Brassica pekinensis). Results indicated that concentrations of PA ranging from 0.03 g L−1 to 0.05 g L−1 inhibited stem browning and decreased the rate of respiration, electrolyte leakage, and weight loss, as well as the level of malondialdehyde (MDA) in freshly harvested mini-Chinese cabbage stored at 25 °C for 5 d. The PA treatment enhanced the activity of antioxidant enzymes (ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), 4-coumarate:CoA ligase (4CL) and phenylalamine ammonia lyase (PAL)), and inhibited the activity of polyphenol oxidase (PPO). The PA treatment also increased the level of several phenolics  and flavonoids . In summary, results indicate that treatment of mini-Chinese cabbage with PA represents an effective method for delaying stem browning and maintaining the physiological quality of freshly harvested mini-Chinese cabbage due to the ability of PA to enhance antioxidant enzyme activity and the level of phenolics and flavonoids during 5 d.The effect of palmitic acid (PA) on stem browning was investigated in freshly harvested mini-Chinese cabbage ( Brassica pekinensis (Lour.) Rupr.) is a green leafy vegetable in the family Cruciferae  × V}/(VS × m × 1000), where V is the total volume of the extracting solution, mL; Vs is the measured volume of the extracting solution, mL; and m is the weight of the sample, g.MDA content was determined by the method of Xu et al. with minor modifications . Samples2O), and extracted for 40 min at 40 °C. The mixture was then centrifuged  at 12,000× g for 25 min at 4 °C. The supernatant was used for measuring total phenolics and flavonoids. Total phenolics were determined using Folin–Ciocalteu (FC)  reagent by the method of Fan et al. [2O and 200 μL FC reagent were prepared and then 400 μL supernatant was added to the test tube, vortexed, and incubated about 3 min at 20 °C. Subsequently, 400 μL 20% (w/v) Na2CO3  and 1.2 mL dH2O were added to the mixture. Then a water bath  was prepared at 20 °C for 60 min. Absorbance of the sample solutions at 760 nm were then measured in a spectrophotometer  and used to determine the level of total phenolics based on the use of a standard curve constructed using different concentrations of gallic acid .Frozen samples of cabbage (2 g) were powdered and mixed with 10 mL 80% ethanol   AlCl3  and 1 mL 5% (w/v) NaNO2  were mixed. After 5 min, 1 mL of 1.0 mol L−1 NaOH  was added to the mixture. Catechinic acid  was used as a standard to calculate the flavonoid content. Absorbance of the resulting sample solution was measured at 510 nm and used to determine flavonoid content. The concentration of total phenolics and flavonoid content were expressed as g kg−1.Flavonoid content was measured using the method by Zhou et al.  with somg for 15 min. The supernatants were utilized in the HPLC analysis. Conditions were as follows: utilization of an Eclipse Plus C18  column, with a temperature of 30 °C. The detector was a UV-detector (280 nm). The mobile phase consisted of 1% of formic acid-water (A) and methanol (100%) (B) and the gradient elution conditions were 0–3 min, 15% to 30% B; 3–35 min, 30% to 45% B; 35–45 min, 45% to 65% B; 45–50 min, 65–15% B.The level of specific phenolics   and specific flavonoids   were determined by HPLC  according to the method by Xu et al. . Briefly−1 phosphate buffer solution  Biotechnology Co., Ltd., Beijing, China)), and then centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was utilized to determine CAT and APX activity.CAT and APX enzyme activity was assessed according to the method by Wang et al. . Brieflyv/v) dH2O2, and 1.9 mL 0.05 mol L−1 PBS (pH 7.8) were mixed. Absorbance of the resulting solution was determined at 240 nm to calculate CAT activity. APX activity reaction mixture included 1.2 mL supernatant, 2.6 mL of 0.1 mmol L−1 EDTA  and 0.5 mmol L−1 AsA  , and 0.3 mL of 2 mmol L−1 dH2O2. Absorbance was measured at 290 nm to calculate APX activity.Amounts of 0.1 mL of supernatant, 1 mL of 0.3% (−1 of PBS (pH 6.4), containing 0.05 g PVPP. The resulting mixture was then centrifugated at 12,000× g for 30 min at 4 °C. Next, 0.1 mL of supernatant was mixed with 0.6 mL of 50 mmol L−1 catechol  substrate to measure PPO activity, while 0.9 mL of 0.2% guaiacol  (v/v) was mixed with 1 mL of 0.3% dH2O2 (v/v) to measure POD activity. Absorbance was measured at 410 nm and used to calculate PPO activity and at 470 nm to calculate POD activity.PPO and POD activity were determined by the method of Zhou et al. with slight modification . Frozen −1 borate buffer  including 5.0 mmol L−1 2-mercaptoethanol  and 0.4 g PVPP. The resulting mixture was then centrifuged at 12,000× g for 20 min at 4 °C. An amount of 0.3 mL supernatant was added to 0.7 mL of 100 mmol L−1 l-phenylalanine  and 3 mL of 50 mmol L−1 borate buffer . The mixture was incubated at 40 °C for 1 h and then 0.1 mL of 5 mmol L−1 HCl  was added to stop the reaction. Absorbance of the solution was measured at 290 nm to calculate PAL activity.The assessment of PAL activity was conducted by Kamdee et al. with slight modification . Brieflyg for 10 min at 4 °C. Absorbance at 333 nm was measured to calculate 4CL enzyme activity.The level of 4CL enzyme activity was determined using a commercial assay kit . Briefly, 2.0 g of sample were homogenized in 10 mL of extraction buffer, and the resulting mixture was then centrifuged at 8000× p < 0.05 were considered significant. All results presented are means ± standard deviation (SD).SPSS ver. 22  software was used to conduct the statistical analyses. Data were subjected to a two-way ANOVA and mean separations were performed using a Pearson’s multiple range test. In the case of single mean comparisons, data were subjected to an LSD analysis in which differences at −1 PA had the lowest BI relative to the control and to cabbages treated with lower concentrations of PA.The visual color of cabbage stems is an excellent indicator of their degree of browning. The level of stem browning in the control group greatly reduced their visual quality over the 4 d of storage A. CabbagL*, a*, and b*. L*, a*, and b* in stems of PA-treated and control groups over 5 d of storage at 25 °C. The level of L* in both control and PA-treated samples decreased during storage; however, in the control group the L* value was lower than it was in PA-treated cabbages during the entire course of storage. In contrast, a* and b* exhibited an increasing trend, with the level of increase in the different treatment groups ranging from the control > 0.03 g L−1 PA > 0.04 g L−1 PA > 0.05 g L−1 PA.The BI is calculated from the values obtained for −1 PA), 11.782 (0.04 g L−1 PA), and 10.625 (0.05 g L−1 PA) mg CO2 kg−1 h−1.The respiration rate exhibited a rapid increase after cabbages were placed in storage, reaching its highest level after 2 d, after which the respiration rate decreased and maintained a relatively stable value A. Peak r−1 PA-treated sample group after 5 d.The percentage weight loss increased in all groups B; howeve−1 PA-treated cabbages exhibited a similar pattern to each other and were higher than they were in the 0.05 g L−1 PA-treated samples. Electrolyte leakage had increased to 0.216%, 0.192%, and 0.181% in the 0.03, 0.04, and 0.05 g L−1 PA treatment groups, respectively, and 0.290% in the control group, after 5 d.Electrolyte leakage also showed an increasing trend in all groups C. The in−1 PA-treated samples were 1.868, 2.111, and 1.213 μmol kg−1, compared to the control group (3.080 μmol kg−1) of mini-Chinese cabbages.MDA levels increased before the first 3 d of storage in all treatment groups, and then declined D. Lower −1 PA-treated group, 0.108 units in the 0.04 g L−1 PA-treated group, and 0.095 units in the 0.05 g L−1 PA-treated group, all of which were lower than that in the control group.PPO activity in the control group was higher than it was in the PA-treated samples A, exhibi−1 PA-treated samples, respectively.The enzymatic activity of 4CL rapidly increased in the first 2 d and then fluctuated from 2 to 5 d. Higher activity was observed in the PA-treated group. After 2 d of storage, 4CL activity was 800.312, 837.459, and 828.569 units in the 0.03, 0.04, and 0.05 g L−1 PA-treated samples exhibited the highest level of activity (93.600 units).Changes of PAL activity in the control and PA-treated groups are shown in −1 PA treatment groups, respectively.An increase in APX activity was exhibited in the control and PA-treated groups during the first 2 d and then declined D. APX acThe data indicated that CAT activity in all groups reached a maximum at 3 d and then decreased E. Althou−1 PA treatment groups, respectively.POD activity reached a maximum after 2 d of storage and then remained stable F. POD ac−1, 1.863 g kg−1, 2.331 g kg−1, and 1.658 g kg−1 in the 0.03 g L−1 PA, 0.04 g L−1 PA, 0.05 g L−1 PA and control treatment groups, respectively.The level of total phenolics increased in both the control and PA-treated groups over the 5 d A. Althou−1) was observed at 3 d in the 0.05 g L−1 PA-treated group.Cinnamic acid content gradually increased in all groups B. The hi−1, 5.067 mg kg−1, 5.615 mg kg−1, and 6.873 mg kg−1 in the control, 0.03 g L−1 PA, 0.04 g L−1 PA, and 0.05 g L−1 PA treatment groups, respectively. The chromatogram and standard curve are in The content of gallic acid exhibited an initial decrease at 1 d of storage and then an increasing trend in all groups from 2 to 5 d C. The co−1 PA-treated samples exhibited a higher level (20.632 and 20.293 mg kg−1) of catechin.Catechin content exhibited an increasing trend in the control and PA-treated groups during the first 4 d and then declined D. At 4 d−1 (0.03 g L−1 PA), 20.866 mg kg−1 (0.04 g L−1 PA), 29.859 mg kg−1 (0.05 g L−1 PA), and 17.276 mg kg−1 (control).Chlorogenic acid content exhibited an increasing trend during storage, with PA-treated groups having a higher content than that in the control group E. Chloro−1 PA treatment group exhibited a change at 3 d (7.640 mg kg−1), a level that was much higher than in other groups.The level of p-Hydroxybenzoic acid showed an enhanced trend during storage in PA-treated groups F. The le−1, 0.04 g L−1, and 0.05 g L−1 PA treatment groups at 3 d of storage when levels increased to 3.818 mg kg−1, 3.822 mg kg−1, and 9.083 mg kg−1, while the level in the control group remained stable  was observed in the 0.05 g L−1 PA-treated group and was higher (1.63 times greater) than in the control group.Flavonoid content exhibited the same trend as total phenolics, increasing with storage time in all the treatment groups A. The hi−1 PA treatment group than it was in the control group during all the storage time.Quercetin content increased before the first 4 d and then decreased B. Querce−1, 0.04 g L−1, 0.05 g L−1 PA treatment groups, at which luteolin content was 6.788 mg kg−1, 7.418 mg kg−1, and 9.109 mg kg−1, respectively.Luteolin content increased during the first day and then decreased in all groups C. Peak cKaempferol levels greatly increased in PA-treated groups D. In con−1 and 0.05 g L−1 PA-treated groups than it was in other groups from 1 to 3 d.Isorhamnetin content exhibited a decreasing trend in all groups E. Isorhaa*, b*, weight loss, electrolyte leakage, MDA content, and 4CL activity. BI levels were negatively correlated with L* (Pearson coefficients were used to determine the correlation between the different measured parameters A. The an with L* A. Total  with L* B.Previous studies have indicated that tissue browning in plants induces an increase in the level of PA, which may explain why exogenous PA can inhibit browning ,15. MiniThe commercial value of mini-Chinese cabbages is dependent on their quality. Thus, it is essential to maintain their quality during storage. Weight loss and high respiration rates are known indicators of quality degradation in mini-Chinese cabbage and reports have indicated that strategies that suppress respiration and reduce evaporation in harvested plants help to maintain their postharvest quality . In our Browning is a complex process that can be affected by many enzymes and chemical compounds, in which tissue color is the most recognizable visual manifestation. In our study, the PA treatment delayed the process of stem browning, as indicated by a reduction in the BI in stems of mini-Chinese cabbage, relative to the control. High levels of PPO activity are known to induce enzymatic browning of plant tissues during storage by catalyzing the oxidation of phenolics to quinones . In our Browning is a great stress in wounding fruit and vegetables during postharvest, a significant postharvest physiological disorder. Plant cells subjected to stress respond by activating two aspects of phenolic metabolism . One invTreatment of mini-Chinese cabbages with PA greatly inhibited weight loss, reduced the rate of respiration, and inhibited stem browning, which collectively served to maintain the overall postharvest quality of mini-Chinese cabbage stored at 25 °C for 5 d. The mechanism underlying the inhibition of stem browning in mini-Chinese cabbage by PA was associated with decreased levels of membrane damage, as evidenced by lower levels of electrolyte leakage and MDA, and an increase in antioxidant metabolism, as evidenced by higher antioxidant enzyme activity, and elevated levels of phenolics and flavonoids. The collective results of the present study determine that the application of PA has the potential to be used as a method to maintain the quality and to extend the shelf life of mini-Chinese cabbage after harvest and during storage.


