IntroductionInflammatory bowel diseases (IBD) are chronic diseases that can affect nutrient absorption leading to micronutrient deficiencies and biochemical abnormalities.This study aimed to assess certain serum micronutrients and nutritionally related biochemical markers levels in patients with pediatric IBD and to compare the actual levels and the prevalence of micronutrients deficiencies and biochemical abnormalities between patients with Crohn’s disease (CD) and those with ulcerative colitis (UC).MethodsA retrospective cross-sectional study reviewing medical records of patients with IBD was conducted in the pediatric department, Salmaniya medical complex, Bahrain, from 1 January 1984 to 31 December 2021. Demographic data and laboratory results related to micronutrients and biochemical markers including full blood count, total protein, albumin, globulin, iron, ferritin, folic acid, vitamin B12, calcium, phosphorous, magnesium, and vitamin D levels were collected upon presentation before starting the treatment. Nutritional deficiencies were compared based on sex, nationality, type of IBD, age at presentation, disease duration, weight at diagnosis, and inflammatory markers levels including erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP).ResultsOf 157 patients with pediatric IBD, 117 (74.5%) were included. Sixty-six (56.4%) patients were males. Sixty-six (56.4%) had CD and 51 (43.6%) had UC. No patient had indeterminant colitis. The mean age at presentation was 10.8±3.8 years. Most patients had one or more micronutrient deficiencies . Anemia was a common finding , with iron deficiency anemia (IDA) being predominant. Low iron levels were noted in 64/77 (83.1%) patients with a median of 5.0 (2.0-9.3) µmol/L ; isolated iron deficiency (ID) in 11/18 (61.1%) and IDA in 53/59 (89.8%) patients. Vitamin D deficiency was the second most common . Serum albumin, ferritin, calcium, phosphorous, and magnesium were deficient in 29.2%, 27.8%, 31.7%, 12.5%, and 10%, respectively. One patient had vitamin B12 deficiency while none had folate deficiency. Patients with CD had significantly lower serum iron  and lower serum protein  but higher serum ferritin (45 (19-110.2) versus 21.3 (10.3-51.2) µg/L, p=0.046) compared to those with UC. Elevated ESR was noted in 62/105 (59.1%) patients while high CRP was found in 67/104 (64.4%). Patients with low iron had higher ESR (28 (17-47) versus 14 (10-33) mm/h, p=0.028) and higher CRP (13.3 (1.6-42) versus 1.8 (0.9-4.6) mg/L, p=0.019) levels compared to those with normal levels.ConclusionPatients with pediatric IBD are at risk of multiple micronutrient deficiencies and biochemical abnormalities. Iron and vitamin D deficiencies are the most frequent. Patients with CD are more prone to have lower serum iron and protein levels than those with UC. ID was associated with elevated inflammatory markers. IBD m 100,000 . For ped 100,000 .Patients with IBD are prone to multiple nutritional deficiencies including macronutrient and micronutrient deficiencies -15. HypoMicronutrient deficiencies are seen more commonly in patients with CD as it mainly involves the terminal ileum and the colon ,14,15,17Despite the advances in the treatment of IBD, micronutrient deficiencies are still frequently seen . The preStudies about micronutrient deficiencies in children with IBD are limited . To dateStudy design and setting January 1984 to 31 December 2021 was conducted.A retrospective cross-sectional study by reviewing all medical records of patients with IBD  diagnosed in the pediatric department, Salmaniya medical complex (SMC), Bahrain, from 1Study populationAll pediatric patients up to 18 years of age with IBD and available results of laboratory investigations related to at least one of the micronutrients or nutritionally related biochemical markers levels at the time of diagnosis were included in the study. Patients who lack the results of these laboratory tests were excluded. Diagnosis of IBD was established using the IBD working group of the European Society of Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) and the North American Society of Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN) criteria ,31.Data collectionData about sex, nationality, type of IBD, age at presentation, weight at diagnosis, duration of the illness prior to diagnosis, medical therapies at the time of diagnosis or during the follow-up course, surgical interventions, and follow-up duration were collected.Results of laboratory investigations related to micronutrients and biochemical markers including full blood count, total serum protein, serum albumin, serum globulin, serum iron, ferritin, folic acid, vitamin B12, calcium, phosphorous, magnesium, and vitamin D levels were collected upon presentation before starting the treatment. In addition, results of the iron profile including transferrin level, transferrin saturation, and total iron binding capacity (TIBC) were gathered. Standard clinical laboratory techniques were used to measure micronutrients and biochemical markers levels. Anemia was defined as hemoglobin level <11g/dL in children six months to five years old, <11.5 g/dL in children five to 11 years old, <12 g/dL in children 12 to 13 years old, <13 g/dL in men, and <12 g/dL in non-pregnant women ,33. PatiTo determine risk factors for micronutrient deficiencies and nutritionally related biochemical markers abnormalities, patients were divided into two groups according to the presence or absence of the two most common micronutrient deficiencies. Both groups were compared based on sex, nationality, type of IBD, age at presentation, pre-diagnosis disease duration, weight at presentation, and inflammatory markers levels.Statistical analysisPatients' data were analyzed using Statistical Product and Service Solutions (SPSS) . Frequencies and percentages were calculated for demographic, micronutrient deficiencies, and biochemical markers abnormalities data. Continuous variables were presented as mean ± standard deviation (SD) or median and interquartile range (IQR) according to the distribution’s normality. Fisher’s exact test or Pearson’s chi-square was used to compare categorical variables while the Mann-Whitney U test or Student t-test was used to compare continuous variables. The confidence interval was set at 95%. P values <0.05 were considered statistically significant.Ethical clearanceThis study was conducted in accordance with the principles of the Helsinki Declaration of 1975 (revised 2013), and it was ethically approved by the secondary care medical research subcommittee, Salmaniya Medical Complex, Government hospitals, Kingdom of Bahrain (IRB number: 76120521).Out of 157 patients diagnosed with pediatric IBD during the study period, 117 (74.5%) patients had the results of laboratory tests available. Despite that, they had laboratory tests performed at the time of presentation, and the remaining 40 (25.6%) patients were excluded due to the unavailability of laboratory results. Demographic data and clinical characteristics of the included patients are shown in Table Most of the patients were males  and Bahraini , while 16 (13.7%) were non-Bahraini . Sixty-six (56.4%) patients had CD and 51 (43.6%) had UC. None of the patients had a diagnosis