Rhabdomyolysis is a potentially life-threatening disease caused by melting or necrosis of skeletal muscle cells and leakage of muscle components into the bloodstream. It has been reported that the interaction of the HMG-CoA reductase inhibitor rosuvastatin with the renal anemia drug vadadustat increases the blood concentration of rosuvastatin in vitro. In this study, we report a case of suspected rhabdomyolysis caused by the drug interaction of rosuvastatin and vadadustat in clinical practice.A 62-year-old male with medical records of hypertension, myocardial infarction, chronic renal failure, renal anemia, dyslipidemia, and alcoholic liver disease. The patient had been diagnosed with chronic kidney disease (CKD) at the Department of Nephrology, and treated by outpatient care with renal support therapy for the past two years. On X-63 day, his prescription was rosuvastatin (10 mg/day) and a continuous erythrocyte-stimulating agent, epoetin beta pegol . X-Day 0, blood tests revealed creatine phosphokinase (CPK) 298 U/L, serum creatinine (SCr) 5.26 mg/dL, and hemoglobin (Hb) 9.5 g/dL; thus, the prescription was changed from epoetin beta pegol 100 μg to vadadustat 300 mg/day. On X + day 80, a prescription for a diuretic (azosemide 15 mg/day) was added for swelling of the lower extremities. On X + day 105, we found CPK 16,509 U/L, SCr 6.51 mg/dL, and Hb 9.5 g/dL. The patient was diagnosed as rhabdomyolysis and hospitalized. After hospitalization, rosuvastatin and vadadustat were discontinued and we administered intravenous fluids. Thereafter, CPK and SCr values of the patient improved. On X + day 122, CPK improved to 29 U/L, SCr to 2.6 mg/dL, and Hb to 9.6 g/dL, and he was discharged on X + day 124. At discharge, rosuvastatin 2.5 mg/day was resumed. A blood test on X + day 133 showed CPK 144 U/L and SCr 4.2 mg/dL.We experienced a case of rhabdomyolysis caused by drug interactions between rosuvastatin and vadadustat. Rhabdomyolysis is a potentially fatal disease caused by melting or necrosis of skeletal muscle cells and the leakage of muscle components into the bloodstream.Previous studies reported that the drug interaction of rosuvastatin , an HMG-A 62-year-old male with medical records of hypertension, myocardial infarction, chronic renal failure, renal anemia, dyslipidemia, and alcoholic liver disease. The patient had been diagnosed with CKD at the Department of Nephrology, and treated by outpatient care with renal support therapy for the past two years. On X-63 day, his prescription was rosuvastatin (10 mg/day) and a continuous erythrocyte-stimulating agent, epoetin beta pegol  (geneticOn X + day 105 at his regular clinic visit, blood tests showed CPK 16,509 U/L, SCr 6.51 mg/dL, Hb 9.5 g/dL, HDL-c 33 mg/dL, and LDL-c 16 mg/dL. The patient was diagnosed with rhabdomyolysis and hospitalized. On admission, the pharmacist suggested to the doctor that the discontinuation of vadadustat and rosuvastatin to the physician as a possible cause of the elevated CPK levels due to drug interactions. As a result, both drugs were discontinued and the patient was administered intravenous fluids. Thereafter, CPK and SCr values improved. The pharmacist suggested to the doctor epoetin beta pegol, which had been previously administered for renal anemia, since the CPK level had decreased. Furthermore, the doctor asked the pharmacist to resume rosuvastatin because of ischemic heart disease. The pharmacist informed the doctor that statins alone have few side effects such as rhabdomyolysis [On X + day 122, CPK improved to 29 U/L, SCr 2.6 mg/dL, and Hb 9.6 g/dL, and he was discharged on X + day 124. At discharge, rosuvastatin 2.5 mg/day was resumed. A blood test on X + day 133 showed CPK 144 U/L SCr 4.22 mg/dL, Hb 8.9 g/dL, HDL-c 57 mg/dL, and LDL-c 64 mg/dL (Fig. Rhabdomyolysis is a potentially life-threatening condition caused by the melting and necrosis of skeletal muscle cells and the leakage of muscle components into the blood .The diagnostic criteria for rhabdomyolysis refer to a CPK level of at least five times the reference value, and symptoms include myalgia and myoglobinuria. Treatment includes discontinuation of the causative drugs, if any, and application of infusion fluids . CongeniIn this study, we experienced a case of rhabdomyolysis that was thought to be caused by the interaction between the statin rosuvastatin and the renal anemia drug vadadustat. Since statins have evidence for secondary prevention of ischemic heart disease, rosuvastatin was taken by this patient aiming for LDL-c < 70 mg/dL . And it Vadadustat taken alone has been reported to have no effect on cholesterol levels . When vaIn conclusion, the pharmacist was aware from the package insert of vadadustat and rosuvastatin as stating “concomitant use is cautioned because the Cmax and AUC of rosuvastatin may increase with concomitant use” , in othe


Matricaria chamomilla L., Asteraceae) ethanolic extract and β-CD/milk thistle  ethanolic extract complexes were obtained by the co-crystallization method with good recovering yields of 55–76%, slightly lower than for β-CD/silibinin or silymarin complexes (~87%). According to differential scanning calorimetry (DSC) and Karl Fischer water titration (KFT), the thermal stability of complexes is similar to β-CD hydrate while the hydration water content is lower, revealing the formation of molecular inclusion complexes. In the Korsmeyer–Peppas model, β-CD/M. chamomilla flower extract complexes reveal Case II transport mechanisms, while the corresponding complexes with leaf extracts indicate non-Fickian diffusion for the controlled release of antioxidants in ethanol 60 and 96%. The same non-Fickian diffusion was revealed by β-CD/S. marianum extract and β-CD/silibinin complexes. On the contrary, almost all model transdermal pharmaceutical formulations based on β-CD/M. chamomilla extract complexes and all those based on β-CD/S. marianum extract complexes revealed non-Fickian diffusion for the antioxidant release. These results indicate that H-bonding is mainly involved in the diffusion of antioxidants into a β-CD based matrix, while the controlled release of antioxidants in model formulations is mainly due to hydrophobic interactions. Results obtained in this study can be further used for studying the particular antioxidants  for their transdermal transport and biological effects in innovatively designed pharmaceutical formulations that can be obtained using “green” methods and materials.This is the first study on the modeling of the controlled release of the estimated antioxidants (flavonoids or flavonolignans) from β-cyclodextrin (β-CD)/hydrophilic vegetable extract complexes and the modeling of transdermal pharmaceutical formulations based on these complexes using an overall estimation by the spectrophotometric method. The Korsmeyer–Peppas model was chosen for evaluating the release mechanisms. β-CD/chamomile ( Matricaria chamomilla L. (chamomile) and Silybum marianum L. (milk thistle) are two medicinal plant species that belong to the same botanical family, Asteraceae. They are important species of medicinal and aromatic plants (MAPs). The area cultivated with MAPs exceeding 200,000 ha in Europe  assay [Chamomile is one of the most cultivated MAPs in the world, with more than 20,000 ha ,4,5. Cha0.8–0.9% . The mai 2.3–46% ,13,14,15traction ,11. The extracts . The maiSilybum marianum L. is widely used for its valuable hepatoprotective effect. This effect is mainly due to the presence of flavonolignans, which are polyphenols derived from flavonoids. A complex mixture of flavonolignans can be found in S. marianum extracts, including silibinins and silychristins. Silymarin is the standardized S. marianum fruit extract. It mainly contains the above-mentioned flavonolignans and up to 25% oil [S. marianum extract is important. Both flavonolignans and flavonoids, such as silibinins, taxifolin, and quercetin, provide antioxidant effects. Among them, chlorogenic acids and tocopherols also have an impact [S. marianum genotypes were in the range of 3086–9499 mg/kg for silibinins, 126.5–395.3 mg/kg for chlorogenic acids, and 3.5–79.7 mg/kg for luteolin, with TFC values of 30–84 mg RE/100 g [S. marianum extracts strongly depends on the plant’s development, growing conditions, and treatments, as well as the extraction procedure. The maximum antioxidant activity was observed in 80-day-old leaves and plants  [S. marianum varieties was determined by various methods. The radical scavenging activity was in the range of 1763–5303 μmol TE/100 g by the DPPH· assay, 2965–14426 μmol TE/100 g by the ORAC assay, and 692–1664 μmol TE/100 g by the FRAP assay [ 25% oil ,19. The  25% oil . The antn impact ,22,23. TRE/100 g . The totAP assay .M. chamomilla or S. marianum extracts [Compositae essential oils [Polyphenols are easily oxidized in proper conditions, especially if they are not appropriately stabilized. Flavonoids such as quercetin and chrysin can be transformed to free radicals and further to quinone methides by various mechanisms, including hydrogen atom transfer (HAT) or single-electron transfer (SET) mechanisms. Metal ions are also involved in the antioxidant mechanism of polyphenols ,27. The extracts ,43,44,45ial oils ,14,46.Matricaria chamomilla L.) or milk thistle (Silybum marianum L.), Asteraceae, hydrophillic extract complexes was performed. It mainly focuses on the applicative aspects regarding the β-CD complexation of the raw ethanolic extracts and the controlled release from the complexes and transdermal pharmaceutical formulations of the main antioxidant compounds  in various media.In this study, a comparison between the thermal stability and controlled release by spectrophotometric evaluation of β-CD/chamomile  and S. marianum (seeds) were obtained at final raw volumes of 27.0–30.5 and 26.5–30.0 mL, respectively  and (2) and M. chamomilla and S. marianum extracts, respectively. The liquid chromatographic technique was not used at this stage due to its main disadvantages regarding such a study, namely its time-consuming nature and possible degradation/hydrolysis during analysis by water/acidic based solvent mixtures.n = 6, r2 = 0.9999, p < 0.00001n = 5, r2 = 1.0000, p < 0.00001n = 6, r2 = 0.9996, p < 0.00001n = 9, r2 = 0.9999, p < 0.00001The extracts of vely see . The extM. chamomilla flowers was obtained using ethanol 96% as extraction solvent . On the other hand, roots and stems had lower flavonoid contents, as determined from extracts obtained with ethanol at 20–96% concentrations (1.14–2.75 mg/g FW as chrysin and 4.01–8.01 mg/g as rutin). Results obtained for the estimated flavonoid content  of M. chamomilla flowers, leaves, roots, and stems using ethanol of various concentrations (v/v) for extraction are presented in the M. chamomilla. They are especially focused on flowers, using liquid chromatographic techniques. Generally, flavonoid contents were determined in dry extracts, such as chamomile ligulate flower extract obtained by subcritical water extraction. Rutin was the main flavonoid glycoside compound identified in extracts at 10 bar pressure (0.23 mg/kg dry extract), while chrysin had the highest concentration in the dry extract obtained at a pressure of 30 bar (1.99 mg/kg). However, higher concentrations of flavonoid aglycones and glycosides were obtained at increased extraction pressure values  [O-glucoside (1101 mg/kg dry extract obtained at 85 °C) and its aglycone, luteolin (97.2 mg/kg at 115 °C and only 55.2 mg/kg at 85 °C) [M. chamomilla ligulate flowers during flowering. Apigenin 7-O-glucoside was determined in the range of 11.38–32.4 mg/g, but the corresponding 6″-malonyl, 6″-caffeoyl, and 4″-acetyl-6″-malonyl-glucosides were also identified [The overall estimation of the flavonoid contents (expressed as chrysin or rutin) revealed that they significantly differed between the flowers/leaves and roots/stems extracts of fresh samples. They were determined using Equations (1) and (2) and various extract dilutions (dilution levels of 0.005 to 0.08). The variation of the estimated flavonoid content  with the ethanol concentration differs for flowers and leaves in comparison with roots and stems a–d. The entified .S. marianum seeds was determined on the basis of the silibinin diastereomer mixture and silymarin mixture standard curves, using ethanol of various concentrations for extraction  and expressed as percent. Recovering yields for β-CD/M. chamomilla extract complexes were in a narrow range of 55.71–61.87%. On the other hand, these yields were significantly higher for β-CD/S. marianum extract complexes (75.72%), which were slightly lower than for the corresponding β-CD/standard silibinin diastereomer mixture and β-CD/silymarin mixture complexes . Quantities, volumes, and recovering yields for all synthesized complexes are presented in β-CD/vegetable extract complexes were obtained by the co-crystallization method using equal volumes of β-CD water solution and ethanolic extract. Only extracts obtained with ethanol 96% were used for β-CD complexation. Due to the complexity of antioxidant compounds in the vegetable extracts, the recovering yields of the β-CD/vegetable extract complexes were determined as the dried complex mass divided by the sum of β-CD hydrate mass and antioxidant mass . Two main regions were identified: (1) the temperature range of 20–150 °C, where the calorimetric effect appears, which corresponds to the dissociation of water and ethanol molecules from the β-CD complex; and (2) the temperature range of ~270–320 °C, where the decomposition of β-CD and encapsulated antioxidant compounds takes place. The β-CD/S. marianum seed extract complex, but with a significantly lower value for the temperature corresponding to water/ethanol molecule release. Moreover, there are two peak temperatures at 64.2 and 82.6 °C, which are due to the release of surface water and strongly retained water molecules [M. chamomilla flower and stem extract complexes . The maximum decomposition rate appears at a slightly lower value of 279 °C, while the transition effects were observed at 192.4 and 220.1 °C ). Both KFT water content/titration behavior and KFT–DSC correlations were in accordance with other studies related to CD/flavonoid, CD/vegetable extract, CD/fish oil, or CD/essential oil complexes [n = 6, r2 = 0.690, p < 0.05, s = 1.05The water content of β-CD complexes can be selectively determined using the volumetric Karl Fischer titration (KFT) . Thus, tomplexes ,55,56,59−1 [−1 [−1 [−1, respectively [−1, while the sesquiterpene β-caryophyllene has a significantly higher value of 23,032 M−1 [M. chamomilla extract complexes and flavonolignans (expressed as silibinin) from β-CD/S. marianum extract complexes was estimated by spectrophotometric analysis. The solvents used for the controlled release studies were ethanol at concentrations of 20, 60, and 96% for the crystalline β-CD/vegetable extract complexes, as well as saline solution  and the above-mentioned ethanol solutions for the studies on transdermal pharmaceutical formulations.β-CD forms stable molecular inclusion complexes with a wide range of compounds having various structural characteristics. Flavonoid aglycones such as quercetin, naringenin, hesperetin, kaempferide, and chrysin are complexed by β-CD with stability constants of 602/3345, 235, 355, 1541, and 574 M−1 ,61,62,63−1 [−1 ,61,64. T [−1 [−1 . Highly ectively ,66. On t,032 M−1 . ConsequM. chamomilla flower and leaf extract complexes was 32.5 μg/mL  in ethanol 96%. On the contrary, these values were 10.4 and 0.8 μg/mL if ethanol 60 and 20% were used. By comparison, the maximum flavonoid content of only 18.0 μg/mL was obtained by controlled release from β-CD/M. chamomilla root and stem extract complexes (S. marianum extract complexes. The estimated maximum silibinin content was obtained by controlled release in ethanol 96% (0.56 μg/mL), compared with ethanol 20 and 60% (<0.24 μg/mL). By comparison, standard silibinin was easily released from the β-CD/silibinin complexes   obtained for the controlled release from β-CD/omplexes . Similar 15 min) .n of 0.5 and 1 reveal Fickian diffusion and Case II transport (relaxation release), respectively. A qvasi-Fickian diffusion  appears if n < 0.5, while the non-Fickian  diffusion appears for n ∈ , which means that compounds are released by both diffusion and relaxation (erosion). Finally, the Super Case II transport is revealed by a diffusional exponent of n > 1 [The mathematical modeling of the controlled release of antioxidants from β-CD complexes and transdermal pharmaceutical formulations can provide information about the releasing mechanism a–d. The of n > 1 ,69.n ≅ 0.92) and super-Case II transport (n > 1) mechanisms were observed for controlled release of estimated antioxidants from β-CD/M. chamomilla flower extract complexes in ethanol 96% and ethanol 60%, respectively (r2 of 0.985 and 0.966). On the contrary, a non-Fickian diffusion (n ≅ 0.76) was observed for the cases of β-CD/M. chamomilla leaf extract complexes studied in ethanol 60 and 96% (r2 of 0.97–0.99). The release mechanisms of the estimated antioxidants from β-CD/M. chamomilla root and stem extract complexes were Case II transport for releasing in ethanol 60 and 96% , or in ethanol 60% for the case of stem extract-based complexes . The silibinin release mechanism from β-CD/S. marianum extract complexes was non-Fickian for all ethanol concentrations (n = 0.66–0.81 and r2 = 0.90–0.98), such as in the case of β-CD/silibinin complexes in ethanol 96% . A comparison between the Korsmeyer–Peppas model and experimental data is emphasized in M. chamomilla flower and leaf extract complexes, β-CD/S. marianum extract, and β-CD/silibinin complexes in ethanol 96%. All values for the kinetic constant, kKP). However, the diffusional exponent had values of 0.5–1 for all other β-CD/M. chamomilla extract complex-based formulations studied in ethanol 60 and 96%, revealing a non-Fickian diffusion for the antioxidant release  were collected from specific locations in the west of Romania. Thus, M. chamomilla L. plants were harvested during autumn seasons of the last years from the Salonta wild region . Flowers, leaves, roots, and stems were carefully separated and stored as fresh samples at −20°C until extractions. Only the seeds of S. marianum L. collected in the same period from the Macea region  were used. The main antioxidant compounds in extracts were chrysin, quercetin, and rutin hydrate. They were used as standard compounds from the flavonoid class and had purities of 97%, 98%, and 94%, respectively . On the other hand, standard compounds from the flavonolignan class, namely silibinin and silymarin, had contents of 98% and 64.7% silibinins, respectively . β-Cyclodextrin hydrate was purchased from CycloLab  and had >98% purity and a water content of 12.4% . Ethanol 96% (v/v) used for CD complexation and controlled release studies was obtained from ChemiCal® Co. .Plant materials  and S. marianum (seeds) were well grounded in a mortar. Approximately 5 g of every sample was immediately weighted into a 150 mL extraction flask. The samples were extracted with 70 mL of ethanol at various concentrations  for 1.5 h by continuous stirring on a water bath at 55–60 °C [Fresh samples of ® filter paper No 1  and washed with 1 mL ethanol 96%. After drying at room temperature, the complex crystals were weighted, sealed, and stored at 4 °C until further analysis.β-CD/vegetable extract complexes were obtained using the co-crystallization method. Approximately 0.5 mmoles of β-CD hydrate were dissolved in 4 mL water in a 20 mL crystallization flask, which was equipped with cooling-heating mantle, reflux condenser, and magnetic stirring system. The solution was heated to 50 °C, and then 4 mL of vegetable extract was added dropwise through the top of the condenser for 15 min. The solution became turbid during the controlled cooling to room temperature (cooling rate of ~7.5 °C/h) and the suspension of the complex was stored at 4 °C overnight. The crystallized complex was filtered through WhatmanThe controlled release behavior of β-CD/vegetable extract complexes was evaluated from the standard transdermal pharmaceutical formulations . The β-CD complex was incorporated into the transdermal cream samples by mixing for 1 h at a complex:cream ratio of 1:20. All determinations were performed against a blank sample (without complexes).M. chamomilla and S. marianum extracts, respectively) using the Wavelength Scan Measurement and/or Time Scan Measurement modules. The monitoring time was set at 30 min for controlled release from the crystalline β-CD/vegetable extract complexes in ethanol solutions of 20, 60, and 96% concentrations (v/v). The controlled release of the estimated bioactive compounds from the transdermal pharmaceutical formulations based on β-CD/vegetable extract complexes was studied for 90 min at the same wavelengths, using even ethanol  or saline solution  as solvent media. The acquisition and handling of the UV–Vis data were performed with the UV–Vis Analyst version 4.67 software from the same manufacturer. In all cases, the appropriate solvent mixture was used as the blank sample.Spectrophotometric analysis was performed for extracts and standard compound solutions at various concentrations, as well as for the evaluation of their release from β-CD/extract complexes and pharmaceutical formulations. This is a fast and non-destructive method for the evaluation/estimation of the overall release of biologically active compounds from nanoparticles and pharmaceutical formulations. A CamSpec 501 equipment  was used. The absorbance values of the samples were monitored at specific wavelengths  :F = Mt/MF) between the antioxidant  amount that is released at the moment t (s) and the total amount of antioxidant in the sample (t/M∞M). This ratio is correlated with kinetic constant kKP)( (expressed as s−n) and the time at the power of a diffusional exponent, nt. Parameters kKP). All values for these parameters, as well as those for the coefficient of determination, r2, can be found in the The model uses the ratio  in order to evaluate the calorimetric effects during heating. A DSC Netzsch 204 F1 Phoenix equipment  was used, with a temperature range of 20–400 °C and a heating rate of 4 °C/min. Nitrogen protection and purge flow were set at 20 mL/min. The sample masses were 4.3–8.0 mg for complexes and 9.8–10.0 mg for β-CD. DSC analysis was performed in open Al crucibles using a reference crucible of 39.4 mg. The Netzsch Proteus-Thermal Analysis ver. 6.1 software  was used for acquisition and handling of the DSC data.