Ultrasound combined with high temperatures (thermosonication) is an alternative to thermal treatments applied for juice preservation purposes. Blend juices, such as orange-carrot juice, are an interesting option for consumers due to their diversity of unique flavors. The main aim of the present study is to investigate thermosonication’s impact on the overall quality of an orange-carrot juice blend over 22-day storage at 7 °C, in comparison to thermal treatment. Sensory acceptance was assessed on the first storage day. The juice blend was prepared based on using 700 mL of orange juice and 300 g of carrot. The effect of ultrasound treatment at 40, 50, and 60 °C for 5 and 10 min, as well as of thermal treatment at 90 °C for 30 s, on the physicochemical, nutritional, and microbiological quality of the investigated orange-carrot juice blend was tested. Both the ultrasound and the thermal treatment could maintain pH, °Brix, total titratable acidity, total carotenoid content, total phenolic compounds, and the antioxidant capacity of untreated juice samples. All ultrasound treatments improved samples’ brightness and hue value, and made the juice brighter and redder. Only ultrasound treatments at 50 °C/10 min and at 60 °C/10 min have significantly reduced total coliform counts at 35 °C. Thus, they were selected along with untreated juice for sensory analysis, whereas thermal treatment was used for comparison purposes. Thermosonication at 60 °C for 10 min recorded the lowest scores for juice flavor, taste, overall acceptance, and purchase intention. Thermal treatment and ultrasound at 60 °C for 5 min recorded similar scores. Minimal variations in quality parameters were observed over 22-day storage in all treatments. Thermosonication at 60 °C for 5 min has improved samples’ microbiological safety and resulted in good sensorial acceptance. Although thermosonication has the potential to be used in orange-carrot juice processing, further investigations are necessary to enhance its microbial effect on this product. Juices are a good option to help increase fruit and vegetable intake by the population ,2. BasedDespite the benefits deriving from juice intake, this product spoils quite fast due to microorganisms’ and enzymes’ action, and this process poses a challenge to the juice industry ,10. Heat+ and OH−) are formed, diffused to the bubbles’ inner part, and released during collapse [Ultrasound (US), also known as sonication, is an emerging technology capable of generating and emitting high-intensity ultrasonic waves at a frequency higher than 16 kHz. Cavitation—i.e., microbubbles’ formation, increase, and collapse—is US’s primary mechanism. Ultrasonic waves induce the formation of alternate compression and expansion zones. Constant changes in this region lead to bubbles’ increase and collapse because of an inner pressure increase that, at a certain time, becomes higher than the pressure outside them. Free radicals  of this technology in fruit and vegetable juice decontamination processes has shown a low microorganism lethality rate; moreover, it failed to reduce the microbiological charge in juices, even after 30-min exposure to it ,20. Howe–25 °C ofOn the other hand, US application in juices should not be limited to ensuring their microbiological quality. This technology must be capable of maintaining juices’ vitamins, anthocyanins, phenolic compounds, and antioxidant capacity, as well as important sensory features, such as flavor and color . Thus, sDespite the aforementioned factors, the impact of this technology on both overall quality during storage and sensory parameters of blended juices still needs to be explored. Thus, the aim of the current study was to assess the effect of thermosonication treatment on orange-carrot juice’s physicochemical , nutritional quality , and microbiological charge  during storage, as well as on its sensory acceptance.p-value > 0.05). Means recorded for pH, °Brix, and TTA reached 4.11 ± 0.03, 8.46 ± 0.06, and 0.40 ± 0.01, respectively , without significant differences between treatments. This finding indicates that the US has maintained these features throughout storage, similar to those of untreated and thermally treated juices. Linear and quadratic regressions were applied to pH, °Brix, and TTA based on storage time. However, there was no adjustment for a regression model. Then, Variations in these parameters were only observed during storage time (Spondias mombin L.) juice thermosonication  preserved its pH, °Brix, and titratable acidity [Hog plum .Color is a critical quality parameter affecting products’ acceptance by consumers. Treatment and storage time have significantly affected orange-carrot juice color , on average, in comparison to untreated samples, after processing. Thermal treatment results were statistically similar to the ones observed for the untreated sample  by 3.13%  was observed between thermosonication and thermal treatment. Still, the herein adopted treatments significantly decreased the hue° value by 3.16%, on average, in comparison to the control sample . All treatments led to increased blended juice chroma values; however, results recorded for the thermal treatment and for US, conducted at 40 and 50 °C (both for 5 min), were statistically similar to the ones recorded for the untreated sample.No difference in hue° values . Linear and quadratic regression analyses were applied to the investigated variables based on storage time. However, no significant models and coefficients were obtained; thus, Color parameters were affected by storage time, with differences between treatments .p-value > 0.05) , although no differences were observed between treatments.All treatments have maintained the juice blend’s total carotenoids (TC), total phenolic compounds (TPC), and antioxidant capacity, based on the DPPH assay ( > 0.05) . Storagep-value > 0.05) TC content on the first day (mean = 121.24 ± 11.23 µg/100 mL). These compounds only underwent more variations on the 18th storage day (Treatments did not affect (rage day . CarotenTreatments have maintained TPC (mean = 59.67 ± 4.51 mg GAE/100 mL) and antioxidant capacity (mean = 20.54% ± 3.95%) after processing (day 1). Modest antioxidant capacity was observed between the 1st and 8th storage days. Minor variations took place over the 22-day storage, without significant differences among treatments . InteresSimilar results were observed after apple-carrot juice manothermosonication , and no significant differences in TPC and antioxidant capacity were observed between treated samples and the control during storage time . Some au2; 10 and 30 min; 10 ± 2 °C). Moreover, US processing increased DPPH values recorded for melon juice, which reached 45%, in comparison to 39% and 29% recorded for pasteurized and untreated juice samples, respectively. However, samples’ total phenolic content decreased by 33% after US processing. Another study [Rodríguez-Rico et al.  recentlyer study  investigDifferent results were observed for bioactive compound contents and antioxidant capacity of juices treated with US. These differences took place because the US effect depends on operating conditions , as well as on sample features.Free radicals  are often formed during ultrasound treatment. This occurs because samples are exposed to oxidation processes that can increase nutrient degradation, depending on the adopted processing conditions. However, cavitation favors the elimination of dissolved oxygen molecules involved in oxidation processes, and it prevents bioactive compounds from being affected during ultrasonic processing. We did not observe an increase in phenolic compounds’ content and antioxidant capacity in the analyzed samples. The hypothesis raised to explain the herein observed result is that ultrasound may have favored oxidation reactions faster than compounds’ release, which led to no difference between samples. Furthermore, oxidation degradation, and decreased bioactive compounds and antioxidant capacity during storage time, were previously described due to the action of pectin methyl esterase (PME) . MoreoveAlthough a longer exposure time and plant cell wall rupture may favor several oxidation reactions and compromise parameters, such as sample color , all ultp-value ≤ 0.05). E. coli. The natural microbial count applied to the blended juice showed higher contamination by yeasts and molds (6.54 ± 0.25) than by aerobic mesophilic (4.75 ± 0.05) and coliform bacteria, at 35 °C (3.24 ± 0.66).The herein applied treatments promoted significantly different microbial reductions in the blended juice , whose aerobic mesophilic bacteria counts decreased to 0.60 and 1.38 log CFU/mL, respectively , in comparison to the untreated sample. However, the highest reduction (1.38 log) in this parameter was observed for the most intense US treatment (60 °C/10 min). Furthermore, only yeast and mold counts conducted after US treatment at 60 °C, for 10 min, reached values statistically different from counts conducted after heat treatment . Only US treatment at 50 °C, for 10 min, and at 60 °C, for both investigated times, differed from the thermal treatment  in coliforms’ inactivation.US treatments conducted at 40 °C, and thermal treatment, slightly reduced coliform counts at 35 °C, and values ranged from 0.24 to 0.58 log CFU/mL. However, they were statistically similar to values recorded for the untreated sample . However, none of the assessed treatments achieved the 5-log reduction recommended by the FDA. Ultrasound at 60 °C promoted a reduction of 1.38, 1.38, and 2.15 log CFU/mL for aerobic mesophilic bacteria, molds and yeasts, and coliforms at 35 °C, respectively. To obtain more expressive results, it would be important to increase the treatment time and/or combine ultrasound with another preservation method, but this could increase energy expenditure, extend the processing time, and would affect juice quality.Different from the less intense treatment (40 °C), the increased processing time of ultrasound treatments conducted at 50 and 60 °C enhanced the inactivation of aerobic mesophilic bacteria, yeasts, molds, and