of indeterminant colitis. The CD was more in males (68.2% versus 41.2%) while UC was more in females (58.8% versus 31.8%), p=0.003. The mean age at diagnosis was 10.8±3.6 years. Six patients (5.1%) had an early-onset disease below five years of age at the time of diagnosis. The mean age at the time of blood collection was 10.79±3.75 years. The median pre-diagnosis disease duration was three (0.5-10) months. Patients with UC had lower median weight (29.0 (20.5-33.0) kg) compared to those with CD (32.5 (25.0-45.9) kg), p=0.042. One patient with UC had concurrent sickle cell disease which was excluded from the analysis of anemia prevalence. No patient had thalassemia. Patients with UC had longer follow-up duration  compared to those with CD.The most frequent medications used were azathioprine, folic acid, mesalazine, and prednisolone. Patients with UC were treated more frequently with mesalazine , omeprazole , iron supplementation , H2 blockers , ursodeoxycholic acid , and fat-soluble vitamins (ADEK)  compared to those with CD. Fat-soluble vitamins were given after the blood collection and as a general support in four patients with UC who had an associated primary sclerosing cholangitis. ADEK was given as 1 mL daily for infants less than one year and 2 mL daily for older children, folic acid 5 mg daily, iron 6 mg per kilogram per day, calcium 250 mg daily (<1 year), 700 mg daily (1-3 years), 1000 mg daily (4-8 years) and 1300 mg daily (9-18 years), vitamin D 400 IU daily, multivitamins 5 mL daily, and vitamin B12 0.5 mcg daily (<1 year), 0.9 mcg daily (1-3 years), 1.2 mcg daily (4-8 years), and 1.8 mcg daily (>9 years) supplementations. Biological therapy was used in 43/100 (43%) patients . Three patients received exclusive enteral nutrition therapy. None of the patients required intravenous iron therapy or total parenteral nutrition and none agreed to nasogastric tube feeding. Eleven patients required packed red blood cell transfusions. Twenty-five (21.4%) patients underwent gastrointestinal-related surgeries , fistula repair (n=4), inguinal hernia repair (n=3), polypectomy (n=2), ileostomy (n=2), gastrostomy and diaphragmatic hernia repair ). These surgeries were performed after the disease diagnosis.The majority of patients had one or more micronutrient deficiencies or biochemical abnormalities  while only seven (5.98%) patients had completely normal tested levels. Frequencies of micronutrient deficiencies and biochemical abnormalities are shown in Figure Anemia was the most common finding , with IDA being predominant. Low iron levels were noted in 64/77 (83.1%) patients with a median iron level of 5.0 2.0-9.3) µmol/L ; isolated ID in 11/18 (61.1%) and IDA in 53/59 (89.8%) patients. Data on hemoglobin, protein, albumin, and calcium levels were normally distributed while data about iron, ferritin, folate, vitamin B12, phosphorous, magnesium, and vitamin D were not normally distributed. A comparison of laboratory parameters between patients with CD and those with UC is shown in Table .0-9.3 µmPatients with CD had significantly lower serum iron  and lower serum protein  but higher serum ferritin (45 (19-110.2) versus 21.3 (10.3-51.2) µg/L, p=0.046) compared to those with UC. Despite those patients with CD had lower levels of iron compared to those with UC, the use of iron supplements was higher among patients with UC (p=0.018), see Table Vitamin D deficiency was the second most frequent micronutrient deficiency . Serum calcium was deficient in 32.1% (n=27/84) of the patients while hypomagnesemia was in 15.2% (n=12/79). In terms of pre-diagnosis disease duration, no significant difference was found between patients with hypoalbuminemia (2.0 (0.3-5.0) months) and those without (3.0 (0.5-12) months), p=0.458. Sixty-two out of 105 (59.1%) patients had high ESR and 67 of 104 (64.4%) had high CRP. A comparison between patients with the two most common micronutrient deficiencies (iron and vitamin D deficiencies) and those without is shown in Table Patients with low iron had higher ESR (28 (17-47) versus 14 (10-33) mm/h, p=0.028) and higher CRP (13.3 (1.6-42) versus 1.8 (0.9-4.6) mg/L, p=0.019) levels compared to those with normal levels. There was no significant relation found between serum ferritin levels and elevated ESR (low ferritin=8/15 (53.3%) versus normal=26/38 (68.4%), p=0.351) or elevated CRP levels (low ferritin=7/15 (46.7%) versus normal=27/38 (71.1%), p=0.120).Anemia in patients with IBD has received little attention from clinicians in terms of diagnosis and treatment, despite that overt or occult gastrointestinal bleeding is a common symptom in this group of patients . In patiAnemia can develop secondary to iron, vitamin B12, and folic acid deficiencies . Low iroFerritin was found to be either normal or high in most of our patients and only 27.8% had low ferritin level while Rempel et al. . ESR wasVitamin B12 deficiency is rare in pediatric IBD diagnosis . Unlike In the current study, all 45 (38.5%) tested patients for folate showed normal or even high levels, 23 51.1%) and 22 (48.4%) patients, respectively. Comparably, Ehrlich et al.  reported.1% and 2The role of vitamin D in calcium homeostasis is well-recognized in pediatric patients with IBD ,14,41,42In the current study, 32.1% of patients were deficient in calcium and 15.2% had hypomagnesemia. Song et al. study ,12. HypoProtein-energy undernutrition is one of the forms of undernutrition in patients with IBD . The serSerum albumin level can be used as a barometer of IBD activity . The curIn the current study, patients with low iron level had significantly higher inflammatory markers including ESR p=0.028) and CRP p=0.019) levels. Song et al. 9 levels. studied 8 and CRPLike any other retrospective study, this study was limited by missing data about patients’ demography, results of laboratory investigations, or medications used in some patients. Accordingly, only patients with available data about laboratory test results were included in the analysis while those missing relevant data we excluded. This selection bias was unavoidable as the aim of the study was to calculate the valid percentage for each nutrient deficiency to avoid underestimation of the true prevalence of each deficiency. Moreover, to the best of our knowledge, no specific hemoglobin norm was published in our country. However, in our region, El-Hazmi and Warsy from Saudi Arabia published hematological references for Saudi healthy children and adolescents aged 1-15 years . Their hThis study showed that iron and vitamin D deficiencies are the most frequent micronutrient deficiencies found in patients with pediatric CD and UC. Moreover, patients with CD showed lower serum iron and protein levels compared to those with UC. Iron deficiency was associated with elevated inflammatory markers. Further studies to assess the effect of micronutrient deficiencies on IBD pathogenesis and on patients’ clinical symptoms and signs are specifically needed. Furthermore, evaluating the long-term impact of using IBD medications on micronutrient levels is warranted. Studies are also required to explore the role of prescribing multivitamins and other micronutrients in altering the disease activity and the quality of life in this group of patients.


