old. This indicates that the overall sequence stratigraphic framework including paleosols decreases the deep aquifer susceptibility to contamination by altering pathways and delaying the vertical transport of contaminants. Therefore, the sequence boundary paleosols have the almost opposite effect on aquifer susceptibility as the IVF does.The BTTPDs for aquifers affected by the boundary paleosols contain heavier late-time tails than those affected by the IVF, and thus we cannot apply the BTTPD the same way as discussed in the previous section. Especially, for the open fan deposits bounded by paleosols, the mean groundwater-age-based susceptibility estimate will be misleading because the mean groundwater age is significantly influenced by the long late-time tail, missing the peak and the main part of a broad age distribution.One could argue that the simulated BTTPDs discussed above based on the single stochastic realization rather than on multiple realizations may not represent the full range of outcomes for transport and susceptibility in this complex aquifer system. Different aquifer realizations may have different distribution properties for both high- and low-permeable materials, resulting in different early-time tail, peak, and late-time tail in BTTPDs. Therefore, the details of BTTPD discussed above, such as the number of peaks and the actual spread length, may vary apparently between multiple aquifer realizations.However, as indicated by the numerical experiments explored by Weissmann et al. , the genGroundwater vulnerability was usually evaluated by a static index without information on long-term water quality evolution and deep-aquifer response to non-point source contamination. To address this historical issue, this study develops three-dimensional transient maps built upon reliable physical models , by extending the backward travel time probability density approach proposed by Fogg et al.  and LaBoFirst, the BTTPD can be used to derive transient, multi-dimensional indexes of aquifer susceptibility to non-point source contamination, which provides much more useful information than the classical, static index. The BTTPD can, if interpreted appropriately, provide the complete information to decipher the long-term evolution of groundwater quality affected by multi-scale groundwater circulation driven by aquifer/aquitard heterogeneity. Particularly, the cumulative BTTPDs with target time intervals can determine likelihood that an aquifer will be contaminated as a function of space and time.Second, the regional-scale incised-valley fill deposits increase the susceptibility of aquifers by enhancing rapid downward solute movement and displaying relatively narrow and young age distributions, due to the coarse-grained nature of the IVF and the incision of the uppermost paleosol layer. The historical record of non-point source pollutants  also implied the relatively more rapid downward movement of pollutants, although the data is not adequate to show the detailed evolution of pesticides across the large area of the study site.Third, the sequence boundary paleosols postpone the contamination of aquifers at all depths by retarding the vertical movement of contaminants and changing the proportions of particle plumes. Therefore, the existence of paleosol boundary decreases the susceptibility of saturated zones to recent contaminants with the price of an increased and a longer-term susceptibility to old contamination.