®  having a titer of 3.6357 ± 0.0195 mg/g at the analysis period. The titer was determined using the water standard 1% apura® . Component 2—Solvent apura®—was used as a working medium and was purchased from the same manufacturer . Sample masses were in the range of 20.0–40.0 mg. The following KFT parameters were used: I(pol) 50µA, end point 250 mV, maximum titration rate 5 mL/min, stop criteria—drift with a value of 15 µL/min. The extraction time was approximately 300 s. All determinations were performed in triplicate.The water content of complexes and β-CD hydrate was determined using the volumetric KFT method. A KF Titrando 701 apparatus having a Metrohm 10 dosing unit and coupled with a Ti Stand 703 mixing unit  was used. The bi-component KFT technique was applied, with the component 1—Titrant 5 apurar, the coefficient of determination, r2, p-values, F-test for the regression equation, and standard errors for both equations and coefficients have been considered.All values obtained for multiplicate determinations  were provided as mean ± standard deviation (SD). The Basic Statistics&Tables and One-way ANOVA modules in Statistica 7.1  software were used. The regression analysis was performed with the Multiple Linear Regression module from the same package. Pearson correlational coefficient, Matricaria chamomilla L.) and β-CD/milk thistle (Silybum marianum L.) extract complexes were synthesized and evaluated for their controlled release properties using a fast, cheap, and non-destructive method, namely spectrophotometric analysis. This is the first study on the controlled release of the estimated antioxidants from such complexes and pharmaceutical formulations containing the above-mentioned complexes. Extracts were obtained with ethanol solutions at various concentrations and moderate temperatures in order to reduce the level of degradation of labile antioxidant compounds. High estimates of antioxidant compounds  in extracts or raw vegetable samples were obtained if ethanol at concentrations of 80–96% was involved. These extracts were used for obtaining β-CD complexes using the co-crystallization method, which provides high recovering yields, especially for the β-CD/S. marianum extract complex. The thermal stability of all complexes is similar to that of β-CD hydrate, according to DSC analysis. However, the hydration water content  is much lower in complexes, which reveals the formation of molecular inclusion complexes.A series of β-CD/chamomile (v/v), which have logP values of −1.14, −0.74, and −0.37  [M. chamomilla flower extract complexes reveal Case II transport mechanisms, while the corresponding complexes with leaf extracts indicate non-Fickian diffusion for the controlled release of the estimated antioxidants in ethanol 60 and 96%. The same non-Fickian diffusion was revealed by β-CD/S. marianum extract and β-CD/silibinin complexes. On the contrary, the controlled release studies for the model transdermal pharmaceutical formulations based on β-CD/vegetable extract complexes that were performed in the same ethanol (20% to 96%) as well as in saline solution (0.9% NaCl) revealed completely different releasing mechanisms. Almost all formulations based on β-CD/M. chamomilla extract complexes and all those based on β-CD/S. marianum extract complexes revealed non-Fickian diffusion for the estimated antioxidant release . This is most likely due to the completely different hydrophilic/hydrophobic characteristics of matrices. Thus, β-CD complexes have more hydrophilic cyclic oligosaccharides and crystallization water as the main matrix components. Flavonoids and flavonolignans are mainly hydrophilic. As a consequence, H-bonding is especially involved in the diffusion of antioxidants into this type of matrix. For transdermal pharmaceutical formulations, fatty acid glycerides and sterol esters with long-chain fatty acids are the main components. Consequently, a competition between the hydrophobic interactions of the hydrophobic components of the matrix and the complexed antioxidants with the hydrophobic inner cavity of β-CD appears. In this case, the controlled release of antioxidants is mainly due to hydrophobic interactions. These differences have been proven in this study by the modeling of the diffusion of the estimated antioxidants into two types of matrices with different hydrophobicities. Results can be further used for studying the particular antioxidant transdermal transport and biological effects of innovatively designed pharmaceutical formulations that can be obtained using “green” methods and materials.The modeling of the controlled release of the estimated antioxidant compounds from β-CD complexes and transdermal pharmaceutical formulations based on these β-CD complexes was an important and innovative part of this study. Different ethanol solutions or saline solutions (releasing solvents with various hydrophobicities) were used for controlled release studies. On the other hand, both β-CD complexes (more hydrophilic) and transdermal pharmaceutical formulations based on β-CD complexes (more hydrophobic) were considered for controlled release studies. First, the release of the estimated antioxidants from β-CD complexes was performed in ethanol of 20, 60, and 96% (ponents) . The sel


Rubus alceifolius Poir (R.A. Poir) leaves are rich in phenolic compounds, offering many health benefits due to their incredible antioxidant potential. In this study, conditions for the ultrasound-assisted extraction (UAE) of phenolic compounds and antioxidant activity from R.A. Poir leaves were optimized using response surface methodology (RSM). This methodology assessed the effects of ultrasound power (X1: 100-500 W), extraction temperature (X2: 30-60°C), and extraction time (X3: 5-55 min). The optimized UAE conditions were then compared with conventional extraction methods (Soxhlet extraction: SE and maceration extraction: ME) for extracting total phenolics. A phenolic profile using GC-MS and antioxidant activity (ABTS) was also compared. According to the RSM, the best conditions for UAE to extract the highest total polyphenol content and ABTS radical scavenging activity were 320 W ultrasound power, 40°C extraction temperature, and 35.5 min sonication duration. Under these optimal conditions, the TPC and antioxidant activity reached 16.68 mg GAE/g dm and 21.9 mg TE/g, respectively, closely aligning with the predicted values. The UAE extraction technique proved to be more efficient in extracting phenolics and antioxidant capacity ) radical scavenging activity, and enzyme inhibition) compared to the conventional extraction methods (SE and ME). A GC-MS analysis identified 12 components, including 5 phenolics and 3 flavonoids, which likely contribute to the antioxidant activity. Consequently, the UAE method improved extraction efficiency within a shorter time frame, suggesting that UAE is a promising, efficient, and ecofriendly technology for extracting bioactive compounds from R.A. Poir leaves. Rubus alceifolius Poir (R.A. Poir) belongs to the Rubus L. (Rosaceae) family and is widely distributed in China, Vietnam, and Malaysia [Rubus alceifolius prefers to grow in humid and shady environments. The fruit of the R.A. Poir is a red or purple drupe, similar in appearance to a raspberry or blackberry, and has a sweet and tangy flavor [R.A. Poir are phenolics, flavonoids, triterpenes, and others [Malaysia . Rubus ay flavor , 2. Thesy flavor –3. Meanwd others . Recent d others , atherosd others . Additiod others . TherefoCelastrus hindsii [and date palm [R.A. Poir leaves is still limited in our current understanding.Soxhlet and maceration are the most common methods used for phenolic extraction from plants , 9. Howe hindsii , and datate palm . HoweverR.A. Poir leaves; and (ii) to compare the extraction efficiency of the ultrasound-assisted extraction with the soxhlet and maceration methods.Several variables affect the UAE process efficiency, such as ultrasound power, extraction time, and extraction temperature –18. It iRubus alceifolius Poir (R.A. Poir) leaves were collected from Song Hinh District, Phu Yen Province. Fresh leaves were prewashed in deionized water and then dried in a Binder ED56 oven (Germany) at 40°C for 10 hours to achieve 5–7% of moisture content. Following this, the dried leaves were ground to a fine powder, which was then sifted through a steel mesh sieve with a pore size of 1 mm. The samples were stored at 4°C until used for further analysis.α-amylase solution  and collagenase were purchased from Sigma. All the solvents and reagents used in this study were of analytical grade.Trolox (97%), ethanol (99.5%), vanillin (99%), acid acetic (99.5%), gallic acid (97%), and perchloric acid (99.99%) were procured from Anpha Chemika, India. Other reagents, such as 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and Folin–Ciocalteu, were from Sigma . Additionally, R.A. Poir leaves. The ultrasonic treatment was carried out using an ultrasonic generator  at a constant frequency of 20 kHz. In each run, approximately 10 grams of sample was mixed with 200 mL of aqueous ethanol , based on the optimal results of preliminary experiments. The mixtures underwent sonication at various ultrasonic powers , temperatures , and durations . After that, the samples were centrifuged at 3000 rpm for 5 min and then filtered using a vacuum pump. The extracts were used to determine total polyphenol content and antioxidant activity  by colorimetric spectroscopy.In this study, ethanol was used for phenolic extraction from R.A. Poir leaf powder. The design consisted of 17 experiments, and each parameter was varied at three levels  coded as -1, 0, and +1 (X1: 100-500 W), temperature (X2: 30-60°C), and sonication time (X3: 5-55 min). The total phenolic content (Y1) and antioxidant activity  were chosen as the responses. A second-order polynomial was used to calculate the predicted responseY1 and Y2 represent the predicted responses . β0, βi, βii, and βij are the regression coefficients. Xi and Xj function as distinct independent variables (i ≠ j). A fitted second-order polynomial was used to generate 3D surface plots, illustrating the correlation between independent variables and the subsequent response [The Box–Behnken design was employed to evaluate the extraction parameters on the recovery of phenolic compounds with high antioxidant activity from , and +1 . The indR.A. Poir leaf powder was extracted with 400 mL of 70% ethanol for 16 hours [For soxhlet extraction (SE), 150 g 16 hours . SubsequμL of the extract was diluted with 1560 μL of water and then mixed with 100 μL of Folin-Ciocalteu reagent. After that, 300 μL of 10% (w/v) sodium carbonate was added and incubated in the dark for 2 h. The solution's optical density was measured at a wavelength of 765 nm. To quantify the total polyphenol content, a standard curve was established using gallic acid (GAE) at concentrations ranging from 0.004 to 0.5 mM. This curve was used to determine the TPC of the sample, which is expressed as milligrams of gallic acid equivalents per gram of dry matter (mgGAE/g dm).The total polyphenol content was determined to the previous study described by Ibrahim et al. with slight modifications . A 40 μLμL extract was mixed with ABTS radical (265 μL at 7 mM) in the screw cap test tube. After that, the tubes were shaken in darkness for 10 min and measured at 734 nm using a microplate reader . For ABTS analysis, Trolox was used as the standard solution (0.1–1.0 g/100 mL in methanol). The ABTS is expressed as mg of Trolox equivalents per g of dry matter (mg/g d.m.).The ABTS radical scavenging activity was determined based on the method of Pham et al. with minor modifications . BrieflyμL of Tris-HCl , and 100 μL of the sample were weighed into tubes. The mixture was then shaken for 1 min before 100 μL of collagenase (200 units/mL) was added. The mixture was incubated at 43°C for 1 h. The samples were centrifuged at 3000 rpm for 10 min to remove the solid. The supernatant liquid was placed on a 96-well plate, and the absorbance was measured at 550 nm. A blank was prepared by adding all reaction reagents without a sample solution.The anticollagenase activity was evaluated using azo dye-impregnated collagen as a substrate based on a previous study by Wang et al. . Brieflyα-Amylase inhibitory activity was determined according to the method described by Savran et al. [μL of plant extract was combined with 50 μL of 0.05% starch solution and 50 μL of α-amylase solution from porcine pancreas  in phosphate buffer (pH 6.9 with 6 mM sodium chloride). The reaction mixture was incubated at 37°C for 10 minutes. To complete the reaction, 25 μL of 1 M HCl and 100 μL of iodine-potassium iodide solution were added. A blank was also prepared by including the sample solution in all reaction reagents but excluding the α-amylase solution. Absorbance was measured at 630 nm using a microplate reader . The α-amylase inhibitory activity was reported as millimoles of acarbose equivalents (mmol ACE/g extract).n et al.  with somμm film thickness). The GC-MS operating conditions were as follows: oven temperature increased from 50°C to 280°C at a rate of 10°C/min and then held isothermally for 10 min. The sample was injected in the splitless mode with 2 μL, and helium, a carrier gas was at 1 mL/min. The mass spectrometer was operated at 70 eV, and the total running time of the GC was 50 min. The compounds identified by GC-MS analysis were compared with compounds in the NIST 17 mass spectral library [GC-MS analysis was conducted following the previous method with slight modifications . The ext library . After tR.A. Poir leaf powder, scanning electron microscopy (SEM)  analysis was carried out. The dried samples were coated with a thin gold film using a sputter coater and then observed in a scanning electron microscope under an accelerating potential of 30 kV.To evaluate the morphological changes of using one-way ANOVA