The electrode/piezoelectric/electrode stack was deposited on a silicon on insulator (SOI) wafer with a 2 μm silicon device layer that serves as the passive elastic layer for bending-mode vibration. The fabricated 32-element PMUT has a central frequency at 1.4 MHz. Ultrasound beam focusing and steering (through beamforming) was demonstrated where the array was driven with 14.6 V square unipolar pulses. The PMUT generated a maximum peak-to-peak focused acoustic pressure output of 0.44 MPa at a focal distance of 20 mm with a 9.2 mm and 1 mm axial and lateral resolution, respectively. The maximum pressure is equivalent to a spatial-peak pulse-average intensity of 1.29 W/cm2, which is suitable for tFUS application.Interest in utilizing ultrasound (US) transducers for non-invasive neuromodulation treatment, including for low intensity transcranial focused ultrasound stimulation (tFUS), has grown rapidly. The most widely demonstrated US transducers for tFUS are either bulk piezoelectric transducers or capacitive micromachine transducers (CMUT) which require high voltage excitation to operate. In order to advance the development of the US transducers towards small, portable devices for safe tFUS at large scale, a low voltage array of US transducers with beam focusing and steering capability is of interest. This work presents the design methodology, fabrication, and characterization of 32-element phased array piezoelectric micromachined ultrasound transducers (PMUT) using 1.5  I.LOW intensity transcranial focused ultrasound stimulation (tFUS) is rapidly growing as a noninvasive neuromodulation treatment with a millimeter spatial resolution compared to modalities such as transcranial magnetic, direct current, or alternating current stimulation –3]. Mul. Mul3]. Conventionally, tFUS experiments utilize single element ultrasound (US) transducers with a relatively large diameter (tens of millimeters) and a fixed focal spot. A precise manipulator is required to mechanically alter the focal spot to stimulate different brain regions, which is a major shortcoming of conventional tFUS systems. However, this limitation can be overcome by leveraging the electronic beam focusing and steering capabilities of optimally designed US phased arrays. In particular, a US phased array enables electronically controllable stimulation over a large tissue volume, i.e., large-scale stimulation.AC with a 100 VDC offset at 183 kHz, yielded a maximum 52 kPa pressure generating a temporal-average spatial-peak acoustic intensity (SPTAI) of ~55.4 mW/cm2. The CMUT successfully stimulated motor cortical areas in freely moving mice . Th. Thμm PZ2 atmosphere at 700 °C for 15 minutes to form TiO2. The Pt was DC sputter deposited at a substrate temperature set at 600°C.Highly {111} oriented Pt bottom electrodes were prepared as described by . In shor0.44Ti0.56O3 sol gel solution  was employed as a seed layer. The seed layer solution was spun at 3500 rpm, pyrolyzed at 200°C for 2.5 minutes and crystallized in RTA at 700°C for 1 min in 2 slpm O2 atmosphere, following the work in . T. T0.44Tiectively . The cryμm thick SPR 955 photoresist. The etching process was adapted from [2 and 40 sccm of Ar at 700 W ICP and 100 W RIE power. The photoresist was then removed by immersing the sample into Baker PRS3000 photoresist remover at 80 °C for 30 minutes, followed by 3 minutes in an oxygen plasma. For PZT etching, 13 μm thick AZ4620 was spun at 4500 rpm for 45 seconds and soft baked at 90 °C and 105 °C for 1 and 3 minutes, respectively. Before exposing the photoresist, the spun photoresist was rehydrated in air for an hour and exposed at 100 mJ/cm2 for 8 cycles with 30 second delay times between each step. Then, the exposed photoresist was developed in 1:4 AZ400K developer for 4 minutes. The ICP-RIE was performed in ICP-RIE system with 3.5 sccm of Cl2, 7 sccm of CF4, and 10 sccm of Ar with 600 W ICP and 150 W RIE power. The finished sample was cleaned using a similar process in a photoresist remover, and an oxygen plasma as mentioned earlier.The Pt top electrode and PZT blanket films were then patterned into individual elements using an inductively coupled plasma – reactive ion etching (ICP-RIE) Ulvac NE-500 system . The top electrode was patterned using a 2 ted from  and condμm deep backside release trenches were achieved by silicon deep reactive ion etching . Before silicon etching, SiO2 on the wafer backside was cleaned by wet etching in BOE 6:1 at room temperature, followed by a deionized water rinse. Afterward, an Al2O3 hard mask was deposited by atomic layer deposition at 150 °C and patterned using an ICP-RIE system with 30 sccm BCl3 and 10 sccm of Cl2 gas at 1000 W ICP and 75 W RIE power. The DRIE was performed immediately after the mask patterning using the Bosch process at 3°C. The BOX layer was used as the stopping layer and the trench depth was confirmed by optical profilometry . Then, the sample was diced into an individual array with 32 active elements. The arrays were glued to a printed circuited board (PCB) and electrically connected via Au wire bonding. The PMUT was waterproofed using a 6 μm thick parylene coating.In the fabrication, 400 B.A custom printed circuit board (PCB) was designed with an on board dual-row 36 position header connector. The ground plane of the thin film array was connected to the ground pads of the PCB with conductive silver paint. Each of the 32 elements was wire-bonded to the excitation pad.AC excitation was measured using a Hewlett Packard 4284A LCR meter. The relative dielectric permittivity and loss tangent of 32 elements array was 1210 ± 12 and 1.9% ± 0.09%. A Radiant Multiferroic test analyzer was employed for analyzing the polarization-electric field hysteresis loop (P–E), which is shown in Pr) of 16.2 μC/cm2 and coercive fields (Ec) of −54.9 and 39.2 kV/cm.As shown in PP pulses, and a delay range of 0-40 μs with 5 ns resolution.For driving the array, a 32 channel beamformer circuit  was used as described in . In shorIV.The electrical impedance of the individual elements was first measured at various frequencies ranging from 0.2 to 3.2 MHz, as shown in F = 20 mm and θs = 0°) was generated by the phased