coliforms, at 35 °C. The most significant reductions were observed after ultrasound application at 60 °C/10 min in all microbial groups  may have compromised heat transfer and cavitation action, requiring more energy. Thus, higher a microorganism inactivation rate was observed in the most intense treatments. Nevertheless, the operating conditions adopted in the present study were not enough to have a sizeable lethal effect on microorganisms, and it may have enabled their growth during storage time. Therefore, future studies should investigate whether an increase in temperature and/or treatment time would affect blended juices’ physicochemical and nutritional parameters. Moreover, the association among ultrasound, temperature, and other technologies, such as microwaves and pressure, among others, should be taken into consideration.n = 103). Participants’ ages ranged from 18 to 56 years, with a mean age of 22.8 years. In total, 77.7% (n = 80) of participants were female. With respect to intake frequency, 40.9% of participants reported to drink juice on a daily basis, whereas 39.8% of them did so on a weekly basis.Sensory analysis is essential to investigate consumer acceptance in advanced studies focused on applying new food processing technologies. In total, 109 evaluators participated in this research stage, but 6 questionnaires were excluded from the analysis due to filling errors. Thus, 103 participants were taken into consideration for the analysis  in color was observed between samples. The mean color value (7.58 ± 0.07) classification ranged from “I moderately like it” to “I like it very much”, and they indicated that the naked eye could not see the variations observed in instrumental color analysis. Aroma, appearance, consistency, taste, global acceptance, and purchase intention scores were statistically equal between the thermally treated and ultrasound-treated samples (at 60 °C for 5 min). This finding indicates that consumers equally accepted both samples. Small scores recorded for flavor, taste, overall acceptance, and purchase intention were observed for ultrasound-treated juice at 60 °C, for 10 min, and they were represented in the hedonic scale as “I slightly dislike it” and “I did not like or dislike it”  were not enough to disrupt covalent bonds. Therefore, compounds accounting for products’ aroma, flavor, and color remained the same . HoweverNo sensory changes were described in the taste, color, aroma, or consistency of strawberry-apple-lemon juice treated with US, at 35 °C, for 10 min . SonicatConsumers’ acceptance of products developed based on new technologies can be a challenging task to accomplish. Therefore, it is essential to conduct analyses focused on ensuring products’ sensory aspects based on consumers’ desire, rather than just on assessing their individual microbiological or physicochemical aspects. Each food’s feature can be positively or negatively affected after ultrasound treatment. Thus, it is crucial to assess different operating conditions to help in developing well-accepted products. The highest temperature and the longest exposure time (US 60 °C/10 min) adopted in the current study enhanced microbial inactivation, but they did not preserve the sensory features of the investigated juice.Citrus sinensis L. Osbeck) cultivar “Pêra” and carrots (Daucus carota L.), without physical damage and free from microbial deterioration visible to the naked eye, were purchased from a local market , washed in tap water, and sanitized with 100 mg/L of sodium dichloroisocyanurate  for 10 min. The orange juice was then extracted . Carrots were peeled and sliced with the aid of a previously sanitized stainless-steel knife. Orange-carrot juice was prepared (700 mL of orange juice and 300 g of carrot) in a blender without the addition of water or any other ingredients to it, according to preliminary tests. Filtered juice samples (400 mL) were stored in previously sterilized glass bottles and subjected to preservation treatments.A completely randomized design was adopted based on following a subdivided plot model conducted in triplicate. Orange  at 40, 50, and 60 °C, for 5 and 10 min. Ultrasound-treated samples were placed at the center of the equipment . Thermal treatment was applied in a water bath at 90 °C, for 30 s, for comparison purposes. Samples were removed from the hot water bath and cooled in an ice bath right away. Untreated orange-carrot juice was used as a control.Samples were subjected to US treatments in bath-ultrasound equipment , away from light, and kept at 7 ± 1 °C until analysis. Sample analyses were carried out after 1, 4, 8, 18, and 22 days of storage, as described in the following sections.®, mPA210, Piracicaba, São Paulo, Brazil), based on using 10 mL of juice, at 25 °C. Total °Brix value was determined with the aid of an analogical refractometer  at 25 °C. Total titratable acidity (TTA) was determined based on using potentiometric titration with 0.1 M sodium hydroxide (NaOH) solution, under agitation, at pH up to 8.2. Sodium (NaOH) volume was used to calculate samples’ TTA, and results were expressed as g of citric acid per 100 mL of juice (g/100 mL) [Sample pH was measured with a calibrated pH meter (Tecnopon/100 mL) .®, model CR-5, Tokyo, Japan). Brightness (L*), redness (a*), and yellowness (b*) values were recorded, whereas hue angle (h°) and chroma (c*) values were calculated based on Equations (1) and (2), respectively [a* and b* are values recorded for the juice’s redness and yellowness components, respectively.Color was measured in a previously calibrated spectrophotometric colorimeter used to determine reflectance and transmittance (Konica Minoltaectively :(1)h°=ta® Series 2000–325 A 1000 nm, Piracicaba, São Paulo, Brazil) at 450 nm. Results were calculated based on Equation (3) and expressed as micrograms of β-carotene per 100 mL of blended juice (µg/100 mL).Total carotenoid content was extracted and measured based on Rodriguez-Amaya , with slv/v) and stirred at 180 rpm, at 21 ± 1 °C, for 15 min. After that, the resulting mix was centrifuged at 1413× g for 5 min, and the supernatant was transferred to test tubes.Total phenolic compound extracts and the juice’s antioxidant capacity were analyzed as described by Bloor . Then, 1®, model AP59, 127/220v, Curitiba, Paraná, Brazil). Sample absorbance was measured at 765 nm , after a 2-hour incubation. Results were expressed as milligrams of gallic acid equivalent (GAE) per 100 mL of blended juice (mg GAE/100 mL), based on the calibration curve , wherein y is absorbance at 765 nm and x is milligram of GAE per mL of juice.Total phenolic compounds (TPC) were analyzed in spectrophotometer, based on the Folin–Ciocalteau method , with sl®, São Paulo, São Paulo, Brazil) assay [®, model AP59, 127/220v, Curitiba, Paraná, Brazil) and incubated at 21 ± 1 °C, for 1 h. Absorbance decrease was measured at 517 nm, in a UV/Vis spectrophotometer . Radical scavenging capacity was expressed as the DPPH radical inhibition rate and calculated based on Equation (4) [control and Asample correspond to control and sample absorbance at 517 nm, respectively.Antioxidant capacity was tested through the 1.1-diphenyl-2-picrylhydrazyl  assay . Aliquottion (4) :(4)DPPH ®, Mumbai, Maharashtra, India) for aerobic mesophilic bacteria enumeration and counted after 48 h at 35 ± 1 °C. Molds and yeasts were analyzed based on the spread-plated technique in Potato Dextrose Agar , and plates were incubated at 25 ± 1 °C for 5–7 days [Escherichia coli were analyzed in Petrifilm plates  and incubated at 35 °C for 48 h. The analysis was carried out in three repetitions, and plating was performed in duplicate. Results were expressed in CFU/mL.The juice was homogenized under aseptic conditions, and appropriate dilutions with 0.1% sterile peptone water were prepared for natural juice contamination analysis. Samples were pour-plated in Plate Count Agar . The sensorial analysis was performed based on the sensory acceptance test and the purchase intention. Juices were prepared one day before analysis and kept at 7 ± 1 °C. Tests were conducted in individual booths under white lighting. A nine-point hedonic scale  was applied to assess attributes such as juice samples’ color, aroma, appearance, consistency, taste, and overall acceptance. The nine-point hedonic scale was also used to assess purchase intent . Consumers received approximately 60 mL of each juice sample in transparent plastic cups coded with three random digits, in random order. Participants also received a glass of water to clean their palates before each assessment.p-value ≤ 0.05), whereas regression analysis was used to assess significant differences  over storage time. Linear and quadratic equation models were tested through regression analysis, based on storage time. Sensory analysis results were subjected to ANOVA and means were compared to each other through the Tukey test (α = 0.05). Data were analyzed based on using SAS® OnDemand for Academics software.Data were subjected to analysis of variance (ANOVA) to assess the influence of treatment and storage time on the investigated variables. Tukey’s test was used to analyze differences between treatments . On the other hand, this treatment condition resulted in consumers’ lower sensory acceptance of the sample.Thermosonication conducted at 60 °C, for 5 min, also reduced the sample’s microbial load, maintained the physicochemical features, color, and bioactive compounds, and resulted in good consumer acceptance, compared to untreated and thermal-treated samples. Therefore, the current study has evidenced that thermosonication has the potential to be used as an alternative for orange-carrot juice blend processing since it contributes to products’ physicochemical properties and bioactive compound content maintenance during storage time. Nevertheless, it is necessary to conduct further studies focused on increasing the processing time and/or temperature or combining it with other preservation methods since US could reduce the product’s microbial load. Still, it did not achieve the 5-log reduction recommended by the FDA.


