Allium saralicum R.M. Fritsch  ethanolic extract to alleviate the adverse effects of streptozotocin (STZ)‐induced diabetes in male BALB/c mice. Seventy male mice were randomly divided into seven groups (n = 10). Diabetes was experimentally induced by STZ (60 mg/kg bw). A. saralicum ethanolic extract with doses 5, 20, 80, and 320 mg/kg was administrated for 20 consecutive days in diabetic animals. Based on the obtained results, the untreated diabetic mice showed high blood glucose level, cholesterol, low‐density lipoprotein (LDL), white blood cells count (WBC), and platelets, as well as liver enzymes, urea, and creatinine. Administration of different doses of A. saralicum extract significantly reduced blood glucose level similar to glibenclamide. Also, the levels of catalase and superoxide dismutase enzymes restored toward normal level. All hepatic and renal function parameters as well as hematological parameters were improved following treatment with A. saralicum extract particularly at high doses. Histopathological studies showed a decrease in hepatic, renal, and pancreatic damage after treatment with A. saralicum extract. The results of the present work indicate that A. saralicum ethanolic extract can attenuate diabetic hepato‐renal, pancreatic, and hematological damages.Medicinal plants can protect different organs against diabetes‐induced oxidative stress due to their antioxidant compounds. The present study was designed to investigate the potential of  Ethanolic extract of Allium saralicum R.M. Fritsch contains antioxidant compounds such as linolenic acid, phytol, and neofitadine. Ethanolic extract of Allium saralicum R.M. Fritsch can improve hyperglycemia caused by diabetes, and attenuated renal, hepatic, and pancreatic complications. Ethanolic extract of Allium saralicum R.M. Fritsch can prevent anemia and blood disorders following diabetes by controlling the hematological parameters. The antioxidant compounds of Allium genus are Allicin [diallyl thiosulfinate], tuberoside M, thiosulfinates, S‐propargyl‐L‐cysteine, quercetin, S‐benzyl‐cysteine, diosgenin, polysulfanes, diosgenin, fisetin, diallylpolysulfides, onionin A, flavonoids, furostanol saponins, allyl sulfides, saponins, glycosides, diallyl tetrasulfide, and garlic oil , Iran in May 2018. AS was identified by the Agriculture Faculty Research Center Herbarium, Razi University, Iran (No. 2738RUH).In this research, 2.2A. saralicum leaves were collected in summer, then milled after drying. 220 g of leaf powder was dissolved in 100% alcohol solution for 2 days. Then, the solution was filtered through paper  and dried in room temperature. Finally, 30 g of A. saralicum ethanolic extract was stored at 4℃  were purchased from the Pastor Institute, Tehran, Iran. The mice were housed in 12 hr of light–dark, at 24–33℃, 45%–65% relative humidity and fed with standard pellet  and water ad libitum. The protocols were approved by the Ethics Committee of Tehran Medical Sciences, Islamic Azad University  and performed completely in line with the guidelines of the Animal Ethics Committee.7‐week‐old male BALB/c mice  dissolved in citrate buffer (0.1 M and pH 4.5), at a dose of 60 mg/kg body weight. The animals with fasting blood glucose more than 350 mg/dl were considered as diabetic  and treated through gavage for 20 days:C: Healthy group treated by 0.5 ml normal saline.UTD: Diabetic group treated by 0.5 ml normal saline.G20: Diabetic group treated by 0.5 ml glibenclamide (20 mg/kg).A. saralicum ethanolic extract (ASEE) (5 mg/kg).ASEE5: Diabetic group treated by 0.5 ml ASEE20: Diabetic group treated by 0.5 ml ASEE (20 mg/kg).ASEE80: Diabetic group treated by 0.5 ml ASEE (80 mg/kg).ASEE320: Diabetic group treated by 0.5 ml ASEE (320 mg/kg).One day after induction of diabetic, the animals were classified into the several groups  from the tail vein and asses by a glucometer. On day 20 of the experiment, 6 mg/kg of xylazine and 38 mg/kg of ketamine HCl were injected into the tail vein for euthanizing the animals. Then, the bloods were extracted from the hearts for biochemical and hematological experiments.2.7The collected samples were centrifuged for 16 min at 12,000 rpm and serum separated. In serum, the levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, total protein, conjugated bilirubin, total bilirubin, creatinine, urea, cholesterol, low‐density lipoprotein (LDL), and high‐density lipoprotein (HDL) were analyzed by using diagnostic kits in Mehr laboratory, Iran.2.8In the hematological section, the blood samples were examined by a hematology analyzer. The parameters including white blood cell (WBC), red blood cell (RBC), hemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were assessed.2.9In this research, the levels of liver and kidney antioxidant enzymes, that is, catalase (CAT) and superoxide dismutase (SOD) were measured according to the Mohammadi et al.,  and Hemm2.10In the histopathological section of the recent study, the pancreas, liver, and kidney samples were collected and investigated after preparing tissue sections. The volume density of the islets and B cells, percentage of B cells, number of islets, and average area of islets were measured. In the liver sections, the enlargement and congestion in sinusoids, central veins, portal veins, and hepatic arteries, sinusoids hyperemia, fibrin and mononuclear cells leakage in pericentral veins and periportal zones, bile ducts proliferation, hepatocytes cellular and nuclear pleomorphism, eosinophilic cytoplasmic bodies and inclusion bodies in hepatocytes, hepatocytes necrosis, and liver fibrosis and cirrhosis were evaluated.In the kidney, the enlargement and congestion in glomeruli, renal veins, and renal arteries, fibrin leakage in periglomerular zone, perirenal veins, and perirenal arteries, proximal convoluted tubules and distal convoluted tubules, cells necrosis, glomerular and tubular atrophy, and renal fibrosis were assessed.2.11p ≤ .05 was considered significant. The values are presented as mean ± SD.The normality of data was determined by Kolmogorov–Smirnov test and followed by one‐way ANOVA test and post hoc Duncan test. All of the statistical analyses were conducted using SPSS 22.0  (IBM Corp.) and a 33.1A. saralicum was 6.25% (18.75g), calculated on the fresh leaves of the plant. Overall, fifteen compounds such as neophytadiene, 2‐hexadecene‐3,7,11,15‐tetramethyl, hexadecanoic acid, phytol, linolenic acid‐methyl ester, hexanedioic acid, bis (2‐ethylhexyl) ester, 1,4,8,11‐tetraazacyclotetradecane, hexatriacontane, nonadecene, ethanol‐2‐tetradecyloxy, γ‐tocopherol, eicosane, vitamin E, 2‐phenyl‐5‐methylindole, and n‐ethyl‐1,3‐dithioisoindoline were identified in the ASEE using GC‐MS, and linolenic acid‐methyl ester (24.39%), phytol (14.19%), and neophytadiene (11.6%) were the most detected compounds  in a time‐dependent manner. However, treatment of the diabetic mice with ASEE at all doses significantly (p ≤ .05) decreased the blood glucose levels similar to the glibenclamide‐treated mice at day 20 of the experiment. ASEE exerted its maximum effect on day 20 of the experiment.The effect of ASEE on fasting blood glucose level in the diabetic mice is presented in Figure 3.3The histological sections of the liver in the untreated diabetic mice showed degenerative changes in the hepatocytes represented by disorganization of the hepatic cords, hyperemia of sinusoids, enlargement and congestion of the central veins, portal veins, and hepatic arteries with mild hepatocellular necrosis, fibrin, and mononuclear cells leakage. The hepatocytes of the untreated diabetic mice revealed morphological changes such as pyknosis, karyorrhexis, chromatolysis, and cytoplasmic vacuolization. However, the liver of the ASEE‐treated diabetic mice indicated significant improvement compared to those of the untreated diabetic ones except the presence of a few mildly degenerated hepatocytes around the central veins and some cytoplasmic vacuoles. In addition, there was no evidence of hemorrhages, inflammatory cells infiltration, or parenchymal cell necrosis in the livers of the ASEE320‐treated diabetic mice. The liver of the control group had normal structure  in the untreated diabetic mice compared to the normal control group. The volume density of pancreatic islets also showed a significant decline (p ≤ .05) following induction of diabetes. The pancreas of the diabetic mice treated with ASEE showed a slight increase in the size of pancreatic islets, having a few cells with hyperchromatic nucleus and regeneration of the beta cells in the center of islets. Also a regeneration process of pancreatic islets was more evident in ASEE‐treated groups. Although the number per square millimeter of the pancreatic islets, the volume density of the islets, and the volume density of the beta cells in the pancreas improved following administration of ASEE320, however, the percentage of beta cells, and the volume density of the beta cells in the pancreatic islets in the ASEE‐treated mice were still significantly (p ≤ .05) lower than those of the control group.The effect of ASEE on histomorphometric findings of the pancreatic tissue in the diabetic mice is presented in Figure 3.4p ≤ .05) as compared to the control group. Several doses of ASEE and glibenclamide could significantly (p ≤ .05) decrease the raised levels of ALP, AST, ALT, cholesterol, LDL, total and conjugated bilirubin and increased HDL, SOD, CAT, total protein, and albumin significantly (p ≤ .05) as compared to the untreated group.The estimated values of the liver enzymes are presented in Figures 3.5p ≤ .05) compared to the control group. Different doses of ASEE could significantly (p ≤ .05) ameliorate the above parameters. There was no significant difference in the above‐mentioned parameters (p ≤ .05) between ASEE320 and control groups.The estimated values of the kidney biochemical parameters are depicted in Figure 3.6p ≤ .05) increased in the untreated diabetic mice. Also, the levels of lymphocytes, monocytes, RBC, MCV, Hb, MCH, MCHC, and PCV significantly (p ≤ .05) reduced in the untreated diabetic group. Treatment with ASEE significantly (p ≤ .05) ameliorated the above parameters. There were no significant differences in hematological parameters (p ≤ .05) between ASEE5, ASEE20, and glibenclamide groups. Also there were no significant differences (p ≤ .05) in the above factors (except for platelet and Hb levels) between ASEE320 and control groups  on streptozotocin‐induced diabetes in male mice from various histopathological, hematological, and biochemical aspects.The present study investigates the efficacy of ethanolic extract of A. saralicum could significantly reduce the blood glucose level in STZ‐induced diabetes. Such hypoglycemic effects of medicinal plants can be attributed to decrease in the rate and amount of intestinal absorption or increase in glucose uptake by peripheral tissues , improved the above‐mentioned parameters. Peelman et al.,  production and damages to the pancreas, leading to increased blood glucose level. These molecules are exacerbating factors in cellular injury, inflammation, cardiovascular diseases, and aging process. Therefore, antioxidants play a significant role in reducing diabetes complications . Mahsa Hadipour Jahromi: Investigation . Zahra Tootian: Writing‐original draft . Nader Goodarzi: Software ; Visualization .This study was approved by the Institutional Review Board of Tehran Medical Sciences, Islamic Azad University .