array being driven with 10 cycles of unipolar 1.4 MHz square pulses (8 V peak to peak). A smaller number of cycles was intentionally used for decoupling electrical interference.F = 20 mm with θs = 0°, 45°, and −45°, respectively. The simulated and measured axial resolutions at θs = 0°, 45°, −45° were 7.9, 9.9, 9.9 mm and 9.2, 10.3, 11.1 mm, respectively. For the lateral (y) resolution, the simulated results were 1.2, 1.3, 1.3 mm while the measurement results were 1, 1.3, 1.4 mm at θs = 0°, 45°, and −45°, respectively. The measured beam profiles closely matched the simulated ones. With 14.6 V driving voltage, the spatial peak pressure measured at θs = 0°, 45°, and −45° were 0.44 MPa, 0.33 MPa, and 0.29 MPa, respectively. Additionally, as expected, there was no significant off target high pressure spot.F = 30 mm. The simulated and measured axial  resolutions at θs = 0°, 45°, −45° were 14.1, 17.4, 17.4 mm  and 13.6, 24.3, 22 mm , respectively. As the targeted focal distance increased, the maximum acoustic pressure dropped to 0.37 MPa, 0.27 MPa, and 0.27 MPa at θs = 0°, 45°, and −45°, respectively.Similarly, y = z = 0) of the beams focused at different focal distances (F). y) pressure profiles of the beams at the axial distances where the peaks shown are corresponding to the maximum pressure output at different F from F = 30 mm, which exceeds the Fmax of 20 mm, the beam becomes comparatively wider . One can note that in this case a higher N  could be used to compensate for the poor resolution. x axis (as defined in F = 20 mm and steered at 0° to 60°. y) pressure profiles of the beams focused at F = 20 mm and steered at 0° to 60°. Although the fabricated array was optimally driven at 1.4 MHz, whereas it was designed for 1 MHz, the phase performance is still good showing no unwanted grating lobes for the maximum steering angle of 60°.SPPAI for the beams focused at F = 20 and 30 mm and steered at θs = 0°, 45°, −45°. SPPAI was calculated using the pulse intensity integral of the spatial peak pressure waveform as described in [ribed in . It shouV.μm thick {100} oriented PZT as the piezoelectric layer and 2 μm of silicon as the passive elastic layer. With a commercial driver board, the 32-element array was driven using calculated time delays for beam focusing and steering at F = 20-30 mm and θs = 0°-60°. The phased array demonstrated a maximum peak-to-peak acoustic pressure output of 0.44 MPa, corresponding to an acoustic intensity (SPPAI) of 1.29 W/cm2, achieved at 20 mm focal depth with 14.6 V unipolar square pulses in an immersion test. This work presents a new route to achieve low voltage US transducers for neuromodulation. Such PMUT US phased arrays can be integrated with a CMOS integrated circuit to drive the transducers.The design methodology of a US transducer array for neuromodulation application was applied to PMUT transducers, with a design goal of exceeding hundreds of kPa acoustic pressure within the operated voltage below 20 V. The linear 32-element phased array PMUT thin film with a 1.4 MHz bending-mode resonant frequency was designed and fabricated with 1.5


Prunus spinosa L. fruits. The effects of different parameters, i.e., ethanol concentration in the extraction solvent (mixture of ethanol and water), operation temperature, and extraction time, on process responses were evaluated. Total phenolic content (TPC), total anthocyanin content (TAC), antioxidant capacity (AC), and contents of protocatechuic acid (PA), caffeic acid (CA), vanillic acid (VA), rutin hydrate (RH), and quercetin (Q) of fruit extracts were selected as process responses. A synergistic effect of obtaining high values of TPC, TAC, AC, PA, and VA was achieved for the extraction in 50% ethanol at 60 °C for 30 min. At a higher level of process temperature, the extraction of protocatechuic acid and vanillic acid was enhanced, but the flavonoids, i.e., rutin hydrate and quercetin, were degraded. A lower temperature should be used to obtain a higher amount of flavonoids. TPC, TAC, AC, and phenolic acid contents  in the extract samples obtained at an ethanol concentration of 50–100%, a temperature of 30–60 °C, and an extraction time of 30 min were strongly directly correlated.This study aimed at establishing the optimal conditions for the classic extraction of phenolic compounds from  Due to the tendency of consumers to choose foods with a positive impact on health, producers have to develop new foods with enhanced beneficial properties. These actions can be carried out in two ways: the former refers to returning to traditional products, while the latter focuses on the production of functional foods by enriching foods with unconventional additives or constituents/ingredients. One method of obtaining the necessary bioactive compounds for functional foods is their extraction from vegetal sources that are not usually valued, such as wild fruits ,2. Prunus spinosa L., which belongs to the rose family (Rosaceae), grows as a shrub in wild areas of Europe and temperate regions of Asia and Mediterranean countries [P. spinosa fruits are known as blackthorn, grater, rasp, sloe, or stickleback and are closely related to plums, cherries, peaches, nectarines, and apricots [ountries ,3,4,5. Papricots . They arapricots . These fapricots .P. spinosa fruits contain different polyphenolic compounds, such as phenolic acids, flavonoids, anthocyanins, coumarins, and proanthocyanins [ocyanins ,8,9,10, ocyanins ,12,13, aocyanins ,15, antiocyanins ,13, and ocyanins  propertiPolyphenols are a class of natural compounds that play an important role in plant development and also contribute to the sensory characteristics of fruits and vegetables ,18. TheyPhenolic compounds represent important secondary metabolites of plants characterized by one or more aromatic rings and several attached hydroxyl groups. These compounds provide protection to the plant against pathogens, UV rays, parasites, and free radicals ,19,20. TP. spinosa fruits and their possible applications in the development of new pharmaceutical products have been evaluated in several studies. An aqueous extract showed antibacterial effects on a strain of Pseudomonas