Additionally, the incorporation of TS extended the IP and enhanced the shelf life of CPSPB. Particularly, the addition of a specific amount of TS (60%) to CPSPB resulted in the highest organoleptic quality. Therefore, these results emphasize the positive impact of TS on the overall quality of CPSPB, highlighting its potential to enhance the nutritional value, sensory attributes, and shelf life.This study aimed to investigate the influence of different ratios of taro slices (TS) on the nutritional quality, sensory quality, and shelf life of Chinese pickled and steamed pork belly (CPSPB). The study examined various aspects of CPSPB, including its proximate components, fat oxidation, fatty acid composition, protein hydrolysis, oxidation reaction, and induction period (IP). Additionally, the sensory quality and texture analysis were compared simultaneously. The results showed that the addition of TS to CPSPB significantly improved water and lipid loss ( The indColocasia esculenta (L.) Schott] is primarily cultivated in the southern province of China  of CPSPB under the addition of 0%–100% mass ratio TS. The sensory quality and texture analysis of these samples were concurrently compared. In line with this, the findings of this study are intended to serve as a valuable reference for domestic and industrial production, providing insights into the benefits of incorporating TS into CPSPB. Additionally, these results can be utilized as a foundation for expanding the utilization of TS, with potential economic benefits in various applications.2.2.1.Taro and pork belly with a lean-to-fat ratio of approximately 1:2 were purchased from a local food market in Yongzhou, Hunan Province, China. They were immediately transported in an ice box to the laboratory within 30 min after purchase. To ensure freshness, the taro and pork belly were stored in a plastic container with crushed ice at the bottom, maintaining a transport temperature of 5°C–10°C. The samples were prepared using traditional Chinese household cooking methods.Firstly, the fresh pork belly was pre-cooked in boiling water for 2 min using a pork-to-water ratio of 1:5 (w/v). After removing any remaining pig hairs from the skin and rinsing off surface impurities and blood with tap water, approximately 200 grams of pre-cooked pork belly were placed in a bowl and cut into pieces measuring 2.5 cm × 3.5 cm × 0.8 cm (length × width × height). These slices were then pickled for 30 min in a pickling jar containing a solution composed of 2% soy sauce, 2% sugar, 2% salt, and 0.1% monosodium glutamate. Next, the taro was carefully selected, peeled, and then cut into fillets of the same size as the pork belly using a slicing machine . It was important for the taro slices to be of uniform size and show no signs of rot on the surface. Different amounts of taro slices, ranging from 0 to 200 g in increments of 40 g, were combined with 200 g of pork in each sample. Finally, all ingredients were thoroughly mixed to ensure that the sample was well-blended for testing purposes.The samples were prepared and placed in a pressure cooker  for 15 min, where they were subjected to high-pressure steaming on an induction cooker . After the steaming process, the samples were left to cool for 5 min before the pressure cooker was opened and the samples were separated. The cooled samples were then either used for textural profile analysis and sensory evaluation or minced using a mincer  to ensure homogeneity and obtain representative samples for further analysis. Finally, the minced samples were stored at −20°C until analysis.For all the steaming experiments, seven groups were formed. These groups include: (I) The control group, which consisted of raw pickled pork belly. Following this, (II) CPSPB alone was examined. Subsequently, CPSPB was treated with different concentrations of TS. These concentrations included (III) 20% TS, (IV) 40% TS, (V) 60% TS, (VI) 80% TS, and (VII) 100% TS. For each determination, three replicates were conducted for all the treatments.2.2.The determination of moisture, crude protein, ash, crude fat, and sodium chloride content followed the guidelines outlined in the AOAC methods (2006) and were followed precisely during the analysis. The standard reference numbers and brief descriptions of the determination methods were as follows: moisture content was determined by oven drying the samples at 105°C by AOAC 950.46. Crude protein was determined using the Kjeldahl method, wherein the nitrogen-to-protein conversion factor of 6.25 was applied, as specified in AOAC 928.08. Ash content was determined by heating 3 g samples in an oven at 550°C until a constant weight was achieved, following the procedure outlined in AOAC 920.153. Crude lipids was determined through Soxhlet extraction, as described in AOAC 991.36. Moreover, NaCl contents was determined by Mohr method . The parameters were measured using the appropriate units specified in the standard, such as percentages or milligrams per gram.2.3.Total lipids were extracted from minced pork belly using the method of Wu et al.  with minThe following GC conditions were used for the experiment: nitrogen was employed as the carrier gas at a flow rate of 9 mL/min and a pressure of 6.6016 psi. The split ratio was set to 5:1. The injector temperature was set to 270°C with an injection volume of 1 μL, while the temperature of the flame ionization detector was maintained at 280°C. The temperature ramp-up procedure was as follows: a five-minute duration at 120°C, followed by an increase to 190°C at a rate of 5°C/min and hold for 12 min. Then, there was a further increase to 210°C at a rate of 2.5°C/min, and the temperature was held at 210°C for 10 min.2.4.The lipid oxidation coefficient was estimated using peroxide value (POV) determinations and thiobarbituric acid (TBA). The TBA determination method was adapted from Sorensen et al.  with min2.5.2.5.1.Two grams of minced pork belly were transferred to a pre-weighed centrifuge tube. Then, 10 mL of 20 mM sodium phosphate buffer (pH 6.8) was added to the tube. The mixture was homogenized twice with a homogenizer at 15,000 rpm and 4°C for 30 s. Afterward, the mixture was allowed to stand for 1 h, and subsequently filtered through Whatman No. 1 filter paper. The filtrate was stored at −20°C until further analysis. Finally, the protein concentration in the filtrate was determined using the BCA kit (Sigma), with bovine serum albumin used as the standard for calibration.2.5.2.The degree of hydrolysis of the protein sample was assessed using the TNBS assay, as described by Adlernissen  with sli2.5.3.Protein oxidation was assessed by measuring protein carbonyl and sulfhydryl levels. Protein carbonyl levels were determined using the method described by Ali et al.  with sli2.6.Texture profile analysis (TPA) was performed using a texture analyzer  equipped with a 5 mm diameter cylindrical probe according to Bourne’s method . Samples2.7.The evaluation team consisted of 16 members, evenly distributed between the sexes (8 men and 8 women), aged 20–25 years. All team members had received professional training in food science and sensory evaluation. For the sensory evaluation, quantitative descriptive analysis following the method established by Morita et al.  was used2.8.The oxidation tests were performed using the OXITEST reactor  as described by Verardo et al. . Each ch2.9.n = 3). Treatment effects were analyzed using SPSS 19.0  and one-way analysis of variance (ANOVA). The separation of means was determined through Duncan’s multiple-range test. Statistical significance was defined as p < 0.05, while p < 0.01 was considered statistically highly significant.Results from all experiments were presented as mean ± standard deviation. These experiments were performed in triplicate, with a sample size of three (3.3.1.p < 0.05) due to the pressure difference from the high-pressure steaming system, which caused lipids melting and liquefaction, and inorganic salt dissolution. Conversely, there was a significant decrease in the total lipids, ash contents and NaCl contents (p < 0.05) to 32.28 ± 0.60%, 2.94 ± 0.03% and 1.97 ± 0.02%, respectively. The alteration in fat content appeared to have the most significant influence on the composition of other components, which aligns with previous studies  in moisture, total lipid, and protein content. This indicates that the addition of 60% or more TS stabilized the major components’ content in the pork belly samples, possibly due to the physical barrier and diffusion-driven physicochemical properties of TS. It is plausible that some of the diffused salt from CPSPB may have transferred to the TS during the steaming process, leading to a reduction in the saltiness and graininess of the pork belly, thereby harmonizing its texture. Throughout the steaming process, the moisture contents of pork belly decreased from 42.74 ± 0.95% to 36.98 ± 0.73%, whereas the total lipids contents decreased from 32.28 ± 0.60% to 27.04 ± 0.34%. This reflects the capacity of TS to bind water and lipids, resulting in reduced moisture and lipid content of pork belly, thereby contributing to the extended shelf life of CPSPB.Additionally, there was a gradual decrease in the moisture, total lipid, sodium chloride, and ash contents of pork belly, leading to a gradual increase in total protein content. The inclusion of 60% or more TS demonstrated no significant difference (3.2.p < 0.05) in both POV and TBA values compared to the original samples. This reduction can be attributed to the exposure of the pork belly samples to the antioxidant agents such as phenolic compounds, resistant starch, and polysaccharides  in the pork belly samples. The increase in medium/short-chain MUFA and SFA may be attributed to the degradation of long-chain PUFA during autoclaving. However, Li et al. (p < 0.01) in the SFA content of pork, as it melted into the broth. Moreover, according to Zhang et al. (p > 0.05) affect the fatty acid composition of grass carp fillets, but led to a slight increase in SFA. Thus, it is suggested that the specifics of the heating process, rather than the sample characteristics and heating method, may influence the fatty acid composition of meat products.i et al.  demonstrg et al. , steaminp > 0.05) when 60% or more TS was added. The alterations in fatty acid composition were generally consistent with the lipid oxidation indices (POV and TBA) observed in this study, further supporting the potential of TS as an effective lipid antioxidant when added to CPSPB.The addition of various percentages of TS influenced the fatty acid composition of CPSPB in this study. The proportion of MUFAs (40.67 ± 0.25%-42.76 ± 0.34%) and PUFAs (17.32 ± 0.17%-23.65 ± 0.02%) gradually increased, while the proportion of SFAs (44.01 ± 1.05%-32.38 ± 0.07%) continued to decrease. The UFA/SFA ratio changed from 1.32 ± 0.07 to 2.05 ± 0.00 with the sequential addition of TS from 0 to 100% of the sample proportion. When TS exceeded 60% of the sample proportion, MUFAs constituted the majority of fatty acids compared to SFAs, while PUFAs remained the least abundant. The predominant fatty acid changes driving this trend were the decrease in C16:0, C17:0, and C18:0, and the increase in C18:1n9c, C18:2n6c, C18:3n3, and C20:2. Along with the Maillard reaction products  of CPSPB with TS, TS contains several naturally occurring antioxidant constituents that are absorbed by the samples during steaming . They co3.4.p < 0.05) (p < 0.05). However, further increasing the TS additions to 80% and 100% did not significantly influence the free amino acid content (p > 0.05). These findings suggest that within a certain range, TS positively affects the hydrolysis process of pork belly proteins. This beneficial effect can be attributed to the Maillard reaction that takes place between the glucose present in TS and the hydrolysate of pork belly protein during the steaming process, facilitating the hydrolysis reaction.The results demonstrated a significantly lower content of free amino groups in raw pork belly compared to the other treatments ( < 0.05) . This su < 0.05) . Additiop < 0.05). The carbonyl value increased from 0.07 ± 0.01 to 0.89 ± 0.13, while free SH decreased from 227.41 ± 1.59 to 94.25 ± 2.61 mmol/g soluble protein. These findings align with previous studies that reported higher protein oxidation in cooked samples compared to raw samples exposed to different time/temperature cooking combinations (p < 0.05). Among all TS treatments, the lowest carbonyl value was 0.71 ± 0.10 (100% TS), and the highest free SH was 166.13 ± 7.46 mmol/g soluble protein (80% TS). These results, consistent with the findings on fat oxidation in this study, strongly suggest that the addition of TS affects protein oxidation in pork belly. It is worth noting that adding more than 60% TS did not contribute to further improvements in the protein oxidation of CPSPB. Therefore, it is plausible that protein oxidation occurs concurrently with lipid oxidation, as previous reports indicated the relationship between the primary and secondary lipid oxidation products as substrates for protein oxidation   of CPSPB compared to the untreated samples ( < 0.01) , as demo < 0.01) , who rep < 0.01) . In conc < 0.01) .3.6.The OXITEST reactor is characterized by its simplicity, as it does not require sample pretreatment . It achiR2 > 0.99). These lines exhibited negative slopes and positive intersections, indicating that as temperature rose, the IP decreased, resulting in reduced oxidative stability and shorter preservation times for the samples. These findings support previous reports , a linear model derived from the OXITEST reactor calculations was employed. The IP at 25°C (IP 25) for CPSPB without added TS was found to be shorter  compared to the raw product . This discrepancy may be due to the induction of fat and protein oxidation during the heating process, as cooked samples generally exhibit lower oxidative stability than raw meat . With in4.Trao (TS) plays a pivotal role in enhancing the nutritional quality, sensory attributes, and shelf life of CPSPB, as a precursor to its industrial production. Our study revealed that the addition of TS resulted in the reduction of moisture content, total fat content, ash content, and NaCl content, while simultaneously increasing the crude protein content of CPSPB. Furthermore, our findings confirmed that TS effectively mitigated fat oxidation in CPSPB, as evidenced by the decreased levels of TBA and POV, along with an elevated ratio of UFA to SFA. Additionally, TS exhibited a certain degree of promotion of protein hydrolysis and inhibition of protein oxidation in CPSPB. The OXITEST analysis demonstrated a prolonged shelf life of CPSPB when higher levels of TS were added at the same induction temperature. Moreover, the chemical composition changes resulting from the addition of TS significantly influenced the organoleptic and nutritional qualities of CPSPB. While the inclusion of 100% TS maximized the shelf life of the samples, the addition of 60% TS yielded the best organoleptic quality of CPSPB, as determined by TPA and sensory evaluations. These results provide a solid foundation for investigating the preservation mechanisms facilitated by TS. More research is required to study changes in odor and taste and determine the optimal organoleptic properties of CPSPB prepared using a 60% TS.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.QQ: Data curation, Methodology, Software, Writing – original draft, Writing – review & editing. YZ: Data curation, Software, Writing – original draft. AN: Methodology, Writing – review & editing. LF: Writing – review & editing. ZQ: Funding acquisition, Project administration, Resources, Supervision, Validation, Writing – review & editing.