Some studies have proved that labrum size is associated with symptoms in patients with hip labral tear. The correlation between the labrum size and the labral tear in asymptomatic volunteers and symptomatic patients is still uncertain.The volunteers with no history of pain, injury, or surgery were recruited from the community. Patients who were diagnosed with labral tear and underwent hip arthroscopic surgery in this period in our hospital were also included. The length and height of the acetabular hip labrum were measured at three separate anatomic sites through magnetic resonance imaging (MRI) along the acetabular rim: lateral, anterior, and anteroinferior.P < .05). In 14 volunteers with unilateral labral tears, length of lateral, anterior, and anteroinferior labrum in the side with tear were significantly larger than normal on the other side. The anterior labral height of volunteers was significantly larger than that of patients (P < .05).A total of 70 volunteers (125 hips) and 70 patients (70 hips) were included in this study. Sixty-six (52.8%) hips had labral tears in all 125 hips of volunteers. The lateral labral length of volunteers with labral tears was significantly larger than those without labral tears (In conclusion, asymptomatic volunteers with larger length of lateral, anterior, and anteroinferior labrum are more prone to present with labral tears. Symptomatic patients with labral tears exhibited thinner anterior labrum. Further studies are warranted to explore the mechanisms of labral tears in asymptomatic people and validate the use of labral size as a guide to differential diagnosis and treatment. The acetabular labrum is a triangular fibrocartilaginous structure attached to the rim of the acetabulum and envelopes the femoral head, which creates a suction seal . It may In our daily work, we noticed that patients with large labrum may be more prone to undergo labral tear. We hypothesized that labrum size may relate with labral tear and show difference in asymptomatic volunteers and symptomatic patients. The purpose of this study was to evaluate the correlation between the labrum size and the labral tear in asymptomatic volunteers and symptomatic patients.Volunteers with no symptoms were recruited from the community. The volunteers with no history of hip pain, injury, or surgery were included in this study. Volunteers chose bilateral MRI or random unilateral MRI of their own accord. Participants were excluded if they had claustrophobia or a contraindication to obtain an MRI. Patients who were diagnosed with labral tear and underwent hip arthroscopic surgery in this period in our hospital were also included. All patients had preoperative MRI of the affected side. All participants signed informed consent. The study was approved by the Ethics Committee of the Third Hospital of Peking University. All methods were performed in accordance with the guidelines and regulations of the Ethics Committee of the Third Hospital of Peking University.The hip magnetic resonance examinations were performed as previously described , with a The measurements of labral size were made independently by two musculoskeletal radiologists, blinded to the clinical information and arthroscopic findings. The length and height of the acetabular hip labrum were measured at three separate anatomic sites along the acetabular rim: lateral, anterior, and anteroinferior , 9. The P values < 0.05 were considered statistically significant. All statistical analyses were performed with SPSS Statistics, version 22 (IBM).Intra- and interclass correlation coefficients were calculated to examine the reproducibility of measurements of labral size. MRI measurement of labral size at three separate anatomic sites was performed twice by one radiologist and once by another radiologist. The interclass correlation coefficient was calculated from the mean of the first two measurements performed by the initial investigator and the single measurement of the verifying investigator. The two-tailed paired t test was used to evaluate significance between labral size of hips in patients with unilateral tear. Continuous variables with a normal distribution in the baseline data between groups were examined using the independent-samples t test. Percentages were compared using the Chi-square test. P > 0.05). There was no significant difference between BMI and labral size both in volunteers group and patients group. There were 36 (51.4%) females and 34 (48.6%) males in the asymptomatic volunteer group, and 30 (42.9%) females and 40 (57.1%) males in the patient group. Sixty-six (52.8%) hips had labral tears in all 125 hips of volunteers. Fourteen volunteers had unilateral tears. Fifty-two patients (74.3%) of the 70 patients had pincer femoroacetabular impingement (FAI), and 61 patients (87.1%) had cam-type FAI and all patients had labral tears. The mean alpha angle of asymptomatic volunteers and symptomatic patients were 49.5 ± 7.1  and 61.5 ± 4.9 , respectively. The mean LCEA of asymptomatic volunteers and symptomatic patients were 28.0 ± 5.7  and 30.9 ± 3.3 , respectively. Asymptomatic volunteers with borderline dysplasia (LCEA 20°–24.9°) had larger lateral labral length compared with asymptomatic volunteers with normal acetabular coverage (LCEA 25°–39.9°) (P < 0.05). There was no significant correlation between alpha angle and labral size.As shown in Table P < 0.05).As shown in Table P < 0.05).The mean lateral labral length, lateral labral height, anterior labral length, anterior labral height, anteroinferior labral length and anteroinferior labral height of hips with labral tear in volunteers were 6.46 ± 1.79, 4.41 ± 1.04, 8.07 ± 1.86, 4.40 ± 0.76, 5.75 ± 2.11, 4.10 ± 0.94, respectively. The mean lateral labral length, lateral labral height, anterior labral length, anterior labral height, anteroinferior labral length and anteroinferior labral height of hips without labral tear in volunteers were 5.44 ± 1.47, 4.45 ± 1.06, 7.92 ± 1.64, 4.14 ± 0.87, 6.10 ± 1.92, 4.27 ± 0.88, respectively. There was significant difference in lateral labral length between volunteers with and without labral tear (P < 0.05). The intraclass correlation coefficients between the two measurements made by the same observer were 0.96, 0.86, 0.81, 0.84, 0.90, and 0.86 for measurements of labrum length at lateral, anterior, and anteroinferior hip and height at these three separate anatomic sites, respectively. The interclass correlation coefficients were 0.92, 0.79, 0.79, 0.76, 0.81, and 0.81 for measurements of labrum length at lateral, anterior, and anteroinferior hip and height at these 3 separate anatomic sites, respectively.There were 14 volunteers who had unilateral labral tears. In volunteers with unilateral labral tears, the mean lateral labral length, lateral labral height, anterior labral length, anterior labral height, anteroinferior labral length and anteroinferior labral height of labrum in the side with tear were 5.86 ± 1.27, 4.36 ± 1.04, 7.57 ± 2.01, 3.77 ± 0.62, 5.88 ± 1.47, 4.55 ± 0.70, respectively. The mean lateral labral length, lateral labral height, anterior labral length, anterior labral height, anteroinferior labral length and anteroinferior labral height of labrum in the side without tear were 5.00 ± 1.18, 4.23 ± 0.98, 6.91 ± 1.46, 3.85 ± 0.92, 6.63 ± 1.38, 4.39 ± 0.71, respectively. There was a significant difference in length of lateral, anterior and anteroinferior labrum between labrum in the side with and without tear in volunteers with unilateral labral tears  hips had labral tears in all 125 hips of volunteers.P < 0.05), which was consistent with previous studies. Garabekyan et al. [Over the past decade, hip arthroscopic surgery has developed rapidly and has been proved to have good clinical outcomes in the treatment of FAI and combined labral tears in both adults and adolescents –15. Hip n et al.  evaluaten et al.  evaluateThere are several studies on the topic of asymptomatic labral tears, but with quite different findings. Lee et al.  performeUntil recently there was no mention of the mechanism of labral tear in nondysplastic hips and hips without FAI , 29. In This study has several limitations. Firstly, we did not have data about intraoperative measurement of the labral length in patients group. Correlation between more accurate intraoperative measurement of labral length and our MRI measurement could not be evaluated. Secondly, this study did not evaluate interobserver reproducibility of MRI measurements. Thirdly, this study did not quantify the relationship between symptoms and the size of the labrum in patients.Asymptomatic volunteers with larger length of lateral, anterior and anteroinferior labrum are more prone to present with labral tears. Symptomatic patients with labral tears exhibited thinner anterior labrum. Further studies are warranted to explore the mechanisms of labral tears in asymptomatic people and validate the use of labral size as a guide to differential diagnosis and treatment.