sp. [Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella abony, and Candida albicans [P. spinosa fruits presented a significant antioxidant capacity and led to an important decrease in glioblastoma from brain cancer cells [The antibacterial, antifungal, and anticancer effects of polyphenolic extracts from onas sp. , whereasalbicans . Also, aer cells . In the last two decades, the interest in investigating these compounds has been shown by numerous papers related to the extraction and separation of phenolic compounds . They arP. spinosa (blackthorn) fruits with enhanced antioxidant properties.Due to its simplicity, high efficiency, and large-scale applicability, solid–liquid extraction is still the most commonly used extraction method . It invoThe aim of this work was to establish the optimal conditions for the classic extraction of polyphenols from Romanian blackthorn fruits. The effects of extraction solvent composition, process temperature, and extraction time on the total phenolic content, total anthocyanin content, antioxidant activity, and chemical profile of extracts  were evaluated.Blackthorn fruits were harvested in October 2019 from Giurgiu County (Romania). After the stones were removed, the fruits were oven-dried at 30 °C until a constant mass was obtained. The moisture contents of fresh and dried fruits were 76 ± 1% and 7.0 ± 0.2%, respectively. The size reduction of dried fruits was achieved using an electric grinder. Dried and ground fruits were stored in a dry place until used.Ethanol, isooctane, and lactic acid were of analytical grade and were provided by Merck KGaA . Folin–Ciocalteu reagent, gallic acid, and sodium carbonate required for spectrophotometric determination as well as copper (II) chloride, ammonium acetate, neocuproine, and Trolox used for cupric reducing antioxidant capacity (CUPRAC) analysis were purchased from Merck KGaA. For chromatographic analyses, several standard substances were used, i.e., caffeic acid , caftaric acid , catechin hydrate , chlorogenic acid , chicoric acid , cyanidin chloride , delphinidin chloride , gallic acid , (−) epicatechin , ellagic acid dihydrate , gallic acid , kaempferol , malvidin chloride , myricetin , pelargonidin chloride (Merck KGaA), protocatechuic acid , quercetin , rosmarinic acid , rutin hydrate , syringic acid , trans-p-coumaric acid , trans-ferulic acid , and trans-resveratrol . v/v) lactic acid were used as extraction solvents. Ethanol concentration in the extraction solvent (etc), extraction temperature (t), and extraction time (τ) were selected as process independent variables (factors). Extraction experiments were performed at different levels of etc , t , and τ , using a heating plate equipped with a temperature control unit and a magnetic stirrer. The solvent-to-fruit ratio of 10:1 (v/w) and a constant stirring rate of 1100 rpm were used for all experiments. After the extraction, the mixtures were centrifuged at 4000 rpm for 10 min, and the supernatants were kept at a temperature of 4 °C before the analysis was performed. Firstly, the lipids found in fruits were removed by extraction with isooctane for 2 h at room temperature. The mixture was filtered, and the solid phase was dried prior to its use for the extraction of polyphenolic compounds. Mixtures of ethanol and water acidified with 0.6% , Folin–Ciocalteu reagent (1 mL), distilled water (3 mL), and 20% Na2CO3 solution (1.5 mL) were added to a 10 mL volumetric flask. The mixtures were kept in the dark for 30 min, and then their absorbance was measured at 750 nm using a Jasco V 550 UV-Vis spectrophotometer . The results were expressed as mg of gallic acid equivalents (GAE) per g of dry matter (mg GAE/g DM) using a standard curve corresponding to 0–1.26 mg gallic acid/mL. The analysis of each extract was performed in triplicate. The TAC, the samples were diluted appropriately with the extraction solvent, and the absorbance was measured at 520 nm using a Jasco V 550 UV-Vis spectrophotometer. TAC was expressed as mg of cyanidin-3-glucoside equivalents (C3GE) per g of dry matter (mg C3GE/g DM) and calculated using Equation (1) [A is the sample absorbance, M the molecular mass of cyanidin-3-glucoside (449.2 g/mol), DF the dilution factor of the extract, V the volume of the extract (L), 1000 the factor for conversion from g to mg, ε the molar extinction coefficient of cyanidin-3-glucoside ), L the cuvette length (cm), and m the mass of the dry matter (g). The analyses were performed in triplicate.To determine the tion (1) , where AAC was determined using the CUPRAC method [x mL of sample, and (1.1 − x) mL of distilled water were added to a vial so that the total volume was 4.1 mL. The absorbance was measured after 30 min at 450 nm. The analysis was performed in quartz cuvettes using a Jasco V 550 UV-Vis spectrophotometer. The results were expressed as µmol of Trolox equivalents (TE) per g of dry matter (µmol TE/g DM) using a standard curve corresponding to 0–0.25 mg Trolox/mL. The analyses were performed in triplicate.The C method . For sam2O/formic acid (100/2.5) (v/v) and ACN/H2O/formic acid (90/10/2.5) (v/v/v), using 1 μL as injection volume for every sample. The gradient elution program as well as calibration curves for standard substances are described elsewhere [High-performance liquid chromatography (HPLC) analyses were performed using a Shimadzu Nexera-2 system  equipped with a photodiode array (PDA) detector (SPD-M30A) with a Nucleosil C18 reversed-phase separation column (2.7 μm × 2.7 μm × 100 mm). The analyses were performed at a flow rate of 0.4 mL/min using gradient elution having as mobile phases Hlsewhere ,33. The 2) pressure of 2.8 bar and flow rate of 3 L/min at a temperature of 200 °C. The spectra were recorded over a mass range between 92 and 1500 AMU at a source voltage of 4700 V.High-resolution mass spectrometry (HRMS) analyses were performed using a Fourier-transform ion cyclotron resonance (FT-ICR) spectrometer  equipped with an electrospray ionization (ESI) source. Each sample was introduced by direct infusion, using negative ion mode ESI and the following parameter levels: sample flow rate of 120 µL/h, nebulizer gas (Np < 0.05) on dependent variables in