Impulse control disorders (ICDs) are common in patients with Parkinson's disease (PD) and strongly linked with dopaminergic medication.Dystonia is a chronic movement disorder that is not considered a dopamine‐dependent nor is treated with dopaminergic medications (with rare exceptions). Similar to PD, though, dystonia is associated with a high burden of neuropsychiatric comorbidity, including for example, depression, anxiety, and sleep disorders, which have been shown to increase the risk of substance use disorders in patients with dystonia.ICDs were evaluated using the Questionnaire for Impulsive‐Compulsive Disorders (QUIP) using a survey e‐mailed personally to the members of the Finnish Movement Disorders Association, as described previously.P = 0.87) or PD − DA , but was significantly lower than in PD + DA  Fig. P = 0.04), younger age , and male sex , but not with dystonia   did not differ from HC  Table .Our results shown that the prevalence of ICDs in dystonia is comparable with HC and PD − DA, but less common than in patients with PD + DA. Our findings support the view that ICDs are linked with dopaminergic treatment, and not with chronic neurological conditions in general.The limitations of this study include the use of QUIP, which as a screening instrument is associated with false positives. In addition, the sample size could be considered low for estimating the prevalence of ICDs in dystonia. However, our aim was not to estimate the actual prevalence of ICDs but to investigate whether dystonia is associated with increased ICD risk. As the proportion of patients with ICDs in dystonia was slightly lower than in PD − DA or controls, it is highly unlikely that the lack of detecting an increased ICD risk in dystonia would be a sample size issue.(1) Research Project: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript Preparation: A. Writing of the First Draft, B. Review and Critique.A.H.: 1A, 1C, 2A, 2B, 3AE.J.: 1A, 1C, 2A, 2B, 2C, 3BJ.J.: 1A, 1B, 1C, 2A, 2C, 3AAnnu Huovinen has received a grant from the Finnish Parkinson Foundation. Elina Jaakkola has received a grant from the Finnish Parkinson Foundation. Juho Joutsa has received lecturer honoraria from Lundbeck; research grants from the Finnish Medical Foundation, Sigrid Juselius Foundation, Instrumentarium Research Foundation, Finnish Foundation for Alcohol Studies, University of Turku, private donation, and Turku University Hospital ; and conference travel support from Abbvie.Appendix S1 Supporting InformationClick here for additional data file.


Rhus semialata Murray syn. Rhus chinensis Mill.), an underutilized fruit crop mainly found in foothill tracks of Eastern Himalaya, India, China, Japan, Korea and other South East Asian countries.Underutilized fruits plays a significant role in socio economic, cultural, nutritional and ethnomedicinal status of tribal people. However, scientific studies on the nutritional and other pharmaceuticals/biological activities of these fruits are meagre. Hence, the present study dealt with the quantification of nutritional quality and deciphering the bioactivity of nutgall  and Water extract (WExt) of the fruit were recorded as 4.05 ± 0.22 and 4.45 ± 0.16 μg/mL, respectively, in the DPPH assay and 5.43 ± 0.37 and 11.36 ± 2.9 μg/mL, respectively, in the ABTS assay as compared to Ascorbic acid . The CUPRAC assay also showed a high antioxidant potential of MExt and WExt . MExt and WExt of the fruit were more active against α-glucosidase  than α-amylase enzyme . In addition, the methanolic fruit extract showed low to moderate pharmacological potential in terms of antihypertensive (Angiotensin converting enzyme-I inhibition), antihyperuricemia (xanthine oxidase inhibition), anti-tyrosinase, and antimicrobial activity. The IC50 values of angiotensin-converting enzyme I inhibition, xanthine oxidase inhibition and tyrosinase inhibition were recorded as 13.35 ± 1.21 mg/mL, 93.16 ± 4.65 mg/mL, and 862.7 ± 12.62 μg/mL, respectively. The study evidently indicates that nutgall fruit is a potential source of phytonutrients, bestowed with commercially exploitable, multifaceted health benefitsThe fruit was rich in essential fatty acids. The presence of linoleic and oleic acids, along with traces of docosahexaenoic acid and eicosapantaenoic acid, revealed the potential food value of the fruit. 59.18% of the total amino acid composition of the protein present was constituted by essential amino acids. The IC Fruits are known to be rich source of antioxidants, soluble fiber, nutrients, phenols, flavones, vitamins, and minerals. Low intake of fruits is connected with unhealthy diets and several chronic illnesses such as aging, cancer, and cardiovascular disease . Edible Recognizing the significance of underutilized food crops, the International Centre for Underutilized Crops (ICUC) promotes their cultivation, usage, and marketing. These crops are also important for sustaining floral biodiversity . FurtherViburnum mullaha (Buch.-Ham. Ex D. Don), an underutilized wild edible fruit of the Indian Himalayan region, had anti-tyrosinase and antiaging activity. Antidiabetic properties have been identified in underutilized fruits found in Indian subcontinent such as Syzygium cumini L.  have been shown to inhibit angiotensin converting enzyme I  N. Robson, and Artocarpus lacucha Buch.-Ham  is one such underutilized fruit crop found in the hills and valley regions of North East Indian Himalayan region. It is also wildly distributed in China, Japan, Java, North Korea, Malaysia, Indo-China Peninsula, Indonesia, Europe, and USA , and phytochemicals such as polyphenols, carotenoids, flavonoids, and others. It also includes vital fatty acids and amino acids, confirming the its nutritional excellence . Aside fon pills .Rhus semialata Murray, an underutilized fruit collected from Manipur, India. There are reports in the literature on the nutritional  and some medicinal qualities of this fruit. However, the current study focused on other aspects of the nutritional profile, such as amino acid content, important fatty acids, and so on. Furthermore, biological properties such as antioxidant, antihypertension, anti-hyperglycemic, antityrosinase, anti-gout, antibacterial activities and cytotoxicity were also assessed.Given this backdrop, the current study sought to investigate the nutritional and biological activity of Matured fruits were collected from five different locations in Purul sub-division, Senapati district, Manipur, India . About 2 kg of fresh fruit was collected from five different plants (400 g each plant) in each location and removed any unwanted non-fruit portions then dried overnight in the oven at 40–45°C. The dried fruit was hand crushed (using a sterile hand glove) to detach the pulp from the seed. Then the pulp was separated by winnowing and ground to a fine powder and stored in an air tide container at 4°C until further used.Ten gram of fruit pulp powder was soaked in methanol (100 mL) at room temperature for 24 h with shaking at 180 rpm . Extraction was repeated for 48 and 72 h and filtered through Whatman no. 1 filter paper followed by drying at 45°C under low pressure . The dried sample was stored at −20°C until used for biological activity assay.Ten gram of fruit pulp powder was soaked in deionized water (50 mL) at room temperature for 3 h with shaking at 180 rpm  then centrifuged at 10,000 rpm for 10 min. Extraction was repeated with the residue for another hour. The supernatants were pooled in a 250 mL flask and kept at −20°C for overnight. The sample was lyophilized at −80°C. The lyophilized sample was stored at −20°C until further use for biological activity assay.viz. moisture (Method No. 934.01), crude protein (Method No. 2001.11), fat (Method No. 2003.05), total carbohydrate, ash content (Method No. 942.05) and micronutrients (Method No. 975.03) were determined according to AOAC  equipped with a diode array and multiple wavelength detector. The fruit sample was hydrolyzed with 6 N HCl, and the hydrolyzed peptide samples were derivatized with OPA  and FMOC (9–fluorenylmethyl chloroformate for secondary amino acids) as reported by Sahoo et al. . Sample fatty acid composition was compared with standard fatty acid composition, and percentages were calculated by normalization of peak areas.The total fat content of the moisture-free sample was extracted in Gerhardt Soxtherm fat analyzer [, Method 517 = 1.1 + 0.01). The mixture was incubated for 30 min in the dark and then read at 517 nm. The radical scavenging activity (RSA) of each extract was calculated as follows (0.1 mL of each of the five different concentrations of the extract was mixed with 1.9 mL DPPH reagent prepared in methanol (A follows .50) was calculated and expressed in μg/mL. IC50 is defined as the minimum amount of the extract required to scavenge half of the initial stable free radical in 30 min in 1 mL of reaction in the dark.From the RSA%, 50 inhibitory concentration (IC734 = 1.0 ± 0.01) then incubated for 30 min in the dark, and the absorbance was read at 734 nm. RSA and IC50 of the extract were calculated as described above.ABTS radical scavenging activity was performed as described by Singh et al. . The rea4 solution (5–30 μg/mL) was used as standard and ferric reducing antioxidant power was expressed as mM ferrous equivalent per gram of the extract (mM Fe Eq/g Ext.) (0.1 mL of each extract dissolved in methanol was reacted with 1.9 mL FRAP reagent and incubated for 5 min and the absorbance was read at 593 nm. FeSO/g Ext.) .0.5 mL sample was mixed with 0.5 mL copper II chloride (10 mM) in a tube. 0.5 mL neocuporine solution was added, and followed by 0.5 mL ammonium acetate solution (1 M). The reaction mixture was incubated for 30 min at room temperature and the color developed was read at 450 nm. Ascorbic acid was used as a standard. Cupric ion reducing antioxidant capacity was expressed in milligram ascorbic acid equivalent per gram of extract (mg AAE/g extract) .The total phenolic and flavonoid content of the fruit extract was analyzed by Folin Ciocalteu and aluminum chloride methods, respectively described in Singh et al. . Phenoli50 was calculated (0.1 mL of the sample extract (5–50 μg) was taken in a 2 mL tube and mixed with 0.1 mL enzyme , incubated at 37°C for 20 min. The enzyme reaction was initiated by the addition of 0.2 mL of starch solution (1%) and continued for 3 min at 37°C. After 3 min, 0.2 mL dinitrosalicylic acid (DNSA) reagent was added and kept incubated at 80°C, followed by 1.4 mL of distilled water. For the control reaction, instead of sample, the buffer was added, and a blank was prepared by the addition of DNSA before the addition of the enzyme. The color complex was read at 540 nm. Acarbose (1–10 μg) was used as a standard positive control. From the percentage inhibition, IClculated .S. cerevisiae), incubated at 37°C for 10 min. To the mixture solution, 0.1 mL 4-nitrophenyl α-D-glucopyranoside (2.5 mM) was added and incubated for 5 min at 37°C. The reaction was terminated by the addition of 0.2 mL sodium carbonate (20%) followed by the addition of 1 mL water. The color complex was measured at 405 nm against a reagent blank (0.1 mL of the sample extract (0.1–50 μg) was taken in a 2 mL tube and mixed with 0.2 mL enzyme  was taken in a tube and the volume was made up to 0.6 mL with buffer , and 0.1 mL enzyme (0.2 U/mL), followed by incubation for 15 min at 37°C. In the reaction mixture, 0.2 mL substrate  was added and then incubated for 30 min at 37°C. After 30 min, 0.2 mL HCl (0.5 M) was added and then read at 293 nm against a reagent blank. Allopurinol was used as a positive control (0 μg was 0.01 mL of enzyme was added to a tube containing 0.140 mL sample prepared in buffer (pH 8.3) and 0.05 mL sodium chloride 300 μM). To the reaction mixture, 0.5 mL substrate (hipuryl-his-leu) was added and incubated for 30 min. After 30 min, the reaction mixture was kept in a boiling water bath for 10 min. After cooling, absorbance was read at 385 nm. Captopril was used as a positive control (0 μM. To 0.340 mL of different concentrations of the sample (0.5–2 mg) was mixed with 0.02 mL enzyme (mushroom tyrosinase) and 0.360 mL substrate (16 mM DOPA) in a