Multimodal tumor treatment settings consisting of radiotherapy and immunomodulating agents such as immune checkpoint inhibitors are more and more commonly applied in clinics. In this context, the immune phenotype of tumor cells has a major influence on the anti-tumor immune response as well as the composition of the tumor microenvironment. A promising approach to further boost anti-tumor immune responses is to add hyperthermia (HT), i.e., heating the tumor tissue between 39 °C to 45 °C for 60 min. One key technique is the use of radiative hyperthermia systems. However, knowledge is limited as to how the frequency of the used radiative systems affects the immune phenotype of the treated tumor cells. By using our self-designed in vitro hyperthermia system, we compared cell death induction and expression of immune checkpoint molecules (ICM) on the tumor cell surface of murine B16 melanoma and human MDA-MB-231 and MCF-7 breast cancer cells following HT treatment with clinically relevant microwaves at 915 MHz or 2.45 GHz alone, radiotherapy  alone or in combination (RHT). At 44 °C, HT alone was the dominant cell death inductor with inactivation rates of around 70% for B16, 45% for MDA-MB-231 and 35% for MCF-7 at 915 MHz and 80%, 60% and 50% at 2.45 GHz, respectively. Additional RT resulted in 5–15% higher levels of dead cells. The expression of ICM on tumor cells showed time-, treatment-, cell line- and frequency-dependent effects and was highest for RHT. Computer simulations of an exemplary spherical cell revealed frequency-dependent local energy absorption. The frequency of hyperthermia systems is a newly identified parameter that could also affect the immune phenotype of tumor cells and consequently the immunogenicity of tumors. Cancer is the first or second leading cause of premature death in over 100 countries around the world .Currently, a combination of the following treatment options is commonly used: surgery, radiotherapy (RT), chemotherapy, and increasingly immunotherapy such as immune checkpoint inhibitors (ICI). Because of their high level of commitment and good results, these methods are often referred to as the four pillars of cancer treatment. With ICI, however, the response rate is mostly dependent on the tumor, its microenvironment, and the treatment scheme. In order to improve the response rate and ultimately the clinical outcome, other methods have come into focus in recent years ,4. A faiTo apply clinical hyperthermia, different technologies can be considered depending on the size and location of the tumor. Often a subdivision in whole-body, regional or local hyperthermia is made first; then further differentiation is made regarding the depth at which it is operated upon. For regions up to a depth of 4 cm under the skin, it is classified as superficial, otherwise as deep hyperthermia. Depending on this differentiation, specific heating means are used to achieve the temperature rise. Conventional heating (CH) takes place by conducting heat through a circulating liquid, which can be applied externally or internally  ,8,9,10. The formation of pores due to an electric field is an example of such an electromagnetic effect , but theTtarget) in the range from 37 °C to 44 °C. To mimic blood perfusion, the flow rate was kept constant at 2 mL/s. Within the closed-loop system, temperature was monitored at the inlet (TIR01) and outlet (TIR02) of the respective heating unit as well as within the cooler (TIR03) and at the end of the variable length (TIR04). Furthermore, the level, i.e., power, of the microwave units was controlled and measured.Ex vivo heat treatment of the cell suspension was performed in a self-designed stainless-steel lab-scale closed-loop system under sterile conditions a,b. The di = 10.2 mm, da = 13.0 mm, tube length 86 mm for media and water load) at the applicator’s closed end, and with manual stub tuners for fine-tuning the energy absorption  . The respective media volume of the heating unit, V1, was kept constant at 6.88 cm3. V2 was set to 25 cm3 and V3 was 8.50 cm3. The total system volume, mV, was set to 129 cm3, and V4+Δ was 88.62 cm3. The effective total volume, Vm,eff, is in fact 119 cm3 as 10 cm3 (bpV) are reserved for bypass and sampling. In case of 915 MHz MH a volume reduction of 13.80 cm3 in the variable length was necessary for the 915 MHz setup as the total media volume in the 915 MHz heating unit was 20.68 cm3 in comparison to 6.88 cm3 in the other system operating at 2.45 GHz. For the self-designed stainless-steel cooling-loop , a total volume of 7.49 cm3 was used to achieve a cooling capacity for all treatment conditions  of the total treatment time, i.e., 60 min of effective heating is applied within 4 h .The 2.45 GHz microwave setup consisted of a 2 kW-microwave generator (TM A20 S1) with a circulator for catching and dissipating the reflected power. For the 2.45 GHz experiments, a custom-made rectangular waveguide  was used with two horizontally aligned quartz-glass tubes (sorption a. For ther load) b. To acher load) b, green.nditions c. During®® Multiphysics© 5.5. In all simulations, the medium flow rate, Ttarget, was varied between 37 °C and 44 °C. In electromagnetic heating processes, the most relevant material parameter is the complex permittivity, consisting of the real part, As the temperature of the cell suspension can only be measured at the inlet and outlet , the rearequency . As a si2 and 90% humidity under sterile conditions. The two human breast cancer cell lines, MDA-MB-231 and MCF-7, were grown in Dulbecco’s modified Eagle’s medium and the murine B16 melanoma cell line in RPMI 1640, each supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 2 mM glutamine, 100 U/mL of penicillin and 100 µg/mL of streptomycin. MDA-MB-231 cell line is triple negative, i.e., ER negative, PgR negative, p53 mutated and caspase-3 intact, while MCF-7 cells are ER positive, PgR positive, p53wt and deficient of caspase-3 [All cancer cell lines were cultivated at 37 °C in 5% COaspase-3 ,18,19,20-1d), the cells were seeded according to the respective cell line, so that the confluency never exceeded 90%. At time point d0 (0′), both for HT and the combinatory arm, i.e., HT + RT (RHT), sample injection of 1 × 107 cells was followed by an effective heating time of at most 60 min (60′). During the (R)HT session at a constant flow rate of 2 mL/s at Ttarget = 39 °C, 41 °C or 44 °C, optional sampling at time points 10 min (10′), 20 min (20′) and 30 min (30′) could be done. In this paper, we only focus on 30′ as optional time point. The total treatment time is four times more than the effective treatment time, i.e., 60 min of effective heating means 4 h of cyclic heating in total. The cells were split into 75 cm2 t-flasks according to their subsequent treatment. For the RHT arms, irradiation was performed for 2 h after HT treatment, as common in multimodal treatments [Inactivation of the tumor cells was performed according to eatments .®® XL™ and Cytoflex™ S) was used to detect cell death forms by AnnexinV-FITC (AxV)/propidium-iodide (PI) staining and to determine immune checkpoint molecules (ICM) by antibody staining. Multicolor flow cytometry . For the EPICS XL system, 400 µL Ringer®® solution stained with 1.0 µg/mL of PI and 0.5 µg/mL of FITC-labeled AnnexinV was resuspended with 100,000 cells/tube. For the Cytoflex S system, 100,000 cells/well were resuspended in 100 µL Ringer®® solution stained with 4.0 µg/mL of PI and 2.0 µg/mL of FITC-labeled AnnexinV.For cell death detection, PI penetrates only into cells that have lost their membrane integrity. Due to the binding mechanism of PI with DNA, the intensity of the PI signals allows a first differentiation between primary necrosis and secondary necrosis, since primary necrotic cells have the full DNA content as viable cells and secondary necrotic cells derive from apoptotic ones that already have degraded and shedded DNA via apoptotic bodies . By co-s®® software. All antibody concentrations were tested according to manufacturer’s instruction and finally titrated for the human and murine cell lines. To distinguish viable from dead cells, Zombie NIR  or Zombie Yellow (murine#2) at a concentration of 0.1 µL/well was used. The mean fluorescence intensity of stained samples was subtracted from unstained mock-treated samples that contained only FACS-buffer and Zombie NIR/Yellow.For analysis of immune checkpoint molecule expression, 100,000 cells/well were incubated with 100 µL of the cell line specific antibody staining solution  for 30 mW, which is used in hyperthermia treatment and better known as the specific absorption rate, SAR in W/kg, can be calculated using Equation (1):f of the applied electromagnetic wave, the density ρ of the corresponding medium, the complex conjugated amplitude 0* of the electric field and 0 as the amplitude of the dielectric displacement field. To solve Equation (1), both, the electric field and dielectric displacement field need to be calculated first. Since both are connected via the permittivity, it is sufficient to work out only one field. As with similar problems, one can use the electric potential as a starting point. The electric potential must satisfy the tensorial Laplace equationAn electric field that penetrates a single cell affects the electric potential inside the cell and the absorbed heat. Both can be described mathematically by Maxwell‘s equations and dielectric boundary conditions. To simplify the highly complex structure of a single cell, only soluble, bulk water and three regions of a dipalmitoyl-phosphatidylcholine (DPPC) phospholipid were modeled including a polar head, linker and tail group a. The voi-th shell is described according to [i, Bi and δi can be directly derived from absorption spectra ϕi spectra a, Ai andished in . Finallyd3) and day5 (d5), for analyses of the effects of normo- and hypo-fractionated radiotherapy alone and in combination with 915 MHz hyperthermia (RHT).The use of incubators or warm-water baths for ex vivo hyperthermia (HT) is easy to perform but does not focus on the clinically relevant radiative-based heating technique. However, radiative-based in vitro hyperthermia has some limitations, such as the use of t-flasks in a microwave chamber. Here, local hot-spots are often observed due to inhomogeneities in the distribution of the electromagnetic field, and sufficiently accurate online temperature monitoring is hardly possible. Further, frequencies of microwaves that are used in clinical devices should be used for the preclinical examinations. We therefore focused in this work on microwave radiation-based HT at 915 MHz and used our self-designed closed-loop media flow system with modular heating devices . After tdi = 10.2 mm, da = 13.0 mm, tube length 247 mm for both the media and water load). For physical reasons, the 915 MHz waveguide is wider by a factor of 2.67 than the waveguide for 2.45 GHz, so a shielding alumina hollow tube  into a resonator cavity a. For th more in . Finally3 larger than in the 2.45 GHz heating unit, and the treatment should remain as similar as possible, modifications were done. On the one hand, an adapted pipeline was installed after the outlet from the heating unit to gain space (red area), and on the other hand, the bypass after the cooling bath was removed. As a result of the modification, the effective total volume, m,effV, as well as the effective heating time, efft, per cycle remained identical to the 2.45 GHz system. Since the volume in the 915 MHz heating unit was 13.80 cm3), the temperature distribution (°C) and finally biological correlations [L(z)/L0. The heat treatment and fluid flow conditions were determined by Ttarget = 44 °C and Clinical treatment planning for hyperthermia includes the distribution of the electromagnetic field (V/m), the specific absorption rate (W/kg or W/melations . For