followed by Duncan's multiple range test. All statistical analyses were processed using Design-Expert software .In this study, each experiment was repeated 3 times, the results are presented as mean ± standard deviation. Data were analyzed R.A. Poir leaves. As depicted in p < 0.05). This decline may be attributed to the high ultrasonic power causing phenolic deterioration, consequently reducing the TPC and ABTS values of R.A. Poir leaf extract [ extract , 23. BasR.A. Poir leaves.p < 0.05). Simultaneously, both TPC and ABTS experienced a minor decline as the duration progressed from 45 to 55 min. The results indicated that with the prolonged ultrasound time, more phenolic compounds were decomposed [Another important factor affecting TPC and ABTS is extraction time. In this study, the samples were ultrasonically treated at 300 W at different times  . Findingcomposed , 24, 25.composed . ConsequX1), extraction temperature (X2), and extraction time (X3). The experiment encompassed 17 runs, featuring three central points. The observed and predicted TPC and ABTS values spanned within the ranges of 6.50–16.56 mg GAE/g dm and 6.56–22.34 mg TE/g dm, correspondingly . The high R2 (0.97-0.98) and adjusted R2 values (0.95-0.96), along with the minimal coefficient of variation (C.V. <10%) indicated the low deviation between the experimental and predicted values of the response [The ANOVA results were used to check the adequacy of the suggested model . The resresponse , 26, 27.X1, X2, and X3) on the TPC. The linear term (X1) has a positive influence on the TPC, while cross terms  and the quadratic terms  exert a substantial influence. The nonsignificant term (X3) was eliminated, and the second-order polynomial equation (Eq. (X1) and extraction temperature (X2) on the TPC. Specifically, the TPC increased with increasing X1 (100-300W) at a fixed X2 (45°C). Similarly, the increase in X2 (30-45°C) at a fixed X1 (300W) also led to a gradual increase in the TPC and nearly reached a peak at the moderate X2 process (40-45°C). A further increase in X1 (300-500W) and X2 (45-60°C) resulted in a reduction in TPC. These might be caused by the acceleration of phenolic degradation with increasing extraction temperature and sonication power. Similar results were also reported for the extraction of phenolic compounds from bitter melon peel [lon peel  and bee lon peel .X1) and extraction time (X3), which affects the extraction efficiency and consequently TPC. As presented in X1 on the TPC is significant at longer X3, while the effect of X1 at shorter X3 is almost negligible. The extraction of phenolic compounds achieved the maximal value after 30 min of treatment and thereafter dropped. A similar trend was found in the UAE in Epimedium brevicornum maxim leaves [X1 (45°C) and increasing extraction time up to 30 min  have a positive influence on the ABTS. Meanwhile, all the quadratic terms  and the cross-term coefficients (X1 × X3 and X2 × X3) have a negative influence on the radical-scavenging ABTS activity. The linear term (X3) and cross-term (X1 × X2) are not significant due to the value of p value greater than 0.05, and are similar to the results visualized in the elliptical contour plot. Figures X1 and X2 and achieved the highest the antioxidant activity value at 323 W and 40.8°C. A further increase in X1 and X2 resulted in the reduction of the ABTS value, consistent with the RSM results of TPC  at any X3 (p < 0.05). The ABTS value also increased with the increase of X3 (5-30min) at a fixed X1. However, higher X1 (400-500W) and extended X3 (40-55 min) resulted in low ABTS values due to the degradation of phenolics. This is similar to what was obtained for phenolic extraction from bitter melon [Celastrus hindsii leaves [X2 and X3 on the ABTS radical scavenging activity. At a fixed X3, the ABTS noticeably increased with the rise in X2 (30-40°C). When the highest ABTS was reached, any further increase in X2 led to a decrease in the ABTS. This might be due to the effect of high temperature on the nature and structure of the phenolic compounds [X2 and X3 for the phenolic extraction were found to be at its central level.In X1 100-0W at anyi leaves .  FigureX1 100-0W at anyX1) of 320 W, temperature process (X2) of 40°C, and extraction time (X3) of 35.5 min. Under the optimal conditions, the experimental TPC and ABTS values were 16.68 mg GAE/g and 21.9 mg TE/g, respectively. These results closely matched the predicted values, demonstrating the models' accuracy with minimal prediction errors. Essentially, these response models can be effectively used to refine the UAE conditions for improved recovery of phenolic compounds and their antioxidant activity.The optimal UAE conditions for maximum recovery of phenolic compounds and ABTS radical scavenging activity, as determined by the Design-Expert software, involved ultrasonic power . As shown in R.A. Poir leaf powder was smooth without cell wall deformation. In ME and SE, the plant cells have several holes, and a few cells were broken  and α-amylase (0.90 mmol ACE/g) compared to the SE (76.34% and 0.45 mmol ACE/g) and ME (75.76% and 0.64 mmol ACE/g) extracts (p <0.05)  . Accordintestine , 29. Meantestine , 31. As R.A. Poir leaf extracts obtained by optimal UAE conditions and conventional solvent extraction were analyzed with GC-MS. As presented in Psidium cattleianum leaves using UAE and conventional aqueous–organic extraction [R.A. Poir leaf extract, which exhibited strong antioxidant capacity and anticancer [The identification of bioactive compounds from the traction . Some prtraction –34. Our ticancer , 36. As R.A. Poir. The Box-Behnken design proved to be a highly beneficial tool for enhancing the parameters of ultrasonic-assisted extraction. The optimum conditions for UAE, as determined by RSM, included an ultrasound power of 320 W, an extraction temperature of 40°C, and a sonication duration of 35.5 min. Under these conditions, the experimental results agreed with the predicted values (p < 0.05). Additionally, the results indicated that the UAE method offered substantial advantages over SE and ME, particularly in terms of superior extraction efficacy and antioxidant activity of the extract in the shortest extraction time. Moreover, the leaf extracts were found to contain 5 phenolics and 3 flavonoid compounds. Hence, our research underlines the potential of the UAE procedure to enhance the extraction yield of phenolics and certain bioactive compounds with potent antioxidant capacities. Nevertheless, additional research is necessary for the full phytochemical characterization of R.A. Poir leaf extracts to confirm their possible applications.The response surface methodology was effectively utilized to optimize the extraction of phenolic compounds from


Whether diurnal variation exists in quantitative MRI indices such as the T1rho relaxation time (T1ρ) of the intervertebral disc (IVD) is yet to be explored. This prospective study aimed to evaluate the diurnal variation in T1ρ, apparent diffusion coefficient (ADC), and electrical conductivity (σ) of lumbar IVD and its relationship with other MRI or clinical indices. Lumbar spine MRI, including T1ρ imaging, diffusion-weighted imaging (DWI), and electric properties tomography (EPT), was conducted on 17 sedentary workers twice (morning and evening) on the same day. The T1ρ, ADC, and σ of IVD were compared between the time points. Their diurnal variation, if any, was tested for correlation with age, body mass index (BMI), IVD level, Pfirrmann grade, scan interval, and diurnal variation in IVD height index. The results showed a significant decrease in T1ρ and ADC and a significant increase in the σ of IVD in the evening. T1ρ variation had a weak correlation with age and scan interval, and ADC variation with scan interval. Diurnal variation exists for the T1ρ, ADC, and σ of lumbar IVD, which should be accounted for in image interpretation. This variation is thought to be due to diurnal variations in intradiscal water, proteoglycan, and sodium ion concentration. Intervertebral discs (IVDs) are fibrocartilaginous structures that form articulation between adjacent vertebral bodies and are present from the second cervical vertebra through the first sacral vertebra . Each inMagnetic resonance imaging (MRI) is widely used to evaluate the integrity of IVD noninvasively. Pfirrmann grade, visually evaluated using T2-weighted images, has long been considered a standard quantifier . HoweverQuantitative assessment by MRI requires normative values . The norThis prospective study aimed to evaluate the diurnal variation in the T1ρ, ADC, and electrical conductivity (σ) of adult lumbar IVD and their relationship with one another and other MRI or clinical indices. σ, derived from electric properties tomography (EPT), was chosen to reflect sodium ion concentration noninvasively. EPT is a recently developed MRI technique that can map the σ of tissues by merely post processing the images of standard MRI sequences . σ is knThis prospective study was approved by the institutional review board of Hokkaido University Hospital, Sapporo, Japan (protocol code 017-0455). Written informed consent was obtained from all participants.Over ten months, sedentary workers engaging in routine nonstrenuous daytime activities were recruited. The exclusion criteria were absolute contraindications to MRI, low back pain that prevented the volunteer from lying still during MRI, and known pathologies of the lumbar IVD. Seventeen subjects (fourteen men and three women) aged 25–48 years (mean age = 33.41 ± 5.70 years) were included in the study. According to an 11-point numeric pain rating scale, the mean low back pain score of the volunteers on the day of the MRI was 1.12 ± 1.32 (range = 0–3) . Their mAll examinations were conducted using a 3T scanner  and a 32-channel torso/radiofrequency (RF) coil. The scan range was set to include all lumbar vertebrae and sacrum.2, in-plane resolution = 1.56 mm × 1.54 mm, NSA = 6), and 2D steady-state free precession EPT  were acquired twice, once in the morning before engaging in the participants’ regular activities  and once in the evening after the activities . The average interval between the two scans was 10.78 ± 0.66 h (range = 9.93–12.23 h). Each scan lasted about 20 min.Sagittal 2D fast spin-echo T2-weighted imaging {T2WI; repetition time (TR)/echo time (TE) = 4000/90 ms}, 3D ultrafast gradient echo T1ρ imaging  = 15°, spin-lock frequency = 500 Hz, spin-lock time (TSL) = 0, 20, 40, 80 ms, in-plane resolution = 1.56 mm × 1.56 mm, the number of signal averages (NSA) = 1), 2D fast spin-echo echo-planar DWI  were co-registered before the reconstruction of corresponding maps at the operator console or using SPM12 .TSL = M0 × e−TSL/T1ρ, where MTSL and M0 denoted T1ρ spin lock prepared magnetization for each TSL and the equilibrium magnetization, respectively. Reconstruction was performed at a standalone workstation .T1ρ maps were reconstructed voxel-by-voxel from the T1ρ imaging data, using the formula Mb = S0 × e−b.ADC, where Sb = signal intensity of co-registered diffusion-weighted images at the given b-value, S0 = signal intensity on echo-planar T2-weighted images without diffusion weighting , and b = diffusion weighting. This reconstruction was performed on the main operator console of the MRI scanner.ADC maps were reconstructed from the DWI data using the formula Sx2 φ/(2μ0 ω), where φ = transceive phase, μ0 = magnetic vacuum permeability, and ω = Larmor frequency (128 MHz for 3T). This formula differs from the general version of EPT, which is based on numerically solving the truncated Helmholtz equation σ = ∇2φ/(2 μ0ω) = (∂x2 + ∂y2 + ∂z2)φ/(2 μ0ω). The partial derivatives in the three spatial directions x, y, and z require a volumetric magnetic resonance (MR) image so that the approximation σ ≅ (∂x2 + ∂y2) φ/(2 μ0ω) can be proposed to allow EPT on fast 2D scans with x and y defining the imaging plane [Using a standalone computer, σ maps were reconstructed from the phase of EPT data, using the formula σ ≅ ∂ng plane ,19. The MR images of the lumbar region can suffer from various artifacts, including flow and motion artifacts. Thus, all images and