tube and then read at 480 nm spectrophotometrically for 3 min for every 30 s. For control (100% activity), instead of sample, 0.340 mL buffer was used. Kojic acid was used as a positive control , Escherichia coli (MTCC 739), Bacillus subtilis (MTTCC 121), Staphylococcus aureus (ATCC 1026), were grown in Mueller-Hinton broth (MHB) at 37°C and 160 rpm and brought to its exponential phase. The sample was prepared by dissolving the MExt in 50% DMSO (at 25 mg/mL w/v concentration) and filtering through a (0.22 μm) syringe filter. Hundred microliter of each bacterial strain, equivalent to 0.5 McFarland turbidity were spread over sterile Mueller-Hinton agar (MHA) plate using a sterile swab. A 20 μL sample (0.5 mg) impregnated in a 6 mm sterile disc was seeded on the freshly spread MHA plates and incubated for 24 h at 37°C. Chloramphenicol 0.025 mg was used as a positive control. One reagent control containing the buffer and solvent used was also included in the experiment. The zone of inhibition was recorded using a Vernier caliper.The Kirby-Bauer disk diffusion method  was usedThe cytotoxic activity of MExt was investigated using Sulforhodamine B colorimetric (SRB) assay against HeLa . The cell was maintained in a Dulbecco’s Modified Eagle Medium (DMEM). The cells were subjected to different concentrations of the extracts (5–100 μg/mL) for 48 h. Doxorubicin (5–100 μg/mL) was used as a positive control. The monolayer cells were then fixed with 10% Trichloroacetic acid (TCA) for 1 h at 40°C followed by washing with water. After drying, the cell plates were stained with SRB for 30 min and then washed with 1% acetic acid. The SRB dye bound to the protein was then dissolved by adding 10 mM Tris base (pH 10.5). The SRB dye released was quantified by taking absorbance at 510 nm in Varioskan Flash Multimode Reader  .50 values of assays were calculated using the Prism software .All the assays were performed with three replications, and each replication had 3 triplicates. The data are presented as mean ± standard deviation (SD). The ICRhus semialata Murray are presented in The nutritional contents of the viz. histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine and valine, constituted 59.18% of the total amino acid composition. Among the EAA, threonine (27.21%) was the highest, followed by lysine (14.64%) and leucine (4.71%), while isoleucine (1.92%) was the lowest. The two EAAs, threonine and lysine, constituted 41.85% of the total amino acid content. The non-essential amino acid content was in the range of 0.71–8.64%. Among the nonessential amino acids, glutamate (8.64%) was recorded as the highest followed by cysteine (7.80%), serine (5.63%), and Arginine (5.43%), while glycine (1.12%) and tyrosine (0.71%) were the lowest.A total of 17 amino acids were detected in the fruit sample . Of thesviz., Linoleic acid methyl ester (C18:2n6c), y-linolenic acid methyl ester (C18:3n6), α-linolenic acid methyl ester (C18:3n), Cis-11,14,17 eicosatrienoic acid methyl ester (C20:3n3), Arachidonic acid methyl ester (C20:4n6), Cis-5,8,11,14,17- eicopentaenoic acid methyl ester (C20:5n3), Cis-13,16-docosadienoic methyl ester (C22:2) and Cis-4,7,10,13,16,19-docosahexaenoic acid methyl ester (C22:6n3). Saturated fatty acids (SFA) were obtained in the ranges of 0.08–28.46% of total fatty acids. The most abundant saturated fatty acid was palmitic acid (28.46%), followed by steric acid (C18:0) (2.92%) and butyric acid (2.38). Monounsaturated fatty acids were obtained in the range of 0.33–11.53%. The highest MUFA was recorded as oleic acid (11.15%), and followed by nervoinic acid (0.35%).The total fat content of the studied fruits was constituted by three different groups of fatty acids: polyunsaturated fatty acid (PUFA) (50.62%), saturated fatty acid (SFA) (35.13%), and monounsaturated fatty acid (MUFA) (12.54%), while 1.72% of fatty acids were unknown fatty acids . The PUF50 value for MExt and WExt was exhibited as 5.43 ± 0.37 μg/mL and 11.36 ± 2.91 μg/mL, respectively. While the IC50 value of standard ascorbic acid was recorded as 3.0 μg/mL. In the DPPH assay, the IC50 value for MExt and WExt was found to be 4.05 ± 0.12 μg/mL and 4.45 ± 0.06 μg/mL, respectively. While the IC50 value of standard ascorbic acid was recorded as 5.40 μg/mL.The fruit exhibited significant free radical scavenging activity in the ABTS and DPPH assay. In the ABTS assay, the IC+3/g Ext. and 1.23 ± 0.07 mM Fe+3/g Ext, respectively. In the CUPRAC assay, the antioxidant activity of the fruit exhibited 1134.84 ± 88.06 mg AAE/g and 456.53 ± 30.02 mg AAE/g for MExt and WExt, respectively  (The inhibition of HeLa cell growth by the fruit extract was observed in a dose-dependent manner. The IC1 μg/mL) .Rhus semialata Murray fruit is its pulp. The pulp contributes 35.39% of the total weight of the fruit. Only the pulp portion was used for analysis in the present study. The proximate analysis of the present study revealed that the fresh fruit contained crude protein (4.25 ± 0.04%), crude fat (19.21 ± 0.76%), total carbohydrate (59.2 ± 1.68%), and ash (4.02 ± 0.13%). Higher amount of protein (8.13 ± 0.38%), lower fat (16.7 ± 0.18%), and carbohydrates (69.2%) content were reported by Loukrakpam et al. . Shi et al.  constituted 92.21% of total PUFA. The essential fatty acids (EFA) such as n6 fatty acids (omega-6 fatty acid) and n-3 fatty acids (omega 3-fatty acid) constituted 49.42 and 1.15% of the total fat of the fruits, respectively. EFA and PUFA derivatives are important structural components of the cellular membrane and play an important role in signal transduction, particularly omega-6 fatty acids. Shi et al.  in Rhus semialata, respectively. Hence nutgall fruit is a potential source of unsaturated fatty acids. Unsaturated fatty acids find applications in the food (for formulation of dips and spreads) and pharmaceutical sectors. Most of the phytochemicals (both polar and nonpolar) are dissolved in methanol; hence it represents one of the most used organic solvents for extraction. However, extraction of bioactive metabolites from plants using water as a solvent has been attempted by many researchers. Moreover, the fruit, simply soaked in water, is used as medicine alone or mixed with other ingredients by local traditional healers, especially in Manipur, to treat various ailments. Hence, the fruit pulps were extracted in two solvents, i.e., methanol and water, and screened for biological activities . Both extracts showed antioxidant activity in all four antioxidant assays. In ABTS and DPPH assay, MExt showed a lower IC50 value, which indicates higher antioxidant activity than WExt. Zhang et al. . For the FRAP and CUPRAC assay, antioxidant activity was expressed as mM Fe Eq/g Ext and ascorbic acid equivalent (AAE) respectively. The higher the value of mM Fe Eq or AAE, the higher the antioxidant activity. The MExt had higher antioxidant activity than the water extract. Similarly, Sharma et al.  and formation of advanced glycation end (AGE) product. They observed that the free phenolic extract had higher α-glucosidase and dipeptidyl peptidase-4 inhibitory activity while the insoluble-bound phenolic extract exhibited higher inhibition on AGE product formation. The two phenolic compounds gvajaverin and quercitrin, made the most significant contributions to the inhibitory effects on α-glucosidase and DPP-IV, while trigalloyl glucose and its isomer may be the primary bioactive substances responsible for the suppression of AGE formation . Hyperuricemia is associated with chemotherapy and calcium oxalate kidney stones in patients. XO inhibitors are a therapeutic option for treating these types of disorders. Current drugs that are available on the market  are associated with adverse side effects . Hence, It is a widely known fact that people with diabetes frequently acquire high uric acid levels and vice versa. Desco et al.  reportedRhus semialata Murray fruit extracts were also screened for XO inhibitor activity. The extracts showed inhibitory activity against XO and their IC50  was comparable with the standard inhibitor allopurinol (77 ± 0.08 μg/mL). Tsai et al.  was screened for the first time in the present study. The crude extracts showed tyrosinase inhibitory activity, though their activity is lesser than the standard kojic acid. It is one of the widely used commercial antityrosinase compounds obtained from different species of fungi and as a byproduct of the fermentation of certain foods  inhibitors are a class of drugs used to control high blood pressure. They’re also used to treat other cardiovascular conditions such as heart failure, diabetes-related kidney disease, and more , 54. SigRhus semialata Murray is being used in traditional medicine and shows promising free radical scavenging activity, there is no report on the ACE inhibitory activity of this plant. Hence, the fruit extracts (MExt and WExt) of Rhus semialata Murray were assayed for ACE inhibitory activity. The extracts were found to be positive for ACE inhibitory activity, indicating that the fruit exhibits potential antihypertensive activity. The IC50 values of MExt and WExt was recorded as 13.35 ± 1.21 μg/mL and 34.57 ± 3.56 μg/mL, respectively. Hence the value-added functional food products prepared using Rhus semialata fruit may be used as therapeutic foods in controlling high blood pressure.Though Rhus semialata Murray, which shows inhibitory capability against these enzymes, might be a significant treatment alternative for degenerative illnesses.Numerous enzymes are responsible for oxidative stress, both directly and indirectly, and the resulting inflammation. For instance, xanthine oxidase increases the generation of superoxide. ROS often stimulates RAS. Several studies have demonstrated an over-activation of the RAS in diabetes problems , and theRhus chinensis Mill. was reported to have antibacterial activity against a wide range of gram positive and gram-negative bacteria such as Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, and Staphylococcus aureus, Streptococcus iniae, Vibrio ichthyoenteri  exhibited antimicrobial activity against Salmonella typhi and Staphylococcus aureus. However, the extract did not show antibacterial activity against Bacillus subtilis or Escherichia coli. The variation may be due to the difference in the solvent used for the extraction. Sreedharan et al. . The 80% ethanolic extract of Rhus javanica exhibited antibacterial activity against three species of Shigella, viz. S. sonnei, S. flexneri, S. boydii, and S. dysenteriae  was evaluated for its in vitro cytotoxicity activity against HeLa cell line. It is the oldest and most commonly studied cell lines obtained from cervical cancer specimens. The inhibition of HeLa cell growth by the MExt was observed in a dose-dependent manner until 60 μg/mL concentration corresponding to 60% growth inhibition. Further increases in concentration did not improve the cell growth inhibition significantly. This might be due to an increase in undesired interactions between the compounds present in the extract at higher concentrations, which ultimately retards the activity of the extract. The IC50 of the MExt against HeLa cells was recorded as 61.55 ± 3.37 μg/mL. Similarly, Lalawmpuii et al. (Rhus javanica L. fruit exhibited cytotoxicity activity against HeLa cells with an IC50 value of 98.28 μg/mL.Numerous plants have been shown to exhibit high ROS-scavenging activity, which is related with cytotoxicity or antiproliferative activity against cancer cells and might thus be employed as therapeutic and preventative agents , 63. Then cancer , 65. Taki et al.  reportedRhus semialata Murray grown in North-east Indian Himalaya. Our research showed that fruits are high in minerals and vitamin C. Interestingly, the fruits were also found to be a potential source of essential amino acids, unsaturated fatty acids, omega-6 and omega-3 fatty acids, all of which are vital for maintaining the normal function of the body. The MExt and WExt of R. semialata Murray fruits demonstrated multifaceted pharmaceutical properties. Fruit extracts showed antioxidant action equivalent to that of ascorbic acid. Furthermore, the fruit extracts inhibited the α-glucosidase and α-amylase