botL/L0 = 0.00), the media temperature is 37 °C. From there it rises while passing the microwave field and reaches an average temperature of 44 °C at the outlet (L/L0 = 1.00). However, not only the fluid temperature is rising under influence of the microwave field but also the quartz glass temperature. As the quartz glass tube is permanently exposed to thermal and microwave load it stores heat despite its low dielectric loss. In the middle of the pipe, at L/L0 = 0.50, the simulations reveal a difference between both microwave frequencies. At 2.45 GHz the quartz glass tube reaches higher wall temperatures than 915 MHz: 45.6 °C compared to 44.3 °C. At the outlet, the wall temperatures are 46.4 °C for 2.45 GHz and 44.6 °C for 915 MHz. Further, there are differences in the distribution of the electric field as shown in +/PI-), primary (AxV+/PI++) and secondary necrotic (AxV+/PI+) cells, were found at Ttarget = 44 °C. By comparing the effective treatment times of 30 min (30’) with 60 min (60’), no significant difference was found for all examined cell lines and temperatures.As shown in Furthermore, a cell line- and frequency-dependent inactivation behavior was found. B16 melanoma showed the highest degree of inactivation at 44 °C with about 80% at 2.45 GHz d and 70%d3) and 5 (d5), we first analyzed forms of cell death after radiotherapy only  ,28. So f For B16 a, no sig®®) within a short time frame (<14 days vs. >14 days) [d3 and d5, the expression of PD-L2, HVEM and Gal-9 was even more upregulated. This was especially found for PD-L2 and Gal9 in B16  . Therefo9 in B16 a on both B16 . The foundations of this approach are described elsewhere .Rcyto = 10 nm) that is enclosed by a single DPPD-bilayer structure (Sbilayer = 6 nm) and a solvent region that is 105 times larger than Rcyto. , illustrating global SAR values for a cell with 4 μm at various microwave and radio frequencies. Global SAR values drop with decreasing frequencies. As mentioned above, our model is very simple as it lacks certain cell components. However, the model demonstrates the role of the DPPD-bilayer and frequency for a single cell’s energy absorption. 9 Hz. Further, As real cellular components such as cell membranes or whole cells are very complex structures, we use a spherical model of a cell with radius 4 μm to 14 μm. The cell has a simplified structure consisting of a cytoplasm region  are sometimes controversial, often due to the inaccurate description of the experimental setup including the temperature distribution and possible hot-spots in the macro- and submicron range. AC current application <300 Hz) has proven to stimulate non-thermal effects and affect DNA synthesis, as demonstrated by Tsai et al. in the proliferation behavior of osteoblasts 0 Hz has ,32. At rOur simulations for a simplified single cell model show that the SAR of the outer monolayer of the DPPD is significantly higher, while the inner monolayer is shielded by the bilayer’s core. This could be a first hint as to why radiofrequency inactivates cells at a much higher magnitude at 44 °C in comparison to conventional warm-water, as described by our group in previous experiments . To obtaTmax) or the median time-average temperatures reached in 20%, 50%, and 90% of all measured sites . In this exemplary phase III study, Tmax was 41.8 °C (41.1–43.2 °C), T20 was 40.8 °C (40.1–42.3 °C), T50 was 40.3 °C (39.5–41.0 °C), and T90 was 39.2 °C (38.5–39.8 °C) [Hyperthermia is probably the oldest method in the fight against cancer. Its immunomodulatory properties have been investigated and demonstrated in various in vitro, in vivo and phase I-III studies ,38. For  °C 41.1–.2 °C, T239.8 °C) , could hSimilar local and systemic effects are known for radiotherapy as for hyperthermia, e.g., HSP70 and ATP release or NK and DC activation . RecentlWe have developed an in vitro system to measure clinically relevant hyperthermia effects at two frequencies, 915 MHz and 2.45 GHz, and at different target temperatures, 39 °C, 41 °C and 44 °C, alone and in combination with normo- and hypofractionation irradiation, to find out how RT in combination with HT alters the immunogenic phenotype of tumor cells and which combination is most beneficial, depending on the microwaves’ frequency. This may also provide the first clues on how to increase the response rate of additional immunotherapies such as immune checkpoint inhibitors (ICI), e.g., by optimizing treatment protocols and taking further into account the individual patient’s tumor ,51,52.In addition to the frequency dependence, a cell line difference was found, i.e., murine B16 melanoma had a higher inactivation rate by microwave-based HT compared with human MCF-7 and MDA-MB 231 breast cancer cells. A possible explanation could be differences in lipid membrane composition, which causes lower stability under thermal stress . In geneTi) for 60 min and 10.1% for 30 min, respectively, while at 2.45 GHz therapy, endocrine therapy, and/or targeted therapies, e.g., hormones or immune checkpoint inhibitors (ICI). The latter have changed the field of oncology in many ways, especially in advanced or metastatic tumors such as non-small cell lung cancer (NSCLC), melanoma, or triple-negative breast cancer (TNBC) . A standn = 116), Malissen et al. also found HVEM to have broader expression than PD-L1 and thus suggest more focus on HVEM as a prognostic marker and potential treatment target [It is generally accepted that RT affects the expression of PD-L1 ,59,60, bt target . The addIn summary, biological variants in tumor tissue are normal ,63,64 anA new focus in preclinical and clinical studies needs to be placed on the combination of hyperthermia and radiotherapy, i.e., temperature, frequency, tumor entity, and timing within multimodal tumor treatments. Our in-silico simulations showed frequency-dependent energy absorption by cellular components, which is currently not considered in hyperthermia. In addition, differences between heating approaches, e.g., by electromagnetic radiation, infrared, or conventional warm-water, could be better understood. The expression of immune checkpoint molecules was highly dynamic and needs to be studied in much more detail to optimize targeted and personalized drugs in the future. The ex vivo results obtained with our system and adapted devices may provide initial clues for tailoring patient-specific treatment. Key analyses of the immunogenic properties of the tumor cells following the treatments by determining apoptosis, primary and secondary necrosis, the latter two tumor cell death forms being indicators for high immunogenicity, were performed in this work. Additionally, the tumor cell surface composition regarding the expression of immune checkpoint molecules was characterized. Future work should additionally include functional immune cells assays, such as testing the activation and T cell stimulating properties of dendritic cells after being in contact with the treated tumor cells . Further


The purpose of the current study was to determine the phenolic composition, antioxidant, and antimicrobial activities in grape cane extracts from typical cultivars of Southern Italy. Aqueous extracts at different pHs (1–13) were prepared from “Aglianico”, “Fiano”, and “Greco” grape canes. The results demonstrated that an alkaline pH (13.00) produced the best polyphenol-rich extracts, as the total phenolic content was more than double when compared to the respective extracts prepared at pH 1.00. “Greco” grape canes gave the highest quantity of phenolic compounds at each pH, ranging from 42.7 ± 0.4 to 104.3 ± 3.0 mg Gallic Acid Equivalents (GAE)/g Dry Extract (DE) from pH 1.00 to 13.00. The Radical Scavenging Activity (RSA) and the Ferric Reducing Antioxidant Power (FRAP) were measured. The highest antioxidant activity was showed by “Greco” extract at pH 7.00. Seventy-five compounds were identified in the extracts by HPLC-MS with six of them described for the first time in grape canes. Procyanidins were highly abundant in extracts at pH 7.00, whereas stilbenoids were the most represented compounds at pH 13.00. Very strong antiviral activity against herpes simplex viruses was recorded for the extracts at pH 7.00 and 13.00 that were active in the early stages of infection by acting directly against the viral particles. The overall results suggest that grape canes, currently underutilized, can be usefully valorised by providing active extracts to use as antioxidant and antiviral agents. Interest in the exploitation of agro-industrial wastes as natural sources for the production of high added-value compounds is constantly growing. The circular economy model proposes the use of materials that were traditionally considered waste, as a resource . Agricultural and industrial processing activities produce large amounts of liquid and solid wastes that can be conveniently utilised before disposal. Usually, these residues are burned or used for composting, even though they still contain valuable bioactive molecules that can be used for a number purposes in a range of sectors ,3.The wine production process generates a huge quantity of solid residues  whose management and disposal presents environmental issues. Several alternatives to the traditional disposal have been investigated, such as composting or removal of heavy metals from aqueous solutions ,5 althouVitis vinifera L.), belonging to the family of Vitaceae, is one of the fruit crops more broadly grown in many areas of the world. In 2020, about 7,231,542 tons of grapes were produced in Italy . In Campania  25,562 ha are dedicated to this cultivation. “Aglianico” (red wine), “Fiano di Avellino” and “Greco di Tufo” (white wines hereafter referred to as “Fiano” and “Greco”), typical cultivars of the territory, produce excellent quality wines pe canes ,33 and ip < 0.001), from 51.51 ± 3.68 to 580.42 ± 0.33 µg/g DE (p < 0.01), and from 136.79 ± 43.31 to 1398.06 ± 23.58 µg/g DE (p < 0.001) for “Aglianico”, “Fiano”, and “Greco”, respectively. The resveratrol dimer, ε-viniferin (peak 70), was particularly abundant in the alkaline extracts with 1657.60 ± 18.80 µg equivalent/g DE (“Aglianico”), 1617.56 ± 199.55 µg equivalent/g DE (“Fiano”), and 1543.28 ± 97.67 µg equivalent/g DE (“Greco”) in contrast to the acid and neutral extracts.The amount of the stilbenoid resveratrol (peak 67) increased from pH 1.00 to pH 13.00 varying from 220.43 ± 16.84 to 1741.01 ± 62.45 µg/g DE , resveratrol (peak 67) and ε-viniferin (peak 70) were identified in all the extracts, together with afzelechin hexoside (peak 8) and viniferol E (peak 68). To the best of our knowledge, afzelechin hexoside and viniferol E were detected for the first time in grape canes. Viniferol E was previously isolated in different Vitis vinifera parts, such as the roots of cultivar “Kyohou” and grape stems of eight French cultivars located in the Loire Valley region − at m/z 419 in the mass spectrum. In the MS2 spectrum, the following fragmentation pattern was obtained: m/z 287, corresponding to the deprotonated ion of dihydrokaempferol (or eriodictyol) after the loss of a pentose moiety [M-H-132], and m/z 269, due to the successive loss of water. HPLC/ESI-ITMSVitis vinifera by-products. To the best of our knowledge, this is the first report describing the presence of dihydrokaempferol pentoside or eriodictyol pentoside in grape canes. Only in the “Aglianico” grape cane extract prepared at pH 1.00, a second compound eluted at the same retention time of eriodictyol pentoside (or dihydrokaempferol pentoside) (peak 53) and was identified as flavonol rutin (quercetin-3-O-rutinoside). Therefore, the compound eluted in peak 53 was tentatively identified as dihydrokaempferol pentoside or eriodictyol pentoside. Dihydrokaempferol and eriodictyol were previously detected as aglycones and/or hexoside in In acid and neutral extracts, the analysis