maps were visually evaluated by a radiologist (23 years of experience) and radiological technologist (15 years of experience) for any artifacts. Only artifact-free images were used for quantitative analysis. The radiologist, unaware of the mapping results, determined the Pfirrmann grade of each lumbar IVD using mid-sagittal T2WI .2), which provided maximum image contrast. Special attention was paid so that each ROI included the entire lumbar IVD while avoiding the inclusion of nearby structures. The ROIs were then superimposed onto the corresponding co-registered maps. The accuracy of the ROI position was confirmed by a radiological technologist (15 years of experience). Example ROIs are given in Regions of interest (ROIs) were placed on each lumbar IVD . ROIs for T1ρ and ADC measurements were placed semiautomatically on the mid-sagittal images of each MRI sequence, using Jim8. ROIs were drawn on T1ρ images at TSL = 0 ms and echo-planar images with no diffusion weighting  performed the measurements on a standalone DICOM viewer . The steps followed those detailed in a previous report .Repeatability of T1ρ, ADC, and σ measurements of IVDs was tested in the volunteer in whom two repeated scans were performed in the same scan session. Intraclass correlation coefficient (ICC) was used to determine the repeatability, and ICC greater than 0.75 was considered repeatable .p < 0.05 was considered statistically significant. Statistical analysis was conducted using Statistical Package for the Social Sciences (SPSS) software version 26 .T1ρ, ADC, σ, and IVD height index of all lumbar IVDs were compared between the two time points, using paired t or Wilcoxon signed-rank tests. The relationships of each index between the two time points and the association of diurnal variation (Δ) of each MRI index, given by the difference of their values between the two time points, with Δ of other quantitative MRI indices, Δ of IVD height index, age, Pfirrmann grade, BMI, IVD level, and the interval between the two scans , were tested using Pearson’s product moment correlation analysis. Variation in the degree of Δ among IVD levels was evaluated using repeated measures analysis of variance (ANOVA). For all comparisons, Altogether, 85 lumbar IVDs were evaluated. Of these, T1ρ measurement was possible in all IVDs. A total of 84 IVDs were valid for ADC, and 83 IVDs for σ. The Pfirrmann grades of the lumbar IVDs were 7, 70, and 8 for grades II, III, and IV, respectively.The ICC values for the T1ρ, ADC, and σ measurements were 0.874, 0.858, and 0.974, suggesting good to excellent repeatability.p < 0.05) (p < 0.001). For all quantitative MRI indices and IVD height index, the morning values correlated moderately to very strongly with the evening values  ( < 0.05) . A signi< 0.001) .p = 0.047) and the scan interval  (p = 0.031) (p > 0.05). For any index, the degree of Δ did not vary significantly with the IVD level .The ΔT1ρ showed a weak negative correlation with age  . ΔADC sh= 0.031) . HoweverThis prospective study reports diurnal variation in the T1ρ, ADC, and σ of lumbar IVDs, in addition to the commonly known diurnal variation in IVD height index . DiurnalA decrease in IVD height in the evenings is a common phenomenon: a reduction in spinal height of up to almost 2 cm or about 1% of total body height can occur throughout the day while recovering during the night ,25. ThisIt is reported that about 20–25% of the IVD’s water content is expressed and re-imbibed during each diurnal cycle . This maT1ρ relaxation time, also known as the spin–lattice relaxation time in the rotating frame, is a time constant for transverse magnetization decay in a very weak B1 field strength produced by a spin-lock RF pulse . InteracIn contrast to other means of determining σ, σ estimation by EPT neither requires the injection of external currents nor the acquisition of different patient orientations. The potential usefulness of σ measured by EPT has been reported in brain and breast tumors and hepatic fibrosis ,23,31. Ip = 0.050). Failure to achieve a significant correlation may be due to a limitation in sample size. The lack of correlation of these changes to Pfirrmann grade or IVD level suggests that the degree of IVD degeneration or the IVD level do not affect diurnal variation. The lack of correlation to Pfirrmann grade can also be due to the inclusion of mostly Pfirrmann Grade III IVDs in the cohort. Finally, BMI has been reported to affect diurnal variation, which was not reproducible in this study [This study indicated a few correlations of the diurnal variation in the quantitative MRI indices with age and scan interval. The correlation of ΔT1ρ with age is thought to reflect the age-dependent diminution in intradiscal proteoglycan concentration, the finding of which is consistent with previous studies ,33. The is study . In thisis study . Anotheris study . A studyis study .Several limitations need to be addressed. First, all measurements were performed using the mid-sagittal imaging plane. This choice was made to limit partial volume artifacts. The inclusion of all slices or volumetric measurements may be ideal. Second, our ROIs included both AF and NP. Separate measurements may enhance the understanding of dynamic changes occurring in the IVDs. However, separating AF and NP is difficult, especially in degenerated discs . ROIs thThis study reports the diurnal variations in the T1ρ, ADC, and σ of lumbar IVDs in sedentary workers. There exists a diurnal variation in these quantitative MRI indices, reflective of daily physiological events. Knowledge of this variation will be important in interpreting these indices for clinical decisions. The results of this study may serve as the reference values for lumbar IVDs. Finally, this study proposed a method to noninvasively assess the σ of IVD.


However, their slow diffusion kinetics is still challenging, restricting practical applications. Here, a one-pot and straightforward method was reported to produce Zn-doped MnOx nanowires with abundant defects and tunable small cross-sections, exhibiting an outstanding specific capacitance. More specifically, based on a facile hydrothermal strategy, zinc sites could be uniformly dispersed in the α-MnOx nanowires structure as a function of composition . Such a process avoided the formation of different crystalline phases during the synthesis. The reproducible method afforded uniform nanowires, in which the size of cross-sections decreased with the increase of Zn composition. Surprisingly, we found a volcano-type relationship between the storage performance and the Zn loading. In this case, we demonstrated that the highest performance material could be achieved by incorporating 2.1 wt.% Zn, exhibiting a remarkable specific capacitance of 1082.2 F.g−1 at a charge/discharge current density of 1.0 A g−1 in a 2.0 mol L−1 KOH electrolyte. The optimized material also afforded improved results for hybrid supercapacitors. Thus, the results presented herein shed new insights into preparing defective and controlled nanomaterials by a simple one-step method for energy storage applications.MnOThe online version contains supplementary material available at 10.1186/s11671-023-03933-2. Struggles to balance the imminent energy crisis and global climate warming are part of long and challenging attempts to develop alternative energy supplies and in-depth research into energy storage systems , 2. At t2, Co3O4, RuO2, etc.) have been demonstrated to be the most promising candidates for pseudocapacitance electrodes :2\d:2\d24]:2x nanowires samples and served as a positive electrode; activated carbon was used as a negative one. The charge stored (q) by each electrode depended on the following Eq.  obtained in the GCD tests for the hybrid supercapacitor, ∆V (V) is the potential range between the cathode and anode, and ∆t (s) is the time of discharge of the device. All the tests were carried out at room temperature.The hybrid supercapacitor tests were performed in a two-electrode system. In typical analyses, the nickel foam was loaded with the Zn-doped MnOwing Eq.  24]:3\d:3\dx nanquations  and 6)  x nanowix nanowires using MnSO4 (reducing agent) and KMnO4 (oxidizing agent) as metal precursors and water as solvent at 110 °C for 24 h. Such a synthetic procedure was chosen once this method leads to defects on the surface of the nanomaterial that could directly impact its electrochemical behavior. Also, this procedure is very simple and can provide "clean" nanostructures without capping ligands or related compounds. The nanowires displayed well-defined shapes and uniform sizes, 51 ± 14 nm in width and > 1 µm in length, as shown in Fig. . In the next step, we modified the classical hydrothermal synthesis of MnOx nanowires by adding different amounts of Zn(NO3)2.6H2O  to the synthesis procedure. We used Zn amounts to obtain 1.0, 5.0, 10.0, and 20.0 wt.% Zn. However, using the ICP-OES technique, the following content was obtained by increasing the Zn precursor: 0.3, 2.1, 4.3, and 7.6 wt.% Zn.Our investigations started with the hydrothermal synthesis of MnOx nanowires presented well-defined shapes and uniform sizes; however, their cross-section diameters depended on the zinc composition. More specifically, the Zn-doped nanowires displayed cross-section diameters of 47 ± 10 nm, 41 ± 11 nm, 39 ± 8 nm, 37 ± 6 nm in width, and > 1 µm in length, as shown in Fig. x nanowires, respectively. Additional file x nanowires, revealing that they are structurally uniform, with mixed growth directions along the [110], [100], and [310] axis. We also performed STEM-EDX spectrum imaging for all the Zn-doped MnOx nanowires to investigate the Mn, O, and Zn elemental distribution, as shown in Fig. x nanowires, in agreement with ICP-OES results.Interestingly, in all the cases, Zn-doped MnOx nanowires, we examined their textural and structural properties, which could influence their storage performances. Thus, the textural properties obtained by N2-physisorption analysis are presented in Table S1. The specific surface area of samples increased with the increase in zinc composition, displaying 105, 107, 132, 144, and 135 m2 g−1 for 0.3, 2.1, 4.3, and 7.6 wt.% Zn-doped MnOx samples, respectively, in agreement with the TEM analyses that showed a decrease in the cross-section diameters of nanowires as the content of zinc increased. The Zn-doped MnOx nanowires also displayed a smaller average BJH pore diameter as the zinc content increased. The representative adsorption isotherms (we have chosen the MnOx and 2.1 wt.% Zn-doped MnOx nanowires samples) – Figure S2 – corresponded to type IV (IUPAC classification), typical of mesoporous materials. The shape of the hysteresis loop corresponded to type H3, with well-defined loops that did not level off at relative pressures close to the saturation vapor pressure. Such characteristics are important for the preparation of supercapacitor electrodes once they can provide enhanced ion accessibility, increased surface area, stability, and durability.After synthesizing the Zn-doped MnOx nanowires are shown in Figure S3. The samples exhibit similar XRD patterns, indexed mainly as α-MnO2 tetragonal crystal phase with I4/m space group (JCPDS Card No: 44–0141) [x nanowires sample. Interestingly, a significant broadening and slight shift of all diffraction peaks could be observed when the composition of Zn increased. Such results suggest that zinc sites could be lightly and uniformly dispersed onto MnOx nanowires’ structure, avoiding the formation of different crystalline phases (only α-MnO2 was detected). Thus, such a procedure avoided the structural transformation of α-MnO2 nanowires during the synthesis. Also, the obtained data suggest that Zn is doped into the oxide lattice, leading to some distortion (evidenced by the observed shifts) and poor structure crystallinity. Poor crystallinity is preferred for supercapacitor applications because the structure can provide an easy way for most active ions to penetrate.The XRD results of the 0.3, 2.1, 4.3, and 7.6 wt.% Zn-doped MnO44–0141) . Specifix and Zn-doped MnOx nanowires were investigated to understand the influence of Zn doping on oxygen vacancy generation and electronic structure modification. Figure 4+. As Kakazey et al. demonstrated, the spectrum's broadening from 0.3 to 2.1 wt.