enzyme, indicating antihyperglycemic activity. Besides, the dose-dependent inhibition of HeLa cell growth by the methanolic fruit extract indicated its anticancer potential. To the best of our knowledge, R. semialta Murray has been reported for the first time globally for its ACE inhibitory and anti-tyrosinase action. Additionally, the fruits extract exhibited low to moderate pharmacological activity in terms of xanthine oxidase inhibitory and antibacterial activity. The findings of our investigation will provide vital information about the food-value and bioactivity of R. semialta Murray fruits. The fruit might be recommended for regular consumption in order to maintain a healthy living. It may also be considered as a component in nutraceutical or functional foods to enhance quality of life or may be researched extensively for innovative value addition. Further investigation into the bioactive compounds responsible for these activities will give deeper insights about this potential fruit.The present study presented thorough scientific evidence for the nutritional and therapeutic potential of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.TSS: experimental, data analysis, literature survey, and manuscript drafting. PK: literature survey, experimental, and manuscript drafting. AKD, PL, KT, TC, CD, TBS, SC, and YD: experimental. HS: experimentation and figure and table preparation. RA: literature survey and manuscript drafting. AK: experimental and manuscript drafting. SGS: experimental and language improvement. SKS: data analysis and interpretation. AD: manuscript editing. SR: conceptualization, supervision, interpretation, and final approval of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer HR declared a shared affiliation with the authors SR, TSS, PK, AKD, PL, KT, HS, RA, TC, CD, TBS, SC, YD, SKS, and AD to the handling editor at the time of review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In anesthetized male rats, we investigated how the blockade of α2- and D2-receptors by atipamezole and raclopride, respectively, modified the firing of noradrenergic neurons in the locus coeruleus (LC) and dopaminergic neurons in the ventral tegmental area (VTA). In freely moving rats, we studied how atipamezole and raclopride modified extracellular noradrenaline, dopamine, and DOPAC levels in the medial prefrontal cortex (mPFC) through microdialysis. When administered alone, atipamezole activated LC noradrenaline but not VTA dopamine cell firing. Combined with raclopride, atipamezole activated dopamine cell firing above the level produced by raclopride. Atipamezole increased extracellular dopamine to the same level, whether administered alone or combined with raclopride. In the presence of the noradrenaline transporter (NET) inhibitor, atipamezole combined with raclopride increased extracellular dopamine beyond the level produced by either compound administered alone. The results suggest that a) the D2-autoreceptor blockade is required for LC noradrenaline to activate VTA cell firing; b) the level of dopamine released from dopaminergic terminals is determined by NET; c) the elevation of extracellular dopamine levels in the mPFC is the resultant of dopamine uptake and release from noradrenergic terminals, independent of dopaminergic cell firing and release; and d) LC noradrenergic neurons are an important target for treatments to improve the prefrontal deficit of dopamine in neuropsychiatric pathologies.Experimental and clinical evidence indicates a deficit of release and function of dopamine in schizophrenia and suggests that α Atipamezole hydrochloride  was administered at 3 mg/kg i.p. or up to 0.5 mg/kg i.v. for microdialysis or electrophysiological experiments, respectively.S(−)-raclopride (+)-tartrate salt  was administered at 0.5 mg/kg i.p. or up to 0.025 mg/kg i.v. for microdialysis or electrophysiological experiments, respectively. Prazosin  was administered at 1 mg/kg i.v. for electrophysiological experiments. Nisoxetine hydrochloride  was administered at 3 mg/kg i.p. for microdialysis experiments.The rats were anesthetized with chloral hydrate , and their femoral vein was cannulated for the i.v. administration of pharmacological agents. The rats were placed in a stereotaxic frame  with their body temperature maintained at 37°C ± 1°C using a heating pad. According to the stereotaxic rat brain atlas by Paxinos and Watson (2007) , the recThe baseline spontaneous firing rate was recorded for 3–5 min; the drugs were administered at 120 s intervals.4, 4.25 g chloral hydrate, 42.8 mL propylene glycol, and 11.5 mL 90% ethanol in 100 mL; 5 mL/kg, i.p.) and stereotaxically implanted with vertical microdialysis probes  in the mPFC , according to Paxinos and Watson  was pumped using a CMA/100 microinjection pump  through the dialysis probes at a constant rate of 1.1 μL/min in freely moving animals. Dialysate samples were collected every 20 min and immediately injected using HPLC. Drugs were administered after stable extracellular levels were obtained, i.e., three consecutive samples with a variance not exceeding 15%. Noradrenaline, dopamine, and DOPAC were simultaneously analyzed using HPLC with electrochemical detection using HPLC systems equipped with 3.0 × 150 mm C18 (3.5 µ) Symmetry columns , maintained at 40°C using Series 1100 thermostats  and ESA Coulochem II detectors . The mobile phase was 80 mM Na2HPO4, 0.27 mM EDTA, 0.6 mM sodium octyl sulfate, 7% methanol, and 4% acetonitrile, adjusted to pH 2.4 with H3PO4, delivered at 0.3 mL/min; the Coulochem analytical cell first electrode was set at +200 mV and the second was set at −200 mV. Quantification was performed by recording the second electrode signal. Under these conditions, the noradrenaline and dopamine detection limit  was 0.3 pg per injection on the column. On the completion of testing, the rats were euthanized by an Equithesin overdose; their brains were removed and sectioned using a cryostat (Leica CM3050 S) into 40-µm-thick coronal slices to verify the locations of dialysis probes.The rats were deeply anaesthetized with Equithesin . Data were analyzed by one-way or two-way repeated measures (RM) ANOVA. The n = 7) produced a dose-dependent increase in the firing rate of LC noradrenergic neurons (p = 0.035).The intravenous administration of atipamezole  nor the bursting activity  = 1.43; p = 0.27; fig. 2B) of dopamine neurons in VTA (n = 6–7).On the other hand, atipamezole at the same cumulative dose modified neither the firing rate  = 1.242; n = 10) produced an additional increase in the firing rate  = 23.64; p <0.0001; p = 0.043; p = 0.042; bursting activity: F = 4.09; p = 0.035; n = 4) reversed the activation produced by the combined administration of atipamezole and raclopride  failed to modify the atipamezole-induced activity of LC noradrenergic cell firing .n = 7), raclopride , and their combination (n = 4) was analyzed by microdialysis in mPFC in freely moving rats , while raclopride failed to modify the extracellular dopamine level  = 1.533; p = 0.293). The combined administration of atipamezole and raclopride increased extracellular noradrenaline to the same level  = 8.277; p = 0.028) as that elicited by atipamezole alone. Two-way RM ANOVA evidenced a significant treatment effect  = 6.93; p = 0.010). The effect of raclopride was significantly different from that of atipamezole at time points from T80 to T160 min and from that of atipamezole plus raclopride at time points from T80 to T140 min; no difference was found between the effects of atipamezole alone and atipamezole combined with raclopride (Šídák’s multiple comparison post hoc test).Atipamezole increased the extracellular noradrenaline levels to a maximum of 240% of the basal values  = 10.99; p = 0.001) as well as combined with raclopride  = 12.49; p = 0.011), while raclopride alone was ineffective  = 0.9247; p = 0.425).The effect of atipamezole, raclopride, and their combination on extracellular dopamine reproduced those on extracellular noradrenaline. Thus, atipamezole increased extracellular dopamine to the same level (250% of the baseline) when administered alone  = 10.60; p = 0.027). The effect of raclopride was significantly different from that of atipamezole at time points T80, T100, T120, and T160 min and from that of atipamezole plus raclopride at time point T100 min; no difference was found between the effects of atipamezole alone or in association with raclopride (Šídák’s multiple comparison post hoc test).Two-way repeated measures ANOVA evidenced a significant treatment effect  = 4.95; p = 0.010; raclopride: F = 12.54, p = 0.026; and atipamezole + raclopride: F = 10.62 , p = 0.030). Two-way repeated measures ANOVA indicated a significant effect of the treatment  = 4.37; p = 0.037), but no difference was found by Šídák’s post hoc test.Extracellular DOPAC levels were increased to 160, 240, and 280% of the basal values by atipamezole, raclopride, and their association, respectively  = 8.723, To verify whether the uptake into the noradrenergic terminal limited the elevation of extracellular dopamine elicited by the combination of atipamezole and raclopride, the effect of atipamezole alone and combined with raclopride was analyzed after the blockade of the noradrenergic transporter (NET) with nisoxetine .n = 6) increased the extracellular dopamine level to 330% of the baseline  = 14.99; p = 0.002). Raclopride coadministered with nisoxetine (n = 6) increased the extracellular dopamine level to 410%  = 17.04; p = 0.0002) and with atipamezole (n = 5) to 530% of the baseline  = 11.61; p = 0.014).n = 11) increased the extracellular dopamine level to more than ten times the baseline. Two-way repeated measures ANOVA evidenced a significant effect of the treatment  = 6.50; p = 0.002), due to the difference between nisoxetine + atipamezole + raclopride co-administration with respect to the other treatments, as no difference was found between nisoxetine alone and nisoxetine–raclopride or nisoxetine–atipamezole combination (Šídák’s multiple comparison post hoc test).Crucially, in the presence of NET blockade, the combination of atipamezole and raclopride . Changes in extracellular DOPAC were correlated with the electrical activity of dopamine neurons, which is in line with the notion that DOPAC represents the intracellular oxidation of dopamine presynaptically recaptured by DAT other than the oxidation of newly synthetized dopamine (A major outcome of this study was that the activation of dopamine cell firing by the combined αdopamine .On the other hand, in contrast to what could be expected from the electrical activity of dopamine neurons, atipamezole produced the same increase in extracellular dopamine levels, whether administered alone or in combination with raclopride. Burst activation of dopamine neurons by electrical stimulation of VTA has been shown to produce a long lasting availability of released dopamine in cortical  and subcIndeed, the rise in extracellular dopamine after NET blockade should represent the amount of dopamine taken up by noradrenergic terminals from the extracellular space and, indirectly, of the amount of dopamine released from dopaminergic terminals and co-released with noradrenaline from noradrenergic terminals.1-receptor overstimulation (The elevation of the dopamine level demonstrates that the uptake into noradrenergic terminals plays a major role in terminating dopamine action, limiting excessive dopamine concentrations in the extracellular fluid and preventing the negative consequences of Dmulation . Mostly,mulation .2 but not α2 receptors, similar to raclopride, stimulated dopamine cell firing but did not increase the extracellular dopamine level (2 and α2 receptors, increased both dopamine cell firing and extracellular dopamine, similar to raclopride/atipamezole in this study. Anesthetics can affect the firing properties of dopamine neurons, meant as quantitative rather than qualitative changes. Yet, dosage adjustments for pharmacological studies are generally producing comparable effects under different anesthesia conditions (Due to the systemic administration of the drugs, an action at the nerve terminal level by atipamezole and raclopride is not excluded; this possibility is not alternative but complementary to an action at the cell level . A limitne level . Conversnditions .2- and D2-receptor activates VTA cell firing and increases dopamine release in mPFC. However, dopamine release from dopamine terminals cannot be monitored by microdialysis because dopamine that escapes the synapse is taken up from extracellular fluid into noradrenergic nerve terminals. By contrast, the elevation of extracellular dopamine produced by atipamezole alone or in combination with raclopride, originates from noradrenergic terminals, independent of the firing of dopamine neurons and the amount of dopamine taken up by noradrenergic terminals.In conclusion, our results demonstrate that the combined blockade αMounting evidence indicates that mesolimbic dopamine neurons release dopamine in the nucleus accumbens and other striatal regions in a phasic and tonic mode to control cognitive and motivational functions, respectively . Accordi