by HPLC/ESI-ITMSn allowed us to detect, for the first time, the occurrence in grape canes of A-type procyanidin dimers, non-galloylated and digalloylated. Grape proanthocyanidins are essentially of B-type, but Passos et al.  reportedO-hexoside (peak 64), identified at all pH values in quantity comprised between 132.93 ± 3.35 (pH 1.00) and 275.35 ± 2.87 µg equivalent/g DE (pH 7.00). As far as we know, this is the first report describing the presence of naringenin-O-hexoside in grape cane extracts. As a matter of fact, only the aglycone naringenin and its disaccharide derivative naringin were previously detected in grape canes from eight varieties located in the Mendoza region, Argentina [Interestingly, some phenolic compounds were distinctive of the cultivar “Aglianico” such as the hydroxycinnamic acid derivative coutaric acid (ester between coumaric acid and tartaric acid) (peak 34) and the aforementioned flavonol derivative rutin, both present in the acid extract, and the flavanone derivative naringenin-rgentina . FinallyThe antioxidant power of natural extracts is due to a mixture of several phytochemicals that can act with several and/or different mechanisms. According to this, two analyses based on diverse action mechanisms were used for determining the antioxidant capacity of the grape cane extracts: the Radical Scavenging Activity (RSA) and the Ferric Reducing Antioxidant Power (FRAP) assays.The deleterious effects of free radicals in oxidative processes involving biological systems, foods, cosmetics, and pharmaceuticals are known. The prevention of the initiation step in the radical chain by scavenging reactive species such as free radicals is considered an important antioxidant mode of action. Thus, the antioxidant power of “Aglianico”, “Fiano” and “Greco” extracts at pH 1.00, 7.00 and 13.00 was investigated through the evaluation of the discoloration of DPPH∙, a free radical that accepts an electron or a hydrogen radical to become a stable molecule.All the tested samples exhibited antioxidant activity, with values of RSA ranging from 40% ± 0.8 (“Fiano” pH 13.00) to 70% ± 0.11 (“Greco” pH 7.00) . RSA rapIn the first five minutes of the assay, the “Greco” extract at pH 7.00 showed a value higher than the reference antioxidant compounds , and after 30 min, its RSA was still slightly higher than Q  B. Even aIn the present paper, it was decided to sacrifice the higher yield and representativeness of the phenolic compounds to the advantage of an extraction method that uses a non-toxic and environmentally friendly solvent. Nevertheless “Greco” extracts at pH 7.00 and 13.00 exhibited a significant radical scavenging activity.The grape cane extracts showed antioxidant capacity when assayed by FRAP, attesting the ability of reacting with ferric ions. The highest FRAP values were measured in extracts at pH 7.00 with “Greco” having reached the value of 39.482 ± 1.287 μg Ascorbic Acid Equivalents (AAE)/mg DE. “Aglianico” and “Fiano” reached 32.058 ± 2.051 and 24.179 ± 0.681 μg AAE/mg DE, respectively.Extracts at pH 1.00 and 13.00 were endowed with lower antioxidant power, ranging from 3.649 ± 0.131 (“Fiano” pH 13.00) to 8.439 ± 0.282 (“Greco” pH 1.00) μg AAE/mg DE. Only the “Greco” pH 7.00 was found to be more antioxidant than BHT used as standard. “Fiano” pH 7.00 and “Aglianico” pH 7.00, however, showed FRAP values comparable to those obtained with BHT, even if slightly lower. The extracts at pH 1.00 and 13.00 showed much lower FRAP values than BHT. Quercetin was found to be hugely more antioxidant than all extracts and BHT. For this reason, the standards are shown in logarithmic scale in p < 0.001) within the same cultivar. The same level of significance was reached in the further increase of pH towards alkaline values (pH 13.00). In this case, the decrease of FRAP values obtained from extracts of the same cultivar (pH 7.00 and 13.00) was statistically significant (p < 0.001).The results indicated that changes of pH in the extraction process from 1.00 to 7.00 lead to a statistically significant increase of the FRAP values  or to ions with high oxidation number (ferric ions), highlighting that “Greco” grape canes, extracted at pH 7.00, can be considered as a valuable resource for the production of natural antioxidants to use in several fields such as food, pharmaceutical and cosmetic industries.The results regarding the antioxidant activity can be further investigated in the future by coupling the traditional antioxidant assays with more recent techniques such as cyclic voltammetry .The cytotoxicity was evaluated in vitro through the metabolic activity of viable cells via 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) assay . Extractp ≤ 0.05). The general results showed that the acidic extraction pH was critical for the cell viability, in particular with “Aglianico” and “Greco” grape canes. It could be justified since when the pH of the extraction varies, the corresponding phenolic composition also clearly changes both in qualitative and quantitative terms, as demonstrated earlier in our work. Consequently, the following antimicrobial assays were performed with extracts prepared at pH 7.00 and 13.00.On the contrary, a considerable reduction of viability was observed with some extracts at pH 1.00, and less marked with extracts at pH 13.00. Setting 50% of cell viability as a threshold line (CC50), “Aglianico” grape cane extract at pH 1.00 showed the highest cytotoxicity up to the lowest concentration tested (1 µg/mL). Furthermore, “Greco” at pH 1.00 was cytotoxic, exhibiting a CC50 at 50 µg/mL, while “Fiano” at pH 1.00 did not cause cellular death except at 1000 µg/mL B. The cyE. coli and S. aureus, respectively , while in the other conditions, no significant variation was obtained compared to the controls .S. aureus, consisting only of a peptidiglycone layer and lacking the outermost lipopolysaccharide (LPS) layer, typical of Gram-negative bacteria. Moreover, no significant results against Candida albicans  were observed with the extracts at any concentration  showed antibacterial activity with minimal inhibitory concentration (MIC) ranging from 0.73  to 10.9 mg/mL (S. mitis ESA65-SWE). The extracts were also active against C. albicans but at high extract concentrations comprised between 2.42  and 16.8 mg/mL  [Vitis vinifera variety, extracted with ethyl acetate, was attested by Gullón et al. [S. aureus and Listeria innocua) to 15 mg/mL (E. coli).Indeed, when 100 µg/mL was used, the bacterial growth decreased by around 40% compared to the respective positive control (only bacterium), while in the other conditions, no significant variation was obtained compared to the controls . This montration . Data weThe antiviral effect of the grape cane extracts was evaluated against HSV-1 and HSV-2. The ability of interfering with HSV-1’s life-cycle through a co-treatment assay was preliminary investigated for extracts at pH 7.00 A. Virus To evaluate the mechanism of action of these extracts more profoundly, further experiments were carried out. A cell pre-treatment test was performed to investigate whether the extracts more active in the co-treatment assay could interact with the cell mechanisms inhibiting the viral infection B. This tThe probability that HSV-1 inhibition could be addressed to an interaction among the extract itself and the virus surface was subsequently investigated. In order to verify this possible mechanism of action, a virus pre-treatment assay was performed C. In thiFinally, we investigated the ability of extracts at pH 7.00 to affect HSV-1 replication through a post-treatment assay D. In thiThe antiviral potential of “Aglianico”, “Fiano” and “Greco” extracts at pH 7.00 against HSV-2, which represents the major causative agent of genital herpes, was also investigated. In the co-treatment assay A, “Agliap ≤ 0.05.The antiviral activity against HSV-1 was also evaluated by extracts at pH 13.00 through the above-described assays. In contrast to what was observed at pH 7.00, “Fiano” showed a significant antiviral effect at 10 µg/mL in co-treatment assay, indicating that some components present in this extract are enriched at basic pH. “Aglianico” and “Greco” extracts also showed higher activity compared to the findings obtained at pH 7.00 by almost totally blocking HSV-1 infection at 10 µg/mL A. The reOur findings showed that the grape cane extracts may act either on virus attachment or directly on virus particle as a virucide, inactivating it irreversibly (virus pre-treatment), excluding that they could stably interact with one or more cellular component (cell pre-treatment) or to affect HSV replication (post-treatment). Furthermore, the extracts exhibited a slightly different response against HSV-1 and HSV-2, in agreement with Leary et al. , who repSeveral authors investigated the antibacterial and antifungal activity of grape cane extracts, but no data about the antiviral effect have been described until now. The first evidence about viral inhibition was reported by Konowalchuk and Speirs  in grape2CO3 for phenolic compounds; NaNO2, AlCl3·6H2O, and NaOH for flavonoids; HCl, Na2MoO4 and caffeic acid for ortho-diphenols; cinchonine hemisulfate and formaldehyde for tannins), 2,2-diphenyl-1-picrylhydrazyl (DPPH∙), 2,4,6-tripyridyl-S-triazine (TPTZ), FeCl3·6H2O, ascorbic acid, butylatedhydroxytoluene (BHT), HPLC standards , brain heart infusion (BHI), agar, glucose, 3-(N-morpholino)propanesulfonic acid (MOPS), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Co. . High-performance liquid chromatography (HPLC)-grade acetonitrile was obtained from Merck . Glacial acetic acid was purchased from Carlo Erba . HPLC grade water (18.2 MΩ) was prepared by using a Millipore Milli-Q purification system . All materials used for cell culture and antifungal assay, phosphate-buffered saline (PBS), trypsin-EDTA, Dulbecco’s modified Eagle medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, and fetal bovine serum (FBS) were purchased from HIMEDIA . The β-Gal assay kit and MTT were acquired from ThermoFisher .Chemicals needed for phenolic families determination . The supernatants, after correction of the pH to 7.00, were lyophilized in an Edwards Modulyo freeze-dryer , and the dry extracts (DEs) were stored at 4 °C until use.Grape canes used in the present study were kindly provided by a winery located in the Campania region . They were collected in February 2017 from the following cultivars: “Aglianico”, “Fiano”, and “Greco”. The material was cut into pieces of 0.5 to 1.0 cm and dried in an oven at 55 °C until reaching constant weight. Then, the samples were powdered with a MF10 IKA mill , sieved to screen particles until 500 μm in size, and used for the extraction of bioactives at different pH (1.00–13.00). Powdered grape canes (2.5 g) were suspended in 20 mM of the following buffers (50 mL): KCl/HCl pH 1.00, citrate/phosphate pH 3.00, 5.00, 7.00, Tris/HCl pH 9.00, NaHCOFor grape cane extract characterization, DEs were dissolved in deionized water at a concentration of 10 mg/mL. All tests were conducted in triplicate and the results were expressed as mean ± standard deviation (SD).w/v) Na2CO3. The reaction was developed at 25 °C for 2 h in the dark, and the absorbance was read at 765 nm against a blank prepared with 150 mL of deionized water . The total phenolic content was estimated by a calibration curve built with increasing quantities of a standard solution of gallic acid (range 1.5 to 10 μg) and the results were expressed as mg GAE /g DE.The phenolic content was measured by the Folin–Ciocalteu method . Aliquotortho-diphenolic content was measured as described by Arnow [2 and 0.4 M Na2MoO4 (solution B), and 400 μL of 1 M NaOH (solution C) were added in sequence. The resulting mixture was immediately read at 500 nm using a blank containing 400 μL of deionized water. The quantification