terms of TPC, TAC, AC, and phenolic compound contents. The values of dependent variables obtained at different levels of extraction factors were processed using principal component analysis (PCA) [r) was used to evaluate the strength of linear correlations between dependent variables. Response surface regression models were used to quantify the effects of process factors on TPC and TAC. Statistical analysis and process factor optimization were conducted using XLSTAT version 2019.1  and STATISTICA version 10.0 .All experimental measurements were performed in triplicate, and the results are presented as mean value ± standard deviation (SD). One-way ANOVA with Tukey’s HSD post hoc test was applied to evaluate whether the process factors had a significant effect (is (PCA) ,35,36. Tetc = 50–100%) on TPC was evaluated. The experiments were performed at 30 °C for 30 min. Lower concentrations of ethanol were not used because a higher concentration of ethanol leads to improved long-term stability and therefore could be considered safer to add in food products. The results presented in TPC with an increase in etc and a significant effect (p < 0.05) of etc. High mean values of TPC were obtained for the hydroalcoholic extracts (21.8–32.2 mg GAE/g DM), and a significantly lower mean value was obtained in the case of the alcoholic extract (4.7 mg GAE/g DM). Accordingly, the optimal value of etc was considered to be 50%. First, the effect of the volume percentage of ethanol in the extraction solvent (τ) on TPC was assessed, keeping constant ethanol concentration (etc = 50%) and process temperature (t = 30 °C). The time range (τ = 5–180 min) was selected taking into account that a short period may not be enough for the depletion of the raw material into compounds of interest, while a high period could lead to compound degradation. The data shown in TPC obtained at τ = 30–180 min (32.21–32.81 mg GAE/g DM) were similar (p > 0.05) and significantly higher (p < 0.05) than those corresponding to short extraction times (5 and 15 min), i.e., 21.38 and 22.23 mg GAE/g DM, respectively. The optimal value of τ was considered to be 30 min. Secondly, the effect of extraction time (t) was studied at etc = 50% and τ = 30 min. As shown in t from 30 °C to 60 °C led to an increase in the mean value of TPC. The mean value of TPC obtained at t = 60 °C (37.23 mg GAE/g DM) was significantly higher (p < 0.05) than those obtained at lower temperatures (32.06–34.00 mg GAE/g DM). Moreover, the extract prepared using reflux heating (82.5 °C) had the lowest mean value of TPC (29.84 mg GAE/g DM). High temperatures are usually associated with phytocompound degradation [t was considered to be 60 °C. Finally, the effect of extraction temperature (TPC (37.23 mg GAE/g DM) was obtained at the following optimal levels of process factors: etc = 50%, τ = 30 min, and t = 60 °C. The values of TPC obtained in this study, i.e., 4.50–37.40 mg GAE/g DM (1.39–11.59 mg GAE/g fresh fruit), were consistent with those reported in the related literature. Opriș et al. [TPC (2.52 mg GAE/g DM) was obtained at an ethanol concentration of 40%, an extraction temperature of 67 °C, and an extraction time of 10 min. For RE, the maximum mean value of TPC (4.01 mg GAE/g DM) was obtained at an ethanol concentration of 30% and an extraction time of 45 min. Tahirovic et al. [TPC using UAE. The mean values of TPC obtained at alcohol concentrations of 50% were higher than those found at alcohol concentrations of 80%, and the maximum mean value of TPC (30.20 mg GAE/g DM) was obtained at an ethanol concentration of 50%. Sikora et al. [TPC mean value of 33.09 mg GAE/g DM (5.992 mg GAE/g fresh fruit) for the methanolic extract of blackthorn fruits. Veličković et al. [TPC for 50% ethanol (20.94 mg GAE/g fresh fruit) than those obtained for 50% methanol (17.69 mg GAE/g fresh fruit), 100% ethanol (15.33 mg GAE/g fresh fruit), 100% methanol (15.33 mg GAE/g fresh fruit), and water (12.17 mg GAE/g fresh fruit).Accordingly, the maximum mean value of ș et al.  applied TPC in the extract samples P1, P2,…, P13, i.e., m,iTPC , which are presented in etc, τ, and t) on total phenolic content were quantified using the response surface regression model described by Equation (2), where the regression coefficients were obtained based on the experimental data specified in pred,iTPC) and related residuals  are also presented in RMSE defined by Equation (3), multiple R, multiple R2, adjusted R2, F, and p, which are also included in The mean values of Y = predTPC. A desirability function, d(Y), is defined by Equation (4) [YL = 4.71 mg GAE/g DM and YU = 37.23 mg GAE/g DM are the lower and upper limits of process response. The predicted values of Y and d(Y) at different levels of extraction process factors, which are shown in Y are those obtained in the experimental study, i.e., etc = 50%, τ = 30 min, and t = 60 °C. Under these optimal conditions, the process response is optY = pred,optTPC = 35.92 mg GAE/g DM and the desirability function is d(optY) = 0.96. Surface and contour plots of desirability, d(predY = TPC), depending on extraction process factors  are shown in The desirability function approach was applied to determine the optimal factor levels to maximize the response tion (4) , where Letc = 50–100%) on TAC at t = 30 °C and τ = 30 min (TPC. TAC and TPC were very strongly correlated (r = 0.993). As shown in TAC (0.297 mg C3GE/g DM) was achieved for etc = 50%, and the lowest mean value (0.034 mg C3GE/g DM) was achieved for etc = 100%. The effect of ethanol concentration in the extraction solvent (= 30 min  was simiτ = 5–180 min) on TAC at etc = 50% and t = 30 °C is shown in TAC increased significantly (from 0.214 to 0.333 mg C3GE/g DM) with increasing τ from 5 to 60 min, whereas the mean values of TAC for τ = 60–180 min (0.329–0.347 mg C3GE/g DM) were similar (p > 0.05). TAC and TPC were very strongly correlated (r = 0.921). Contrary to the results obtained for TPC, the anthocyanins require a longer time for a more efficient extraction. The effect of extraction time (t = 30–82.5 °C) on TAC at etc = 50% and τ = 30 min is shown in TAC increased significantly (from 0.297 to 0.407 mg C3GE/g DM) with