Marsupellamicrophylla from Brazil is reassessed and described as a new species, M.brasiliensis. The new species is characterized by paroicous inflorescence, bispiral elaters, scale-like, commonly unlobed leaves and very small leaf cells. Descriptions and drawings are provided along with a corresponding discussion of the morphological peculiarity of the new species. Marsupellabrasiliensis belongs to sect. Stolonicaulon, and the distribution of Marsupellasect.Stolonicaulon in the New World is confirmed. The infrageneric position of M.microphylla remains unresolved, and whether it belongs to the same section is still unclear.The specimen previously identified as  Marsupellasect.Stolonicaulon (N. Kitag.) Váňa is a mysterious section that includes the smallest taxa of the genus and is characterized by a disjunct range, mainly in the tropical and subtropical mountains of Asia to Melanesia, as well as in the Venezuelan Andes and the mountain range of SE Brazil of Latin America  or deeply dissected conduplicate leaves, resemble small Schizophyllopsis Váňa & L. Söderstr. . The cited revision  collected a specimen identified by J. Váňa as Marsupellamicrophylla (referred to in the report as specimen ASV15033) was studied by traditional morphological techniques, and plant morphology was compared with other taxa of sect. Stolonicaulon (N. Kitag.) Váňa, originally described and well known in Asia, and further treatments of Marsupellamicrophylla by The specimen identified by J. Váňa as trnL‒F) from the specimen ASV15033 and added these sequences to the dataset from Eremonotusmyriocarpus (Carrington) Lindb. & Kaal.). Specimen voucher details, including GenBank accession numbers, are listed in Table To compile the dataset for molecular phylogenetic analysis, we sequenced two loci . Ampliﬁcation was performed using an Encyclo Plus PCR kit  with the primers listed in Table trnL–F)/12.4 µl (for ITS 1–2) of double-distilled water , 1 µl of dimethylsulfoxide/DMSO (for ITS 1–2) and 0.4 µl of each primer . Polymerase chain reactions were carried out using the following program: 180 sec initial denaturation at 94 °C, followed by 35 cycles of 30 sec denaturation at 95 °C, 20 (for trnL–F) – 30, 35 sec (for ITS 1–2) annealing at 58 °C (trnL–F and ITS 1–2) and 60 °C (ITS 1–2) and 30 sec elongation at 72 °C. Final elongation was carried out in one 3-min step at 72 °C. Amplified fragments were visualized on 1% agarose TAE gels by EthBr staining and purified using the Cleanup Mini Kit . The DNA was sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit  with further analysis of the reaction products following the standard protocol on an automatic sequencer 3730 DNA Analyzer.  in the Genome Center .The polymerase chain reaction was performed in a total volume of 20 µl, including 1 µl of template DNA, 0.4 µl of Encyclo polymerase, 4 µl of Encyclo buffer, 0.4 µl of dNTP-mixture (included in Encyclo Plus PCR Kit), 13.4 µl  with IQ-tree ver. 1.6.12 (BA) with MrBayes ver. 3.2.7  (For the IQ-tree) . Consensth generation. The first 2,500 trees in each run were discarded as burn-in; thereafter, 15,000 trees were sampled from both runs to produce a resulting tree. Bayesian posterior probabilities were calculated from the trees sampled after burn-in. The average standard deviation of split frequencies between two runs reached 0.0021 before the analysis was stopped.Bayesian analyses were performed by running two parallel analyses using the GTR+I+G model. The analysis consisted of four Markov chains. Chains were run for five million generations, and trees were sampled every 500Marsupellasect.Stolonicaulon we used TCS network inference method (http://popart.otago.ac.nz/), accessed on 08 November 2019 ; variable sites ‒ 420 (29.81%); and parsimony-informative sites ‒ 205 (14.55%).Newly obtained ITS 1‒2 and ML criterion recovered a bootstrap consensus tree with a log-likelihood = -6121.943. The arithmetic means of the log likelihoods in Bayesian analysis for each sampling run were -6145.17 and -6144.03.The ML topology with an indication of bootstrap support values (BS) and Bayesian posterior probabilities (PP), specimen ASV15033 formed a clade within sect. Stolonicaulon with high support of BS = 96% and PP = 1 (or 96/1). The TCS haplotype network . Stem 70–90 µm in diameter, branching ventral, but new branch always arose near to the ventral base of the leaf and may be regarded as deeply postical-intercalary; cross section nearly orbicular, outer cells with outer wall thin, other slightly thickened (10-)12–14 µm in diameter, inward cell walls become thicker, with large triangular trigones, while cell size become smaller, 8–10 µm in diameter. Leaves obliquely to suberect spreading, slightly narrower to twice wider than stem, 50–180×60–190 µm, reniform to widely ovate, smaller constantly entire, sometimes with obtuse apex, larger divided by U-shaped sinus descending to 1/10–1/5 of leaf length, lobes acute. Underleaves absent. Midleaf cells strongly collenchymatous with large and slightly convex trigones, 10–14 µm in diameter to shortly oblong 10–16×8–12 µm, cuticle virtually smooth. Paroicous. Perianthous branches distinctly clavate, perigynium high, ca. 1250×550 µm, with 3 pairs of leaves whose lower pair composed by strongly ventricose bracts containing antheridia; perianth hidden within bracts, conical, eroding from the mouth, and completely disappearing after sporophyte emergence. Female bracts slightly wider than long, to 500×550 µm with gamma-shaped sinus descending to 1/4–1/3 of bract length, lobes triangular, acute, with somewhat diverging apices; bracteoles absent. Androecial bracts in 1(-2) pairs in lower part of perigynium, monandrous. Capsule wall bistratose, outer cells with nodular thickenings present on vertical walls and only sometimes present in horizontal walls; inner layer of rectangular cells with small nodular (not semicircular) thickenings. Spores brownish, 8–10 µm in diameter, faintly papillose, elaters bispiral, (-75)100–130×7–8 µm brown, with narrow, but never homogenous ends.Plants wiry, ascending in loose patches, densely intermixed with 22°24'S, 44°41'W, Serra de Itatiaia, Hochgebirgsvegetation auf der Hochfläche bei Abrigo Rebouças, an exponierter, zeitweise sickerfeuchter Felswand , 2420 m, 15. Oct. 1991, leg. Schäfer-Verwimp & Verwimp 15033 ).Brazil, Rio de Janeiro, ca. Fig. Marsupellabrasiliensis seems to be a rather rare species, hitherto known only from the Itatiaia mountains of Southeast Brazil between 2300–2420 m. M.microphylla (type of M.brasiliensis), and subsequently, a second specimen was cited from the same region by Cephalozielladivaricata (Sm.) Schiffn. In the type specimen, shoot fragments of a Metasolenostoma Bakalin & Vilnet species have been detected . Marsupellabrasiliensis was found only along the main road in the National Park on exposed rock faces, and further associated species are not known to us. It may have been overlooked due to its very small size and may rarely occur elsewhere around Itatiaia or Serra da Mantiqueira. Climate change may pose a threat to high-altitude bryophytes confined to Southeast Brazil.The newly described Marsupellamicrophylla previously and treated here as M.brasiliensis belongs to Marsupellasect.Stolonicaulon; therefore, the distribution of the section in Latin America is definitely confirmed. At the same time, the morphological study of the material revealed that the plants are not conspecific with M.microphylla, which has been described from the Venezuelan Andes . On one side, the high peaks of Itatiaia and the “páramo” characteristic of the “Campos de Altitude” benefit the establishment of large numbers of Andean species, such as Aureolejeuneafulva R.M. Schust., Diplasiolejeuneapauckertii (Nees) Steph. , Drepanolejeuneagranatensis (J.B. Jack & Steph.) Bischler, Herbertusoblongifolius (Steph.) Gradst. et Cleef, and others  is retained: larger and thin-walled  cells on the outer layer and smaller and thick-walled cells inward, the cell size of M.brasiliensis is much smaller. In the latter, the outer cells are 12–14 µm in diameter and the inner cells are 8–10 µm in diameter, versus 12–25 µm in the outer layer and 12–15 µm in the inner layer in M.microphylla. Third, the leaves of M.microphylla are consistently bilobed, while in our species, they are entire on smaller shoots and are only shortly incised on larger shoots. In addition, M.microphylla type and the specimens described here as M.brasiliensis. He found that the seta cross section consists of 10–14(-16) rows of cells in the outer layer with 4–8 rows in the inner layer, whereas that of M.microphylla possesses 8–10 outer rows and 3–4 inner rows. Marsupella.The present study showed that this specimen identified as d others , 2018. Mla Smith . TherefoMarsupellasect.Stolonicaulon, based on taxa whose position within the section is genetically confirmed, sporophytes are only known in M.stoloniformis N. Kitag. The elaters of this species are 2–3-spiral ”. Therefore, it is unclear if Váňa actually saw the 3-spiral elaters or was referring to the 3-spiral elaters previously described by M.microphylla: capsule wall bistratose, with nodular thickening in both the outer and inner layers. Within sect. Stolonicaulon, M.brasiliensis is distinguished from other species by paroicous inflorescence . The morphology of sterile plants of M.brasiliensis is similar to that of M.vermiformis (R.M. Schust.) Bakalin & Fedosov and M.microphylla; the haplotype network . The stem cross-section characteristics of M.vermiformis are distinctly different from those of M.brasiliensis , but soeva (cf. ).Marsupellasect.Stolonicaulon in Latin America, but was unable to determine whether M.microphylla belongs to sect. Stolonicaulon. Taking into account that the number of elater spirals and the shape of the elaters may be a rather important taxonomic feature, there are doubts that M.microphylla can be referred to as the same section, and the subgenus Nanocaulon may require re-evaluation, at least at the rank of section. Marsupellasubg.Nanocaulon was synonymized with M.sect.Boeckiorum Müll. Frib. ex R.M. Schust. (M.sect.Boeckiae Müll. Frib.) in M.microphylla. However, taking into account data in an integrative systematic study by Boeckiorum is strictly different from sect. Stolonicaulon, and considering the distribution pattern, we suggest that even if M.microphylla does not belong to sect. Stolonicaulon it also seems hardly possible that M.microphylla belongs to sect. Boeckiorum, which contains north Holarctic taxa and has a sister position to the Marsupellasect.Marsupella, which is also composed of North Holarctic species.Therefore, the present study has confirmed the occurrence of Marsupellabrasiliensis is thus far the only known species of the sect. Stolonicaulon with paroicous inflorescence and occurring in Latin America whose position has been confirmed genetically. All other species are dioicous (with adjustment for the fact that the inflorescences are not known for M.praetermissa and M.anastrophylloides). Finally, we may assume that the mountain systems of Latin America may hide additional taxa belonging to Marsupellasect.Stolonicaulon, and purposeful research in this field should be continued.