was carried out by a calibration curve obtained with increasing quantities of a standard solution of caffeic acid (range 5–50 μg). The results were expressed as mg CAE /g DE, and subsequently converted, through a conversion factor, to mg GAE in order to compare the amount of ortho-diphenols with the total phenolic content. One milligram of CAE corresponded to 0.86 mg GAE.The by Arnow . Brieflyw/v) NaNO2. After 5 min, 150 μL of 16% (w/v) AlCl3 were added. After 1 min, 500 μL of 1 M NaOH and 275 μL of deionized water were added, and the resulting solution was vigorously mixed. The absorbance was read at 510 nm versus a blank containing 250 μL of deionized water. The flavonoid amount was determined by a calibration curve obtained with increasing quantities of a standard solution of catechin (range 5 to 75 μg). The results were expressed as mg CE /g DE and subsequently converted, through a conversion factor, to mg GAE in order to compare the amount of flavonoids with the total phenolic content. One milligram of CE corresponded to 0.93 mg GAE.The flavonoid content was determined following the method of Barreira et al.  with somw/v) cinchonine hemisulfate in a 2 mL Eppendorf tube. The solution was mixed and left overnight at 4 °C in order to achieve a better precipitation. After centrifugation at 13,200 rpm for 5 min at 4 °C, a supernatant containing the non-tannin fraction, and a pellet representing the tannin fraction were obtained. The Folin–Ciocalteu assay was carried out on the supernatant in order to calculate the non-tannin content. The total tannin fraction was determined by the difference between the total phenolic content and the non-tannin content, and results were expressed as mg GAE/g DE. The total tannin content was estimated according to Peri and Pompei , with sov/v). Then, 0.5 mL of this solution were mixed with 0.25 mL of 37% HCl/water (2:5 v/v) and 0.25 mL of 4.8% formaldehyde. The resulting mixture was vigorously mixed, incubated overnight at room temperature, and centrifuged at 13,200 rpm, for 30 min at 4 °C. The supernatant (150 μL) was assayed by the Folin–Ciocalteu method, and the value obtained represented the hydrolysable tannin fraction. The condensed tannin content was determined by the difference between the total tannin content and the hydrolysable tannin content. Results were expressed as mg GAE/g DE.Hydrolysable and condensed tannin fractions were determined applying the procedure of Peri and Pompei  followed® system manager data acquisition software . Individual compounds were separated on a Luna C18 (2) column  equipped with a SecurityGuard™ pre-column containing a C18 cartridge, at a flow rate of 700 μL/min; solvent A was 0.5% acetic acid, and solvent B was 0.1% acetic acid-acetonitrile (1:1 v/v). After a 5 min hold at 5% solvent B, elution was performed by a linear gradient from 5 to 55% solvent B in 55 min, and from 55 to 95% solvent B in 10 min, followed by 10 min of maintenance. The column effluent was split into two by means of a “T junction” placed after the chromatographic column and analysed “on-line” both by UV and ESI/MS; 80% of the effluent was sent to the UV detector (detection 280 nm) while 20% of the effluent was analysed by ESI/MS. Mass spectra were recorded from a mass-to-charge ratio (m/z) of 120 to 2000 in negative ionization mode. The capillary voltage was set at −20 V, the spray voltage was 3.5 kV, and the tube lens offset was −10 V. The capillary temperature was 275 °C. Data were acquired in MS, MS/MS, and MSn scanning modes.Selected grape cane extracts  from “Aglianico”, “Fiano” and “Greco” cultivars were reconstituted in deionized water and analysed by HPLC/ESI-ITMSn on a Surveyor MS micro HPLC coupled with a LCQ DECA XP Max ion trap mass spectrometer, equipped with Xcalibur® 3000, equipped with a quaternary pump and an UltiMate® diode array detector  was used. The HPLC separation column and the elution program are described in the previous paragraph 3.3.6. The dry extracts were dissolved in deionized water (10 mg/mL), filtered through a Chromafil syringe filter, pore-size 0.45 μm , and 50 µL were loaded onto the column. For the identified phenolic compounds for which a commercial standard was not available, the quantification was performed through the calibration curve of the most similar available standards. Results were expressed as μg of phenolic compound/g DE.For quantification of the identified compounds, the HPLC system Dionex UltiMateThe free-radical scavenging activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH∙) assay according to Barreira et al. , with so3·6H2O (C), was mixed in 10:1:1 (A:B:C) ratio at the time of use. Samples containing 1 mg of solubilized DEs were diluted to 50 μL and added to 1.5 mL of the FRAP solution. After 4 min incubation, the absorbance was read at 593 nm against a blank made of FRAP solution. FRAP was calculated using a calibration curve built with amounts of ascorbic acid ranging from 0.5 to 6 μg, and compared with FRAP values obtained with 1 mg of the standards Q and BHT. The results were expressed as μg AAE/mg DE for extracts and μg AAE/mg for standards.FRAP assay was performed according to Fernández-Agulló et al. . BrieflyE. coli ATCC 11219, S. aureus ATCC 6538, and C. albicans ATCC 90028. To standardize the bacterial cell suspension, the fresh colonies of each strain, grown on BHI Agar plates, were inoculated into BHI broth and cultured overnight at 37 °C in a shaking incubator . Subsequently, the bacterial inoculum was resuspended in the fresh medium and further incubated at 37 °C until reaching the exponential growth phase (6 × 108 CFU/mL). Serial dilutions and subsequent plating were performed in order to determine the final concentration of bacteria (1 × 106 CFU/mL).The bacterial and fungal strains used were C. albicans were inoculated in 3 mL of sterile saline, and its turbidity was adjusted to 0.5 McFarland standards (5 × 106 CFU/mL). Serial dilutions, conducted in RPMI 1640 added to 2% glucose and MOPS (0.165 mol/L), were performed in order to determine the final concentration of strains (1 × 105 CFU/mL).For the antifungal activity assay, colonies of 5 CFU/mL. The antibacterial activity was expressed as the relative percentage (%) of bacterial growth after 20 h of incubation at 37 °C [Susceptibility testing was performed using the broth micro-dilution method outlined by the National Committee on Clinical Laboratory Standards (NCCLS) using sterile 96-well microliter plates. The dilutions of each extract (in a range of concentration from 1 to 100 μg/mL) were prepared in BHI broth at a volume of 100 μL/well. Each well was inoculated with 50 μL of the standardized bacterial suspension, corresponding to a final test concentration of about 5 × 10at 37 °C ,55. All 5 CFU/mL. The antifungal activity was expressed as the relative percentage (%) of growth observed after 48 h of incubation at 37 °C. All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation (SD).Antifungal susceptibility assay was performed using EUCAST guidelines in sterile 96-well microliter plates. The dilutions of each extract (in a range of concentration from 1 to 100 μg/mL) were prepared in RPMI 1640 at a volume of 200 μL/well. Each well was inoculated with 100 μL of the standardized fungal inoculum, corresponding to a final test concentration of about 1 × 10Cercopithecus aethiops) supplied by ATCC (CCL-81) were grown in DMEM supplemented with 10% FBS. HSV-1 (strain SC16), and HSV-2 (strain 333), both containing a lac Z gene under the control of the CMV IE-1 promoter for the expression of β-galactosidase, were propagated on Vero monolayers [Vero cells  and incubated overnight at 37 °C/5% CO2. Then, the extracts, positive (only medium) and negative (DMSO) controls were added to the wells. In detail, the same increasing concentrations were tested for the different samples . After 24 h, the MTT (blue) was added for 3 h and cells converted it into Formazan (purple). Finally, DMSO was used to re-solubilize the formazan and the viability was assessed at 570 nm through a Bio-Rad microplate reader . All assays were performed in triplicate and the results were obtained in three independent experiments. The data were reported as a percentage of cell viability compared to the positive control.MTT assay was performed to evaluate the cytotoxicity of the extracts. Vero cells were plated in a 96-well flat-bottomed plate . The peptide gH493-512 was used as a positive control in co-treatment and virus pre-treatment assays, while dextran sulphate and acyclovir were used for cell-treatment and post-treatment assay, respectively . In orde(a)5 cells/well) in 12-well plates and incubated overnight at 37 °C/5% CO2. The day after, the extracts and HSV-1 or HSV-2 were added simultaneously to the cell monolayer at multiplicity of infection (MOI) of 0.01 for 1 h at 37 °C.Co-treatment assay. A preliminary assay to assess if the compound has antiviral activity. Vero cells were seeded (3 × 10(b)Virus pre-treatment assay. The assay allows us to evaluate if the extract affects HSV infectivity acting directly on the viral particles. The extracts were incubated in the presence of HSV-1 or HSV-2 (MOI 0.1) at 37 °C for 1 h. Then, the mixtures were diluted with medium and were titrated on Vero cell monolayers.(c)Cell pre-treatment assay. To assess if the extracts could act on the target cells, pre-chilled Vero cells were incubated with the samples for 1 h at 4 °C. Subsequently, they were removed and the cells were infected with HSV-1 or HSV-2 (MOI 0.01) for 1 h at 37 °C.(d)Post-treatment assay. The assay allows us to assess if the extract acts on HSV replication. For this purpose, Vero cell monolayers were firstly incubated with HSV-1 or HSV-2 (MOI 0.01) for 1 h at 37 °C. Then, the extracts were added for an additional incubation period of 1 h at 37 °C.For all above treatments, non-penetrated viruses were inactivated by citrate buffer, pH 3.0 after the incubation time of 1 h at 37 °C with cells. The infected cells were washed with PBS, covered with fresh culture medium supplemented with 5% carboxymethyl cellulose (in 1:2 ratio), and incubated for 24 h. After, monolayers were fixed, stained with X-gal , and plaques were microscopically scored.p ≤ 0.05.All tests were performed in triplicate and expressed as mean ± standard deviation (SD) calculated by Microsoft Excel. Experimental data were analysed using GraphPad Prism (version 5). Significant differences were determined by two-way analysis of variance (ANOVA) completed by Bonferroni post-tests. Mean values were considered significantly different at In the present study, the valorisation of grape canes, a by-product of vine processing produced in large amounts, was achieved through the production of active aqueous extracts. The importance and dimension of the wine industry worldwide justify the need to redirect this waste, currently not valued, to more significant and environmentally friendly uses. Grape canes from “Aglianico”, “Fiano” and “Greco” cultivars were used to produce extracts with the dual purpose of providing the phenolic profile of each cultivar and testing the extracts as potential antioxidant and antimicrobial agents. The method of extraction here applied is simple, cost-effective and easily scalable. This could make it spread quickly and allow grape canes to have a more noble use. The extracts produced could be safely used as antioxidants in several fields, such as food and cosmetic industries, thus representing a response to the growing attention of consumers to health and the environment, and to the increasing trend of preferring natural preparations to achieve health benefits. Moreover, the remarkable activity registered towards HSV-1 and HSV-2 may facilitate and guide further studies to develop new antimicrobial compounds from agro-industrial residues currently underutilized.