increasing t from 30 °C to 50 °C, were similar for t = 50 °C (0.407 mg C3GE/g DM) and t = 60 °C (0.415 mg C3GE/g DM), and decreased significantly (from 0.415 to 0.375 mg C3GE/g DM) with increasing t from 60 to 82.5 °C. The effect of extraction temperature , were consistent with those reported in the literature. The anthocyanin content obtained by Tahirovic et al. [TAC for an extraction with 50% ethanol (0.238 mg C3GE/g fresh fruit) compared to an extraction with absolute ethanol (0.11 mg C3GE/g fresh fruit) and water (0.12 mg C3GE/g fresh fruit).The values of c et al.  by UAE wc et al. . StankovTAC in the extract samples P1, P2,…, P13, i.e., m,iTAC , which are presented in etc, τ, and t) on total anthocyanin content were quantified using the response surface regression model described by Equation (5), where the regression coefficients were estimated based on the experimental data summarized in pred,iTAC), corresponding residuals , and characteristic statistics of the regression model, i.e., RMSE defined by Equation (6), multiple R, multiple R2, adjusted R2, F, and p, are also included in The mean values of d(Y = predTAC) calculated with Equation (4), where YL = 0.034 mg G3GE/g DM and YU = 0.415 mg G3GE/g DM, surface and contour plots of desirability depending on extraction process factors are similar to those shown in For etc = 50–100%) and extraction temperature (t = 30 °C and t = 60 °C) on AC in the extract samples obtained at τ = 30 min are shown in etc was similar to those on TPC and TAC. Moreover, AC  was very strongly correlated with TPC (r = 0.997) and TAC (r = 0.988). The highest mean value of AC at t = 30 °C (56.82 µmol TE/g DM) was achieved for etc = 50%, and the lowest mean value (20.30 µmol TE/g DM) was achieved for etc = 100%. For etc = 50%, the mean value of AC at t = 60 °C (67.36 µmol TE/g DM) was significantly higher (by 19%) than that obtained at t = 30 °C. The effects of the volume percentage of ethanol in the extraction solvent  and t (30 °C and 60 °C), are summarized in Using HPLC-PDA analysis of the prepared extracts, up to five polyphenols from the twenty-three standard substances were identified , five inPA) in the extracts were in the range of 2.08–4.47 mg/100 g DM; the highest mean value was found in the extract prepared at 60 °C with 50% ethanol, whereas the lowest one was found for the extract obtained at 30 °C with 100% ethanol. The effects of etc and t on PA were similar to those on TPC, TAC, and AC. Moreover, PA was very strongly correlated with TPC (r = 0.965), TAC (r = 0.914), and AC (r = 0.961). The mean values of protocatechuic acid content (CA) in the extracts ranged from 1.05 to 3.36 mg/100 g DM (0.32–1.04 mg/100 g fresh fruit). The mean value of CA obtained at etc = 50% and t = 30 °C (3.34 mg/100 g DM) was significantly higher (up to 3 times) than those obtained under other experimental conditions. Lower CA values (0.34 ± 0.04 mg/100 g DM) were reported by Pozzo et al. [CA values of 0.44 ± 0.02 mg/100 g fresh fruit for extracts prepared in methanol (70%) by UAE at 25 °C for 60 min, probably due to a higher solubility in methanolic solutions, the presence of an ultrasound field, or a longer extraction time.The values of caffeic acid content  in the extracts, i.e., 1.36–2.77 mg/100 g DM (0.42–0.86 mg/100 g fresh fruit), were lower than those found in methanolic extracts by Cosmulescu et al. [VA obtained in this study at etc =100% and t = 30 °C (1.46 mg/100 g DM) was significantly lower (up to 1.8 times) than those obtained under other experimental conditions. The values of vanillic acid content (RH) in the extracts were in the range of 1.87–3.77 mg/100 g DM (0.58–1.17 mg/100 g fresh fruit). They were lower than those reported by Cosmulescu et al. [RH obtained in this study at etc = 50–75% and t = 30 °C (3.42–3.56 mg/100 g DM) were significantly higher (up to 90%) than those obtained under other experimental conditions. The values of rutin hydrate content  in the extracts obtained at t = 30 °C varied from 0.25 to 1.03 mg/100 g DM (0.08–0.32 mg/100 g fresh fruit). For etc = 50–75%, the mean values of Q (0.26–1.02 mg/100 g DM) increased with increasing etc. For t = 60 °C, the degradation of quercetin occurred, and its concentration decreased below the detection limit of the apparatus. The maximum values of Q obtained in this study (1.02 ± 0.01 mg/100 g DM) were consistent with those reported by Pozzo et al. [The values of quercetin content  provides high-resolution predicted isotopic patterns, which can be compared to experimental spectral data. The predicted and measured values of the mass-to-charge ratio (m/z) for some phenolic compounds from extract samples P1 and P2 are summarized in m/z (percent errors from −0.00007% to 0.00006%). The results presented in m/z = 153), caffeic acid (m/z = 179), and quercetin (m/z = 301), in blackthorn extracts. Applying an HPLC-PDA-ESI-MS3 method, Magiera et al. [An FT-ICR-MS method was applied for the additional identification of the phenolic compounds quantified by HPLC-PDA. This method involved the direct infusion of the extract sample (without chromatographic separation) and was used only for qualitative analysis. FT-ICR spectrometers are the most advanced mass analyzers with resolving power with sub-ppm mass accuracy ,44. The a et al.  identifietc and t) on TPC, TAC, AC, PA, CA, VA, RH, and Q in the extract samples obtained at τ = 30 min. The PCA results highlighted that only the eigenvalues corresponding to PC1 (5.84) and PC2 (1.89) were >1, and PC1 and PC2 explained 96.6% (73.0% + 23.6%) of the total variance. PCA was applied to evaluate the effects of the extraction conditions  and P12 (etc = 50% and t = 60 °C) had higher values of TPC, TAC, AC, PA, CA, and VA than sample P4 (etc = 100% and t = 30 °C) (discrimination on PC1 highlighted in Extract samples P1 (etc = 66.67% and t = 30 °C) and P3 (etc = 75% and t = 30 °C) had higher values of RH and Q than samples P4 (etc = 100% and t = 30 °C) and P12 (etc = 50% and t = 60 °C) (discrimination on PC2);Extract samples P2 ; TPC, TAC, and AC were inversely correlated with Q (−0.748 ≤ r ≤ −0.634); RH was directly correlated with CA (r = 0.642) and Q (r = 0.515).The results