% Zn is due to exchange interactions between Mn4+ ions and can also be ascribed to the partial substitution of Mn4+ for Mn3+ ions [3+/Mn4+ ratio was achieved for the sample 2.1 wt.% Zn-doped MnOx nanowires. In addition, as previously demonstrated by Dang et al., the higher the Mn3+/Mn4+ ratio in MnO2-based structures, the better performance towards tailored supercapacitors due to increased MnO2 conductivity [3+-O-Mn4+). In this mechanism, there is an interaction between Mn and O orbits in which electrons from Mn3+ could migrate to Mn4+ using O 2p orbits as a bridge. It was also demonstrated that Mn4+ to Mn3+ substitution in the MnO2 lattice induces the formation of oxygen vacancies [The EPR spectra of the MnOn3+ ions . Therefouctivity . They deacancies , 29.Fig.2+ becomes more evident, as shown in Figure S4 [3+/Mn4+ ratio and oxygen vacancies concentration are increasing, which could play a pivotal and synergistic role in Zn-doped MnOx nanowires' improved performance up to 2.1 wt.% Zn. Hereafter, as Zn content increases, an adverse effect on the supercapacitor's performance associated with Mn2+ presence becomes significant.Interestingly, as Zn content addition increases, the EPR signal and linewidth decrease, and so does the sextet pattern; the characteristic hyperfine splitting of Mnigure S4 –32. Thusx nanowires and 0.3, 2.1, 4.3, and 7.6 wt.% Zn-doped MnOx counterparts as electrodes for storage applications. CV curves were performed to analyze the materials' electrochemical performance, as shown in Fig. −1 KOH.26 There are two faradaic peaks for all the electrodes, one related to an oxidation process, observed during the positive going scan, centered around 0.35 V, and another, a reduction one, observed during the negative going scan, centered around 0.20 V. The potential peak is not the same for all materials and is related to the redox couple Mn2O3/MnOOH. The fact that the peaks are separated by at least 140 mV  shows that the reaction is not happening just on the material surface; there is at least a mass transport step in which some species approach or leave the electric double layer. In addition, the process cannot be called reversible due to this large peak separation, which is larger than the ∽60/n mV separation window. However, for the 0.3 and 4.3 wt.%, the ratio between the current peaks is close to 1, indicating that the process can be quasi-reversible. The lower concentration of Zn ions in the MnOx nanowires structure hampers its clear identification in the curves for the Zn-doped MnOx materials, which reflects the XRD results that considered a single manganese oxide phase. However, once ICP-OES confirmed the existence of Zn species, it makes us believe that the following reaction occurred:In the next step, we focused our attention on applying the MnOx nanowires, indicating that the doping process enhanced the electrical conductivity of the materials. Interestingly, however, this current gain does not follow an expected trend, i.e., the increment in the Zn content did not provide a current increase. Instead, the 0.3 wt.% Zn-doped MnOx material showed higher values and potential shifts , followed by the 2.1 wt.% Zn-doped MnOx counterpart, with a shift to a more positive potential for oxidation and reduction processes. The other two materials presented lower currents, suggesting that the doping affected their conductivity. It is important to note that the peak position correlates with the population of oxygen vacancies. For instance, the material containing 0.3 wt.% Zn exhibited the lowest vacancy population, resulting in the most negative peak potential. Conversely, the material with 2.1 wt.% Zn, possessing the highest vacancy concentration, displayed the most positive potential peak. Consequently, the other two materials, with lower vacancy levels approaching those of the 0.3 wt.% Zn material, exhibited intermediate potential peaks during the positive potential scan. Given that oxygen vacancies indicate an excess of electrons or, in other words, act as n-type dopants, it is reasonable to expect that the most heavily "doped" material  would have the most positive oxidation potential, creating a significant barrier for oxidative behavior. Additionally, the same material showed the most positive reduction potential, indicative of the lowest reductive barrier, as anticipated for an n-type semiconductor.One can notice that the CV currents for the doped materials are much higher than the observed for pure MnOx nanowires, indicating a quasi-reversible feature of the redox couples. Figure S6 shows the peak current density versus the square root of the scan rate for all the materials, which suggests that diffusion-controlled reactions occur during the electrochemical process. For most materials, the slopes are similar, meaning that the diffusion of the same species limits the reaction. In the case of 0.3% material, the slope is 10 times higher, meaning the diffusion is facilitated in this electrode. The diffusion-limited species is probably the K+(aq), responsible for the charge compensation process. The small porous diameter observed for these materials, around 2.5 nm, is in the same order of magnitude as the solvated K+ diameter, around 0.7 nm.We also performed the CV of the materials to evaluate the effect of different scan rates  for the dopped materials. We could observe reduction peaks shifting to more negative potentials with the scan rate increase, while the oxidation peaks shift to more positive ones for all the Zn-doped MnO−1 KOH electrolyte at different discharge current densities of 1.0, 2.0, 5.0, 10.0, and 20.0 A g−1. All the samples have only one plateau, consistent with the CV curves in Fig. x nanowires with 432.6 F g−1 (1 A g−1), 379.6 F g−1 (2 A g−1), 316.3 F g−1 (5 A g−1), 265.3 F g−1 (10 A g−1), and 204.1 F g−1 (20 A g−1); 0.3 wt.% Zn-doped-MnOx nanowires with 497.7 F g−1 (1 A g−1), 451.2 F g−1 (2 A g−1), 372.1 F g−1 (5 A g−1), 302.3 F g−1 (10 A g−1), and 139.5 F g−1 (20 A g−1); 2.1 wt.% Zn-doped-MnOx nanowires with 1082.2 F g−1 (1 A g−1), 928.8 F g−1 (2 A g−1), 600.0 F g−1 (5 A g−1), 340.9 F g−1 (10 A g−1), and 266.7 F g−1 (20 A g−1); 4.3 wt.% Zn-doped-MnOx nanowires with 608.9 F g−1 (1 A g−1), 560.0 F g−1 (2 A g−1), 511.1 F g−1 (5 A g−1), 466.7 F g−1 (10 A g−1), 400.0 F g−1 (20 A g−1), and; 7.6 wt.% Zn-doped-MnOx nanowires with 345.2 F g−1 (1 A g−1), 204.8 F g−1 (2 A g−1), 178.6 F g−1 (5 A g−1), 166.7 F g−1 (10 A g−1), and 142.9 F g−1 (20 A g−1). The Coulombic efficiencies achieved were higher than 93% for the samples in all the current densities. Figure −1. The curves are virtually symmetrical, representing reversible pseudo-faradic reactions between the electrode and electrolyte.Figure Interestingly, a volcano-type relationship between the storage performance and the Zn loading was observed . Then, it is clear that we achieved an optimum Zn content for the improvement of the storage properties of the material. Above its quantity, there is a decrease in the capacity of the material. It is important to highlight that we decided not to perform characterizations after using the electrodes once previous literature showed that similar materials, after electrochemical cycles, undergo carbon deposition and Mn, O-containing nanoflakes over it, which hinder a conclusive observation of the modification of the as-prepared nanowires .x nanowires in the literature at 1 A g−1 are lower than the one presented herein for our Zn-doped MnOx nanowires. For example, for pure MnOx nanowires, Yin et al. obtained 180 F g−1, while Mahmood et al. acquired 337.5 F g−1 [x nanowires. Thus, the Zn species appear to aid electron transport due to synergetic issues, as observed when Co ions are used to synthesize MnOx nanowires [At this point, we could observe that supercapacitors for MnO.5 F g−1 . Undoubt.5 F g−1 , 36; howanowires .x and 2.1 wt.% Zn-doped-MnOx nanowires were also investigated by XPS to shed some light on their differences. Figure S8 shows the low-resolution spectrum of the doped material, showing only the expected elements: Mn, O, Zn, and C . Figure S9A shows the MnOx nanowires' high-resolution XPS spectrum, which shows peaks centered at 653.8 and 642.2 eV, related to Mn 2p3/2 and Mn 2p1/2, respectively. An asymmetric Gaussian/Lorentzian fitting deconvolved them into four sub-peaks; two are assigned to the B.E. of Mn3+ (642.1 and 653.6 eV) and two to Mn4+ (643.5 e 654.9 eV). As shown in Fig. 3/2 and Mn 2p1/2, respectively, are observed for the 2.1 wt.% Zn-doped-MnOx, very similar to the observed for the unmodified MnOx nanowires. Also, the four sub-peaks ascribed to the B.E. of Mn3+ (642.1 and 653.7 eV) and two to Mn4+ (644.2 e 654.6 eV) were detected. However, The ratio of Mn3+/Mn4+ was increased from 1.6 (MnOx nanowires) to 2.9 (2.1 wt.% Zn-doped-MnOx nanowires), indicating that oxygen vacancy concentration increases after in the sample with Zn doping, which follows the EPR, that shows an increasing content of Mn3+ in the material with 2.1 wt.% Zn. For the 2.1 wt.% Zn-doped MnOx sample, we evaluated the Zn 2p region of the XPS, which showed two peaks centered at B.E. of 1021.4 and 1044 eV, ascribed for Zn 2p3/2 and Zn 2p1/2 of Zn2+, respectively  and 2.1 wt.% Zn-doped MnOx nanowires , oxygen vacancy (Ov), and lattice oxygen (OL), respectively, for the doped sample. Thus, remarkable differences in the oxygen XPS show how the dopping process changed the surficial material characteristics.However, it is essential to analyze the O1s spectra for the MnOres Fig. D. The Mnv vacancies could be explained as follows: during the doping and formation of manganese oxide, the Mn4+ species present in MnOx can be replaced by Zn2+ sites, leading to charge disparity in the oxide structure. This phenomenon leads to a weakening of the Mn–O bond. Therefore, punctual defects such as oxygen vacancies are expected to form in the crystal lattice to compensate for this charge inequity and maintain the nanomaterial's electrical stability. These conclusions directly relate to the XPS data presented here: the Zn2+ can contribute to the material's electrochemical performance.However, XPS is not the most accurate technique to evaluate oxygen vacancies, so the EPR was performed herein. Thus, based on the results of EPR and XPS analyses, the formation of O2 nanowires' cycling stability by performing 1000 charge–discharge cycles at 10 A g−1. According to Fig. −1 (the first test performed 340.9 F g−1). Thus, the material remained at 47% of its initial capacitance. Some relevant considerations are necessary regarding these results: during the cycles, irreversible redox reactions can occur, causing damage, inactivation, or loss of active sites; mechanical stress from electrolyte ions intercalation/deintercalation to counterbalance the overall charge can happen, lowering the efficiency of the material; also, insufficient active species utilization can occur due to high current densities reached in the experiments [By choosing the optimized doping, we obtained further Information on the 2.1 wt.% Zn-doped-MnO−1 KOH electrolyte before (as-prepared), during (after 500 cycles), and after the 1000 cycles  would be needed to interpret this system. For the "as-prepared" electrode, it can be seen that the data tend to have higher imaginary parts and smaller real ones, as expected for a high capacitive contribution. After some cycling, a linear region can be seen, and the slope for this is equivalent to 0.5, meaning that a mass transport limitation due to diffusion is gaining importance. The capacitance/resistive contribution is changing, as seen in Fig. To evaluate these issues further, we performed EIS analyses at open circuit potential in 2.0 mol Lles Fig. B. None ox nanowires' performance, we assembled a hybrid supercapacitor, using our material as the positive and activated carbon as the negative electrodes. The possible operating voltage window of the hybrid device was studied by CV , 87.1 F g−1 (2 A g−1), 75.0 F g−1 (5 A g−1), 71.4 F g−1 (10 A g−1), and 71.4 F g−1 (20 A g−1). The Coulombic efficiencies were higher than 90%. The hybrid device yielded a maximum energy density of 91.7 Wh kg−1 at a power density of 2520 W kg−1 at 1 A g−1. The energy density obtained here is higher than conventional electrochemical double-layer capacitors, which is highly interesting for future applications [−1 up to the end of the 1000 cycles , avoiding the formation of different crystalline phases or depositing zinc particles at the surface of the α-oxide nanowires. The EPR results showed that oxygen vacancies achieved a higher value for the optimized material, which could help to explain its performance. More importantly, a hybrid capacitor based on the Zn-dope nanowires and activated carbon electrodes supplied an energy density of 91.7 Wh kg−1 and a power density of 2520 W kg−1 at 1 A g−1. Once the synthesis of the Zn-doped nanowires is simple and one-pot, we believe this material can be a promising electrode for low-cost storage devices.Herein, we prepare one-dimensional Zn-doped nanowires with a simple hydrothermal synthesis method, varying the quantity of Zn content. The material presented a high specific capacitance of 1082.2 F gAdditional file1 Additional data regarding the material characterizations and performance can be found in the supplementary file