Thymus mastichina L. was studied as a potential food ingredient. In this context, dried (DR) and fresh (FR) samples were submitted to carbon dioxide (CO2) supercritical extraction (SFE) and hydrodistillation (HD) methods. The extracts were characterised according to their volatile composition by GC-MS, cytotoxicity against a non-tumour cell culture, and sensory attributes (odour threshold and olfactive descriptors). The most abundant aromas were quantified, and the analysis performed by GC-MS revealed an abundance of terpenoids such as thymol chemotype, followed by the precursors α-terpinene and p-cymene. DR and FR extracts (EX) obtained from SFE-CO2 show the highest content of thymol, achieving 52.7% and 72.5% of the isolated volatile fraction. The DR essential oil (EO) contained the highest amount of terpenoids, but it was also the most cytotoxic extract. In contrast, SFE-CO2 products showed the lowest cytotoxic potential. Regarding FR-OE, it had the lowest extraction yield and composition in aroma volatiles. Additionally, all samples were described as having green, fresh and floral sensory notes, with no significant statistical differences regarding the odour detection threshold (ODT) values. Finally, FR-EX of T. mastichina obtained by SFE-CO2 presented the most promising results regarding food application.Humans often respond to sensory impulses provided by aromas, and current trends have generated interest in natural sources of fragrances rather than the commonly used synthetic additives. For the first time, the resulting aroma of a selected culture of  Lipophilic volatile compounds play a notable influence on the olfactory marketing strategy in the food, cosmetic and fragrance industries. Selective oils from natural origins are recognised as one of the main approaches to product improvement, whether to extend shelf life, in alternative medicine treatments or for sensory purposes ,2. The cp-cymene molecule, an aromatic monoterpene and precursor of the thymol and carvacrol (both isomers) compounds [2 as a solvent at a critical pressure of 73.8 bar and a minimum temperature of 32.1 °C [2 can change its solvent capability by changing pressure and temperature, thus affecting the density of the fluid and the extraction efficiency [2 as a supercritical fluid is highly selective and favours the extraction of thermolabile bioactive compounds [Chemically unstable and easily isomerized, volatile fractions obtained by conventional extraction procedures , maceration (MAC), and Soxhlet (SOX)) show significant chemical alterations. The presence of light, heat and the existence of an organic solvent are some of the conditions that promote the decomposition of unsaturated or esterified molecules . The theompounds ,7. This ompounds ,8. Howevompounds . SFE is  32.1 °C . In addi 32.1 °C , CO2 canficiency . Generalficiency , CO2 as 2 process with gradual decompression, different compounds can be collected in order of their molecular weight in different separators. In this work, the first separator was designed to capture the heavier substances (mainly sesquiterpenoids such as β-caryophyllene), and the second separator was designed for lighter components such as monoterpenoids . Depending on the system pressure, no EX was recovered in both separators simultaneously, as shown by García-Risco et al. [As shown by Costa et al. , by usino et al.  using pr2 conditions are around 80 to 100 bar and 40 to 50 °C [Comparable results were shown for several raw materials ,15,16,17to 50 °C ,16. MoreT. mastichina were studied. T. mastichina is an endemic and undershrub Iberian Peninsula herb, reaching more than 50 cm in height and blossoming from April to June [Thymus [T. mastichina shows a few explorative works, especially in the early 2000’s [T. mastichina collected from 20 different populations in Spain.Aiming to present these well-known concepts for traditional plant material from Northern Portugal, the main genotypes and sensory attributes of  to June . Between [Thymus , Portuguy 2000’s ,19,20,21y 2000’s  in 1997,y 2000’s  studied Thymus spp. aromatic plants, the chemical composition of its main molecules is listed in Regarding the numerous studies found in the literature about Staphylococcus aureus) [p-cymene) show desirable improvements in foods as flavouring agents [Thyme’s dual activities highlight the herb for edible applications. First, the plant is widely used as a natural additive due to its expressive antimicrobial property against common food pathogens  . On the g agents . Its pleg agents . In factT. mastichina extracts obtained from different extraction methods, namely SFE-CO2 and HD. Descriptive and quantitative sensory assays (QDA) determine the fragrances attributes and odour detection threshold of samples and, finally, the cytotoxicity potential is defined within a safe concentration range for the addition of extracted oils in food products.This work compares the performance of DR and FR T. mastichina extracts from SFE-CO2 and the HY fraction obtained in the HD extraction have been studied. Finally, this work aims to present a proposal for the use of T. mastichina EX as a compound rich in odours and which can be used as a flavouring additive for food products.To the authors’ best knowledge, this is the first time that the n-hexane  were obtained from Supelco .Carbon dioxide  was supplied from Linde . Thymol , eucalyptol  and camphor  were acquired from Alfa Aesar . α-pinene , thyme oil  and alkane C8–C40 (ref. 40147-U) standard solution were purchased from Sigma Aldrich . Verbenone  and T. mastichina herb was cultivated under controlled conditions at Deifil Technology . After the development of micropropagated seedlings from the same mother plant, the aerial material of the plant was collected in May 2020 and subjected to an air-drying process at 40 °C  until constant weight, achieving 82.06 ± 1.14% (w/w) of water content.Thymus are evaporated by heating a mixture of water and plant materials followed by the liquefaction of the vapours in a condenser [The HD procedure was conducted with 75 g of FR and DR raw plant in duplicate and independent assays. The extraction was carried out using a conventional extraction apparatus. In this methodology, the essential oils of ondenser . The extondenser . The exp2 process, and following this work, the volatile compounds were obtained under fixed conditions of pressure and temperature, namely 80 bar and 50 °C. A stainless-steel extraction cell capable of containing 1 L of CO2 and designed to perform experiments in static mode was inserted into the system with 30 g of T. mastichina plant using FR and DR samples. To guarantee full recovery of the extracted content after 2 h, a CO2 re-alimentation cycle was performed. The products were collected through the regulation valves (VR1 operated at 40 bar and VR2 at 6 bar) inside 2 separators (S1 and S2). The recovered EX was stored at 4 °C for further characterization.The supercritical extractor developed by Gomes et al.  was usedThe extraction yields for both extraction methods was obtained byT. mastichina products were analysed by gas chromatography (GC) with an ion-trap mass spectrometer (MS)  using a cross-bonded fused column  for low-polarity phases . The GC-MS instrument operated with a splitless injector and automatic sampler (AOC-20s+i). All samples were diluted in n-hexane (GC grade), and a volume of 1 µL was injected at 290 °C, carried at 1 mL·min−1 by ultrapure He.The volatile compounds of −1, followed by an increase to 250 °C for 2 min at 15 °C·min−1 and finally to 280 °C for 1 min at 20 °C·min−1. The mass scanning range was kept at m/z 40–500, while the ion and interface temperature were at 250 °C and 260 °C, respectively.The isothermal oven program was initially set at 40 °C for 1 min, then raised to 200 °C for 2 min at a rate of 7 °C·minThe resulting aroma fractions are presented in relative concentration (%), obtained by the GC peak areas for each identified compound. Molecules were discriminated based on the National Institute of Standards & Technology  mass spectra. To confirm the identification, the retention time of the homologous series of alkanes (C8–C40), analysed under the same chromatographic conditions, was used to calculate the linear retention indices (LRI) by the Kovats retention index equation . The comOlfactory evaluations are affected by vapour pressure, relative composition in the gas and liquid phases, atmospheric pressure and temperature conditions, in addition to the psychophysics background .T. mastichina on the perception of minimum odour concentration, ODT, and index their respective sensory notes by Quantitative Descriptive Analysis (QDA). Subsequently, the commercial standard was incorporated from this study phase to evaluate the achieved products.In accordance with the recommendations of the International Standard ISO 8586:2012 , a panelTo carry out the experiments, a clean, ventilated and well-lighted room was used with a controlled temperature of 20 °C. This room was also free from annoying noises. Furthermore, in the face of the SARS-CoV-2 crises, social and hygienic precautions against the virus were also adopted.−5 to 1.0 × 10−2 µg·mL−1 covered with cotton and conditioned in polypropylene flasks were presented to each panellist. The odour provided by the solutions was properly inhaled, and the minimal odour perception was identified.Odour analysis was performed for the individual products following the instructions of ISO 13301:2018 [3 µg·mL−1 were all evaluated individually by the panel members. The descriptors used in the sensory analysis were carefully selected in the literature according to the main compounds of T. mastichina. An in-depth discussion indicated woody, green, fresh, citrus, floral, sweet, spicy, fruity and oily as the appropriate odour notes. The methodology was based on ISO 11035:1994 [Aqueous samples with a concentration range of 1.0 × 10035:1994 , and panv/v) at a final concentration of 8 mg∙mL−1 and further diluted in the range of 400 to 6.25 µg∙mL−1. Following the protocol previously described by Barros et al. [All aqueous and lipophilic-based products were subjected to a cytotoxic assay using a monkey non-tumour cell line, Vero . Kidneys play an important role in vertebrate organisms due to their osmoregulatory and excretion functions. Therefore, as the envisioned application is for food purposes, the potential cytotoxic effect of the extracts was evaluated through these cells. The samples were dissolved in aqueous DMSO  performed in Statistica StatSoft .2 and HD) and between types of samples (FR or DR) , followed by DR-EX with 3.02 ± 0.39%, determined in DW (Dry Weight). These results are an effect of the high water content in the raw material [w/w); this value was considered in the extraction yield calculations. Additionally, the differences can be explained by three possible phenomena: (i) the dissolution of water in the supercritical solvent increased the solubility of the Thymus compounds; (ii) the hydration of the samples decreased the mass transfer resistance by the diffusion mechanism of intracellular compounds to the particle surface of the Thymus; and (iii) there were significant losses of matter during the drying process [T. mastichina of 1.3% [Significant differences were observed in the values of extraction yields between the two methods (SFE-COR or DR) . The SFEmaterial , which r process . Statist of 1.3% , 2.3% [2 of 1.3%  and 1.4  of 1.3% , far froThymus family goes beyond the extraction method and offers a remarkable diversification according to the species and origin of growth [2 method showed the highest selectivity in the extracted compounds in relation to the EO-HD results. The EO had a large source of different molecules, with a total of 39; this is as compared to 25 of the FR-EX from SFE-CO2. Similar results were obtained in the DR raw material, with 30 molecules for the EO in comparison to 22 compounds from the DR-EX obtained by SFE-CO2. Usually, HD extractions show a lower selectivity than SFE-CO2. For example, Costa et al. [T. lotocephalus extracts obtained by HD and SFE-CO2 methods, respectively.The chemical variation of the f growth . Howevera et al.  identifiT. mastichina samples obtained in this work, and 2-EX, representing 72.4% and 52.7% or 1511.3 ± 602.3 μg∙g−1 and 282.1 ± 7.5.7 μg∙g−1 of FR and DR samples, respectively. In relation to the lyophilized HY, a relative composition of 98.6% to FR-HY and 11.5% to DR-HY was obtained. The high relative concentration of thymol was previously reported for T. vulgaris and T. zygis HY, 77.1% and 61.9%, respectively [ectively .p-cymene also showed significant values, mainly in the EO samples. The FR sample was composed of 30.6% of γ-terpinene and 10.3% of p-cymene, while the DR-EO presented 35.5% and 17.6%, respectively. In contrast, the representativeness of both molecules was lower in SFE-CO2-EX, reaching 13.6% (FR-EX) and 28.8% (DR-EX). Following the chemical route, the relative influence of thymol increases as the contribution of γ-terpinene and p-cymene decreases. Tohidi et al. [p-cymene does not present itself as a final product.The precursors γ-terpinene and −1 of thymol, 609.4 ± 28.3 μg∙g−1 of γ-terpinene and 337.0 ± 20.3 μg∙g−1 of p-cymene molecules. In total, the sample presented an amount of 3700.7 μg∙g−1. As observed through the yield results, FR-HY had a higher total mass concentration (2429.1 μg∙g−1) than the FR-EO fraction (524.8 μg∙g−1), which suggests a considerable amount of the oil fraction solubilized in hydrophilic fraction. On the other hand, the HY of the DR plant was composed only of 4.3 μg∙g−1. Furthermore, the methodology to concentrate the organic products in HY is limited in terms of energy and time costs, although it is easy and cheap to produce them [As previously mentioned, the most abundant molecules present in the obtained extracts were quantified, and the results are shown in uce them .T. mastichina herbs. Eucalyptol, linalool and camphor stand out in EO obtained by the HD method [T. vulgaris plants collected in Algeria [T. zygis species from Portugal [Different volatile compounds were previously referenced in Portuguese D method ,19,21,22D method . Althoug Algeria , Italy [ Algeria , Portuga Algeria , Romania Algeria  and Spai Algeria ,24, or ep-cymene precursors. Both thymol and carvacrol isomers are indicated as the end of the biosynthetic chain catalysed by the two-step oxidation of γ-terpinene under the action of the cytochrome P450 enzyme (γ-terpinene synthase), which is able to modify the polarity of the compounds and make them more water-soluble [p-cymene molecule [p-cymene is a by-product of the premature release of substrate from the active site [The thymol chemotype is a natural phenolic monoterpene group synthesized by the transformation of γ-terpinene and -soluble . Geranyl-soluble . Throughmolecule . p-cymenive site ,47, and,ive site . At the ive site .T. mastichina plant, mainly in DR samples. S-verbenone was only found in FR-EX (0.4% or 61.8 ± 21.1 μg∙g−1), while notable amounts of eucalyptol were measured in DR-OE (0.5% or 123.8 ± 9.7 μg∙g−1) compared to the other lipophilic fractions. The resulting mass composition demonstrates that even a low relative concentration can indicate the presence of a compound rich in the molecules of interest.Moreover, α-thujene, β-myrcene, 3-carene and β-caryophyllene had some influence on the chemical profile of 2-EX, the GI50 values ranged from 235 ± 7 µg·mL−1 to 271 ± 2 µg·mL−1 in the FR and DR samples, respectively. Instead, the commercial EO was statistically equal in both EO obtained in this study, which reveals the average toxicological profile of 83.5 ± 0.1 µg·mL−1, while FR-EO and DR-EO reached 122 ± 12 µg·mL−1 and 65 ± 1 µg·mL−1, respectively. Nikolic et al. [T. serpyllum (7.02 µg·mL−1 < GI50 < 52.69 µg·mL−1) and T. algenensis EO (62.12 µg·mL−1 < GI50 < 64.79 µg·mL−1). Furthermore, the results for T. vulgaris EO (76.02 µg·mL−1 < GI50 < 180.40 µg·mL−1) ranged around the FR-OE cytotoxic potential [−1).The most promising products were selected to perform the successive odour and cytotoxicity characteristics. c et al.  showed hotential , and the−4 µg·mL−1, while the DR-EO had the highest concentration value (5.0 × 10−3 µg·mL−1). FR- and DR-EX showed a concentration of 6.3 × 10−4 µg·mL−1 and 3.0 × 10−4 µg·mL−1 as minimum values to reflect some sensory perception, respectively.The ODT results are expressed in T. mastichina herb is comprised of volatile terpene compounds. Its influence on odour description was evaluated under controlled conditions to ensure the accuracy of human measurement, and the results were compared with the literature. The most pronounced attributes mentioned by the mean scores of 12 panellists were green, fresh and floral , spicy (1.1) and fresh (0.8) aromas. The intensity results agreed with the respective ODT values.The human nose is a powerful tool to predict the dominant odours in a mixture , and, asd floral . The DR-T. vulgaris plant were related with spicy, pungent, herbaceous and phenol-like odour characteristics. The product was obtained with supercritical CO2 under 167 bar and 40 °C. Changing the pressure to 104 bar, the descriptors’ intensity decreased, while the EO extracted by HD presented lower note values plus the burn attribute [Thymus, ranging from a sweet herbaceous fragrance, phenolic herbaceous odour and fresh camphoraceous notes [EX of ttribute . Furtherus notes .Thymus families. According to the data, herbaceous nuances prevailed in the samples, in addition to citrus, woody, floral and phenolic sensory attributes.Moreover, using validated models with ODT results, relative odour intensities and resulting odour values (OV), Teixeira et al.  classifiT. mastichina, the description of the odour presented is akin to the profile of the compounds previously mentioned. Furthermore, the obtained products revealed a promising opportunity to enhance the flavouring trajectory of processed foods in a safe range of cytotoxicity, especially to the FR-EX.In fact, the herbaceous term codifies the aromatic characteristics provided by most Lamiaceae species ,51 and o2 appears most promising. The sample reached a balance between the extraction yields, mass composition of the main compounds and toxicity value, and it has been rarely studied previously [T. mastichina species has been sensorially described.Scanning all samples and features analysed in this work, FR-EX obtained by SFE-COeviously . To the T. mastichina herb by means of green technologies, namely HD and SFE-CO2. The predominant thymol chemotype and its precursors, γ-terpinene and p-cymene, were the main molecules responsible for the sensory profile described as green, fresh and floral fragrance.Terpinene-rich extracts were isolated from 2 products achieved the lowest toxicity among the lipophilic products. Moreover, the FR-HY fraction showed a higher selectivity of the compound, although with a low yield on a DW. The analysed characteristics of the extracted products suggested the FR-EX obtained through SFE-CO2 as the most promising sample to use as a flavouring additive for food applications.Additionally, no ODT results showed significant differences, and both SFE-COT. mastichina was studied for the first time under supercritical conditions as well as regarding its sensory profile. Hydrodistillation and supercritical fluid extraction were applied to the respective final extraction yields for fresh and dry samples of thymus plant material. The extracts’ chemical composition, sensorial attributes and cytotoxic potential were evaluated. Safe concentration ranges of the most promising T. mastichina products were defined by cytotoxicity measurements and ODT values, and the significant differences between the extraction yields, sensory ODT values and cytotoxicity analysis were achieved using statistical analysis, namely ANOVA and Tukey tests.According to the authors’ best knowledge, the species T. mastichina extract as compound rich in odours and which can be used as a flavouring additive for food products.The resulting research presented an unexplored point of view for one of the most studied aromatic plants in recent decades and gives a suggestion for the use of