Development of clinical-grade, cell preparations is central to cGMP (good manufacturing practice compliant) conditions. This study aimed to investigate the potential of two serum/xeno-free, cGMP  culture media to maintain “stemness” of human minor salivary gland stem cell (mSG-SC) cultures compared to a complete culture medium (CCM). Overall, StemMacs resulted in higher proliferation rates after p.6 compared to the conventional serum-based medium, while StemPro showed substantial delays in cell proliferation after p.9. The mSG-SCs cultures exhibited two distinct cell populations at early passages a mesenchymal subpopulation and an epithelial-like subpopulation. Expression of several markers  variably decreased with prolonged passaging . The percentage of SA-β-gal positive cells was initially higher for StemMacs compared to StemPro/CCM and increased with prolonged passaging in all cases. The telomere fragment length decreased with prolonged passaging in all three media but more pronouncedly for the CCM. Expansion under serum-free conditions caused pronounced upregulation of ALP and BMP-2, with parallel complete elimination of the baseline expressions of LPL  and ACAN (serum-free media), therefore, showing a preferential shift of the mSG-SCs towards osteogenic phenotypes. Finally, several markers  decreased with prolonged culture, indicating successive loss of “stemness”. Based on the findings, it seems that StemPro preserve stemness of the mSG-SCs after prolonged culture. Nevertheless, there is still a vacant role for the ideal development of clinical-grade culture conditions. The rationale for the clinical application of salivary gland stem cells includes the regeneration of the salivary gland parenchyma in cases of severe or irreversible damage, caused by several pathologies or conditions. For instance, these include the therapeutic radiation of the head and neck area; drug-induced xerostomia; the presence of Sjögren’s syndrome or other autoimmune sieladenitis; metabolic diseases, such as diabetes mellitus; or surgical removal of the salivary glands in case of neoplasms. All these conditions are associated with salivary gland dysfunction, leading to xerostomia, which affects a large portion of elderly individuals. Oral dryness (another term for xerostomia) leads to dysphagia, difficulties in speaking, increase of the prevalence of dental caries and periodontitis, decreased resistance to infections , and loss of dentures cohesion, and overall, it compromises the patients’ quality of life-QoL . A search in the + salispheres) in radiation-damaged salivary glands, which resulted in the development of amylase-producing tissues and partial regeneration of the gland  have been used in different mouse models, including transplantation of the cell populations (cell-based therapy) or transplantation of salivary organoids  were microscopically negative for Sjögren’s syndrome or other type of sialadenitis. The study and all the experimental methodology were performed according to Helsinki Declaration II and were approved by ethical committee of the School of Dentistry, Aristotle University of Thessaloniki, Greece (reference number 322/15–4-2013).Human minor salivary glands were isolated from lower lip biopsies taken for Sjögren’s syndrome investigation, as described previously  and immersed in a solution with yeast collagenase I (3 mg/ml) and dispase (dispase) II (4 mg/ml)  following incubation at 37 °C for 1 h with occasional agitation to achieve the breakdown of individual cells. Later, cells were placed in 25-cm2 flasks (p.0) and incubated at 37 °C in 5% CO2 when cells reach 70–80% confluency and were transferred to 75 cm2 flasks for further subculturing. The medium was changed every other day, and the cells were used until passage 10. For salivary stem cell expansion, 3 different culture media were used (CCM vs StemMacs vs StemPro): specifically, (1) α-MEM  supplemented with 15% FBS , 100 μM ascorbic acid phosphate  and 1% antimicrobial solution  . The cells were detached using 0.25% trypsin/1 mM solution EDTA for 5 min at 37 °C and replated at a density of 5000 cells/cm2. (2) StemPro MSC SFM XenoFree  was prepared according to the manufacturer instructions; 98 ml StemPro MSC SFM basic medium was supplemented with 1 ml of StemPro supplement, 1 ml L-glutamine solution GlutaMAX – I CTS, and 50 μl gentamicin solution (50 mg/ml). Plastic surfaces were coated with CELLstart CTS . Cells were detached with TrypLE™ Select CTS™  as an alternative to porcine trypsin used elsewhere for 4–5 min at 37 °C and replated at a density of 5000 cells/cm2. (3) Miltenyi StemMacs MSC expansion media kit XF  was prepared according to the manufacturer instructions; 98.6 ml of StemMacs MSC basal expansion media XF was supplemented with 1.4 ml StemMacs MSC expansion media XF supplement and 50 μl gentamicin (50 mg/ml). For cell detachment and further subculturing, TrypLE™ Select CTS™ was used as mentioned previously for 5 min at 37 °C, and cells were seeded at a density of 5000 cells/cm2.Minor salivary gland stem cells (mSGSCs) were isolated from biopsies of minor salivary glands of lower lip from patients aged between 22 and 67 years old. All healthy individuals were non-smokers and systemically healthy. After clinical and laboratory evaluation, they were confirmed negative for Sjögren’s syndrome. The process of isolating mSGSCs was carried out by means of the enzymatic separation, as already described previously . A total of 50,000 events per sample were recorded, and data were analyzed with the software GuavaSoft 3.1.1 and Summit 5.1. In addition to determining the percentage of cells positive for each marker, the cell size and cell internal complexity (granularity) distribution profiles were analyzed by forward scatter (FSC) vs side scatter (SSC) fluorescence intensity plots, respectively.The mSGSCs were characterized based on the 3 different culture media for mesenchymal, endothelial, fetal, and hematopoietic cell membrane markers of stem cells, such as CD90/Thy-1, CD73, CD146/MUC18, STRO-1, CD105, CD106, CD34, and the embryonic marker SSEA-4, as well as glandular epithelial cell markers  by flow cytometry as described previously  was calculated as PD = log2 (Nx / No). At p.0, the total number of cells initially attached in the flask after 48 h were defined as No. Lastly, PDT for each passage was estimated as t/n, where t stands for time and n was the number of PD through passage.Cell morphology was studied in reverse phase contrast view finder  equipped with digital camera and an analysis software (Carl Zeiss Axiovision 4.6 software). Images were taken from selected areas, representative of the cell growth in cell culture. The proliferation potential of mSG-SCs was evaluated by measuring the population doubling time (PDT) over passages based on the following formula: 22, X-GAL 1 mg/ml in DMSO) for 14–16 h at 37 °C. Positive (blue stained) and negative cells were measured using a light microscope in random positions, and the total percentage of positive cells was evaluated.The expression of senescence was measured based on expression of β-galactosidase  at different representative passages (p.2–3), (p.6–7), and (p.10–11) using a beta galactosidase chromogenic assay kit (Sigma-Aldrich). Cells were fixed with 4% PFA, washed with PBS and incubated with staining β-galactosidase  was isolated from cells with the Nucleospin tissue DNA isolation kit . The relevant telomeres’ length in different cells was measured with the TeloTAGGG telomere length assay kit  according to manufacturer instructions; specifically, 2 μg gDNA/sample was initially digested with the Hingl/RSA1 system, then electrophoresed in 1% agarose/TAE gel, and transferred for southern blot on positively charged nylon membrane at 20 X potassium citrate buffer. The gDNA hybrid was tagged with a non-isotopic marker labeled with digoxygen (DIG) incubated with an antibody against DIG covalently linked to alkaline phosphatase and visualized with an optical analysis system chemiluminescence (ECL) . The length of the telomeres was evaluated with final restriction fragment analysis  calculated as follows: TRFs = Σ (ODi) / Σ (ODi / Li), where The protein expression of several markers pluripotency  was investigated with the Proteome Profiler array: human pluripotent stem cell array kit , after long-term culture in the serum-containing medium (CCM), as this medium showed the most pronounced effect in reducing the telomere length of the mSG-SCs.Briefly, the nitrocellulose membranes incubated with different antibodies were further incubated with a blocking buffer solution for 1 h at room temperature; cell lysates were placed in contact the membranes and further incubated at 2–8 °C in a stirring platform overnight. The following day, the membranes were washed and further incubated with a streptavidin-HRP secondary antibody for 2 h in a stirring platform. Then, a chemiluminescent visualization reagent (Chemi Reagent Mix) was applied to the membrane. Finally, the membrane was visualized using an optical chemiluminescence analysis (ECL) system . Image J software  combined with the dot blot micromodule tool was used to quantify positive expression of the membrane.The upregulation/downregulation of different osteogenic , chondrogenic , and adipogenic  markers were evaluated with qPCR. The primers of these genes are summarized in Table mSG-SCs were induced with 3 different culture media (CCM vs StemMacs vs Stem-Pro) towards osteogenic, chondrogenic, and adipogenic phenotype to evaluate their differentiation capacity at p.2–3, p.6–7, and p.10–11. For osteogenic and chondrogenic differentiation, 300,000 cells were seeded in 6 well plates until reaching confluency, and the medium was changed . After 14 days of continuous culture, the RNA of the cells was isolated to perform qPCR as described in previous section, and the expression of different osteogenic markers , and chondrogenic ) was analyzed. Furthermore, mSG-SCs were evaluated for bone formation potential. Therefore, the wells were exposed to osteogenic medium for 21 days and further were evaluated by histochemical staining with 1% red of Alizarin (Alizarin Red S) followed by measurement of mineralized tissue by its dissolution coupled staining with cetyl pyridinium chloride (CPC), as already described previously  with multiple comparisons between the different parameters such as 3 different types of cell culture media (CCM vs. StemMacs vs. StemPro) and early vs middle vs late passages. Tukey’s post-hoc test was used for correlations with Prism 6.0 software . The normal distribution was confirmed by the D’Agostino and Pearson tests. The data were expressed in mean values with constant deviations .Primary mSG-SCs from minor salivary glands were cultured in the three different media , and their proliferation was evaluated in different passages from 1 to 10. A first key parameter evaluated is the population doubling time (PDT) of cell cultures. PDT was calculated by measuring the number of cells at the beginning and end of each passage. The time required from the initial phase of mSG-SCs culture after the enzymatic digestion step to p.1 was 8.9 (± 2.3), 9.3 (± 3.2), and 9.1 (± 1.4) days for culture of mSG-SCs in CCM, StemMacs, and StemPro, respectively. This suggests a similar growth potential of mSG-SCs at the initial stage. fA shows the PDT replication time (in days) of the mSG-SCs for each culture media from p.2 to p.10. In Fig. n = 3 donors/culture medium), in experiments repeated three times . According to Fig. p < 0.01), whereas from p.9 onwards, StemMacs had a statistically significantly greater PDT compared to CCM (p < 0.01). This observation indicates that the mSG-SCs in StemMacs retain their proliferation capacity even after many subcultures compared to CCM; in contrast to StemPro, a significant downregulation of the cell proliferation rate indicates the possible loss of the stemness and the onset of cellular aging. Figure p < 0.05) were observed in p.8, p.9, and p.10 between StemMacs and StemPro, with the latter achieving a greater number of replication doublings. Overall, the total number of PDs after ten subcultures (passages) was 33.9 (± 2.4) for CCM, 32.7 (± 1.9) for StemMacs, and 35.9 (± 1.2) for StemPro. After the completion of the first two subcultures (p.2), the total number of cells were approximately 15–20 million, and after three subcultures, 4–60 million .PDT in days is the mean for mSG-SCs  in early p.2), middle p.6), and late (p.10) passage for the expression of a range of stem cell membrane markers  and cytoplasmic epithelial cell markers  by flow cytometry as shown in Figs. , middle , and latp < 0.01). This increase was statistically significantly higher in all passages for StemMacs media compared to the other two culture media CCM and StemPro (p < 0.01). Overall, StemMacs has shown the greatest number of aging cells and where gradually increased during subculturing at later passages followed by CCM and StemPro. These results coincide with the findings on cell proliferation  and the length of chromosomes telomeres. As shown in Fig. a, SA-β-gion Fig. b. The anion Fig. c. In Figp < 0.01) and Goosecoid (GSC) (p < 0.01) demonstrated high expression in p.2 with a decrease in p.10. Also, the expression of transcription factors such as SOX2 (p < 0.01), GATA-4, PDX-1/IPF1 (p < 0.01), Otx2, VEGF R2/KDR/Flk-1, HCG (p < 0.05), and α-Fetoprotein (AFP) was notable in stem cells. In addition, there was an elevated expression of E-cadherin  in mSG-SCs of p.2 with significant decrease in p.10 (p < 0.01). On the other hand, Snail which suppresses E-cadherin and is a marker of stem cells was overexpressed from at p.2 to p.10, (p < 0.01). Finally, oncoprotein p63/TP73L/TP63 (p < 0.01) was downregulated indicating a decrease in the proliferative potential of the mSG-SCs during passaging.To complete the characterization of mSG-SCs cultured with CCM in p.2 and p.10 using different markers, as shown in Fig. p < 0.05). For StemPro, the expression of BMP-2 showed a statistically significant increase already from p.6 (p < 0.01), while in StemMacs, the baseline of BMP-2 was already increased compared to the other two culture media in p.2 (p < 0.01) and showed a further statistically significant increase up to p.10 (p < 0.01). In Fig. p < 0.05 for CCM and p < 0.01 for StemMacs and StemPro). For StemPro, it was observed a statistically significant difference in expression compared to CCM at p.6 (p < 0.05) and p.10 (p < 0.01). Similarly, for StemMacs, ALP expression demonstrated moderate expression values between CCM (lower) and StemPro (higher). Especially in p.10, ALP expression was statistically higher compared to CCM (p < 0.01). For ACAN  for CCM. For the marker SOX-9  and higher in relation to CCM (p < 0.05). At p.6, SOX-9 demonstrated an increase in the expression for StemMacs compared to p.2 and even statistically higher compared to CCM (p < 0.05) and StemPro. Finally at p.10, there was an overexpression of SOX-9 for StemPro compared to CCM/StemMacs (p < 0.05).The results for the potential for osteogenic, chondrogenic, and adipogenic differentiation vary significantly among three different culture media , as shown in Fig. p < 0.05) and StemPro (p < 0.01). In addition, there was a statistically significant increase in LPL expression for StemMacs compared to StemPro (p < 0.01). Ultimately, the transcription factor PPAR-γ2  and StemPro (p < 0.05). Finally, at p.10, the expression of PPAR-γ2 for StemMacs was statistically higher compared to CCM  f-γ2 Fig. f was uprp < 0.01 for p.2 and p.6 and p < 0.05 for p.10 with maximum expression on day 7 for the latter). For StemMacs, no change in baseline expression was observed at all passages. Also, for StemPro, there was a statistically significant increase in ALP expression  at day 7. In Fig. p < 0.05). Finally, for p.10, there was no statistically significant changes between days 0, 7, and 14. For StemMacs, p.2 and p.6 demonstrated similar expression pattern for BMP-2 at days 0, 7, and 14. Similar pattern was observed in p.10 with statistically significant decrease at day 0 to days 7 and 14 (p < 0.05). For StemPro, in p.2, there was increase in the expression at day 0 to day 7, and then, there was a statistically significant decrease up to day 14 (p < 0.01). In p6 and p.10, there was a statistically significant increase in the expression of BMP-2 between day 0 to 7 and 14 (p < 0.01). In Fig. p < 0.01). Finally in p.10, there was a non-significant increase at day 7 and later a decrease at day 14. For StemMacs, there was no significant expression for BGLAP in all passages. For StemPro, there was a notable increase in BGLAP expression at days 7 and 14 for all three passages  (p < 0.01). To further evaluate the osteogenic differentiation, Alzarin staining (AR-S-based assay) is used as shown in Fig. p < 0.01, and StemMacs > CCM, p < 0.01) at early passages. In p.6, there was an increase in in vitro biomineralization for CCM and in contrast a decrease for StemPro and StemMacs. Finally, in p.10, there was a decrease of the in vitro biomineralization for StemMacs, whereas for CCM, there was no apparent difference. Also, there was a statistically significant increase for CMM (p < 0.01) and StemPro (p < 0.01) compared to StemMacs. As far as chondrogenic differentiation is concern, several markers were evaluated such as ACAN and SOX-9. In Fig. p < 0.01, p.6, p < 0.05, p.10, p < 0.01). For StemMacs, there was a statistically significant increase in the expression in p.2 at day 7 and day 14 compared to day 0 (p < 0.01). Similarly in p.6 there was a gradual increase at day 7 and day 14 compared to day 0 (p < 0.01). Finally, in p.10, there was no statistically significant expression between days 7 and 14 compared to day 0, indicating the inability of mSGCs to differentiate towards a chondrogenic phenotype after several subculturing. For StemPro, there was an apparent expression of ACAN in all passages. Specifically, in p.2, there was a gradual significant increase between days 0 and 7 and day 14 (p < 0.01). In p.6, there was a statistically significant increase between day 0 and day 7 (p < 0.01) and then a slight decrease at day 14. Finally, in p.10, there was a significant increase in ACAN expression at day 7 and then a decrease at day 14 (p < 0.01). In Fig. p < 0.01) in p.2. However, in p.6, there was an increase in expression at day 7 followed by a stabilization in the expression at day 14 (p < 0.05). Finally, in p.10, there was a non-statistically significant increase between days 0 and 7 and day 14. For StemMacs, there was a non-statistically significant increase in the expression in all passages  and at all days . For StemPro, there was a statistically significant increase in the expression in p.2 at day 7 followed by a decrease at day 14 compared to day 0 (p < 0.01). In p.6, there was a statistically significant increase between days 0 and 7 and day 14 (p < 0.01). Finally, in p.10, there was a non-statistically significant increase in the expression at day 7.These assays intended to elucidate if there were considerable variations in gene expression patterns through extended growth, as well as indicate changes of the differentiation potential of mSG-SCs at early, middle, and late passages. The evaluation of the differentiation potential towards osteogenic and chondrogenic phenotype is crucial for the determination of “stemness” of the cultured cell populations under variable microenvironmental conditions. Several osteogenic and chondrogenic markers are evaluated in mSG-SCs cultures  at different passages  in a time response experiment , as shown in Fig. The salivary glands’ pathology leads to a negative outcome in patients’ QoL, which makes the need for rehabilitation crucial  may be found throughout the acinar and ductal epithelium  or found at the epithelial lining of a specific type of ducts named intercalated ducts  cells have gained increasing attention given that they are stem/progenitor cells that are actively involved in salivary gland development, homeostasis maintenance, and regeneration following injury  followed by a significant decrease at a later passage (p.10), in combination with simultaneous increase of Snail expression during the same passages (p.2–p.10), confirmed the morphology of the cell populations but also the profile expression of the keratins. This observation coincides with the fact that initially the stem cells of the salivary glands co-exist with an epithelial population of glandular cells that over time they are replaced by mesenchymal stem cells which eventually become the majority.The increase in the oncoproteins p63/TP73L/TP63 indicated the extensive transition of these cells to a mesenchymal stem cell population with strong proliferative potential. Further study is needed in cell cultures with culture media without FBS as in the proteomic technique that was used in this study; we were limited to culturing salivary gland stem cells in CCM as control. Criterion for the efficiency of a stem cell culture system after subculturing in prolonged incubation periods is the aging (cellular senescence). The evaluation of aging of stem cells is important since several morphological, metabolic changes occur. Furthermore, it is noticed secretion of inflammatory factors  with a negative impact on the possibility of their clinical application  and epithelial characteristics (expression of specific keratins and E-Cadherin) of glandular epithelium. In addition, the analysis of our results showed the strong potential of these cells for trilineage differentiation. This needs further investigation because the potential of multilineage differentiation of mSGSCs to other glandular tissues, for example, pancreas, liver, kidneys, or thyroid among others, could be of therapeutic value for tissue regeneration and clinical applications. In the second part of the study, the characteristics of mSGSCs, in terms of maintaining their stem cell properties (stemness), during prolonged incubation periods had been investigated, as it represents a critical aspect for regenerative medicine applications. These technical characteristics involved the isolation, proliferation, and evaluation of aging (cellular senescence) with different culture media (StemPro and StemMac) to meet cGMP requirements towards a safe and effective future stem clinical application of mSGSCs according to international guidelines. Based on the results, it appeared that the isolated mSGSCs have a good proliferation rate in serum-free media  compared to conventional serum medium (CCM) and maintained their stem cell properties (stemness) and their differentiation capacity after prolonged incubation periods (multiple subculturing) especially in the StemPro medium. Future approaches may include the investigation of the integration of additional factors in this type of culture media that may enhance media’s effectiveness substituting the absence of serum with a—in parallel—further investigation by means of pre-clinical studies with experimental animal models to recapitulate the in vivo analogue.