shown in etc = 50% and t = 60 °C (τ = 30 min) had the highest values of TPC (37.23 ± 0.21 mg GAE/g DM), TAC (0.42 ± 0.00 mg C3GE/g DM), AC (67.36 ± 0.62 μmole TE/g DM), PA (4.47 ± 0.06 mg/100 g DM), and VA (2.65 ± 0.11 mg/100 g DM), but lower values of RH (1.87 ± 0.00 mg/100 g DM) and Q (0.00 ± 0.00 mg/100 g DM) than those obtained under other experimental conditions. Accordingly, at higher levels of process temperature, the extraction of protocatechuic acid and vanillic acid was enhanced, but the flavonoids (rutin hydrate and quercetin) were degraded. The extract sample P3 obtained at etc = 75% and t = 30 °C (τ = 30 min) had the highest values of flavonoid content (RH = 3.56 ± 0.02 mg/100 g DM and Q = 1.02 ± 0.01 mg/100 g DM).The extract sample P12 obtained at This study highlighted that the optimal extraction of different types of phytocompounds requires special experimental conditions depending on their chemical and thermal stability as well as molecular mass. etc), process temperature (t), and operating time (τ), affected the performance of the extraction process. A synergistic effect of obtaining high total contents of polyphenols (TPC) and anthocyanins (TAC), high contents of protocatechuic acid (PA) and vanillic acid (VA), and a good antioxidant capacity (AC) was achieved for the extraction with 50% ethanol at 60 °C for 30 min. At a higher level of process temperature, the extraction of protocatechuic acid and vanillic acid was enhanced, but the flavonoids (rutin hydrate and quercetin) were degraded. If a high amount of flavonoids is desired, a lower temperature should be used. TPC, TAC, AC, and phenolic acid contents  in the extract samples obtained at etc = 50–100%, t = 30–60 °C, and τ = 30 min were strongly directly correlated. Flavonoid contents (RH and Q) as well as RH and CA were directly correlated, whereas TPC, TAC, and AC were inversely correlated with Q.All tested parameters, i.e., ethanol concentration in the extraction solvent (


Compound with cancer stem cell (CSC)-suppressing activity is promising for the improvement of lung cancer clinical outcomes. Toward this goal, we discovered the CSC-targeting activity of resveratrol (RES) analog moscatilin (MOS). With slight structural modification from RES, MOS shows dominant cytotoxicity and CSC-suppressive effect.2-DA staining. CSC-rich populations of A549 cells were generated, and CSC markers, and Akt signaling were determined by Western blot analysis and immunofluorescence. Molecular docking and molecular dynamics (MD) simulations were used to predict the possible binding of the compound to Akt protein.Three human lung cancer cell lines, namely H23, H292, and A549, were used to compare the effects of RES and MOS. Cell viability and apoptosis were determined by the MTT assay and Hoechst33342/PI double staining. Anti-proliferative activity was determined by colony formation assay and cell cycle analysis. Intracellular reactive oxygen species (ROS) were measured by fluorescence microscopy using DCFHIn this study, we evaluated the effects of RES and MOS on lung cancer and its anti-CSC potential. Compared with RES, its analog MOS more effectively inhibited cell viability, colony formation, and induced apoptosis in all lung cancer cell lines . We further investigated the anti-CSC effects on A549 CSC-rich populations and cancer adherent cells (A549 and H23). MOS possesses the ability to suppress CSC-like phenotype of lung cancer cells more potent than RES. Both MOS and RES repressed lung CSCs by inhibiting the viability, proliferation, and lung CSC-related marker CD133. However, only MOS inhibits the CSC marker CD133 in both CSC-rich population and adherent cells. Mechanistically, MOS exerted its anti-CSC effects by inhibiting Akt and consequently restored the activation of glycogen synthase kinase 3β (GSK-3β) and decreased the pluripotent transcription factors (Sox2 and c-Myc). Thus, MOS inhibits CSC-like properties through the repression of the Akt/GSK-3β/c-Myc pathway. Moreover, the superior inhibitory effects of MOS compared to RES were associated with the improved activation of various mechanism, such as cell cycle arrest at G2/M phase, production of ROS-mediated apoptosis, and inhibition of Akt activation. Notably, the computational analysis confirmed the strong interaction between MOS and Akt protein. MD simulations revealed that the binding between MOS and Akt1 was more stable than RES, with MM/GBSA binding free energy of − 32.8245 kcal/mol at its allosteric site. In addition, MOS interacts with Trp80 and Tyr272, which was a key residue in allosteric inhibitor binding and can potentially alter Akt activity.Knowledge about the effect of MOS as a CSC-targeting compound and its interaction with Akt is important for the development of drugs for the treatment of CSC-driven cancer including lung cancer.The online version contains supplementary material available at 10.1186/s12906-023-04016-6. Despite advances in early diagnostics, effective drugs, and surgical, and radiotherapy improvement, lung cancer remains the major cause of cancer-related mortality , 2. FailDendrobium moschatum, which is a natural trimethoxylated RES analog , and H1 H NMR  δ: 7.43 , 7.36 , 7.30 , 6.95 , 6.86 , 6.82 , 5.16 , 4.61 , 3.91 .3)diethyl (4-(benzyloxy)-3-methoxybenzyl)phosphonate (2Cl2 (44 mL) was added dimethylsulfide  at 0 °C over 7 min. The reaction mixture was stirred at this temperature for 10 min. A solution of 2  in CH2Cl2 (44 mL) was cooled at − 18 °C and was added above solution. The reaction mixture was stirred at − 18 °C for 3 h. The reaction mixture was warmed to 0 °C and diluted with H2O and extracted with CH2Cl2 (80 mL×3), washed with saturated NaHCO3 solution and H2O, dried over anhydrous Na2SO4, and concentrated. The crude product was dissolved in triethyl phosphite . The reaction mixture was stirred at 140 °C for 4 h. After cooling to RT, the reaction mixture was evaporated under vacuum. The residue was purified over SiO2 column (n-Hex. : EtOAc = 1 : 9) to give 3  as a yellow oil.A solution of NBS  in CH1 H NMR  δ: 7.29–7.44 , 6.89 , 6.82 , 6.75 , 5.13 , 3.93–4.11 , 3.89 , 3.04 , 1.20 .4)4-(benzyloxy)-3,5-dimethoxybenzaldehyde (3OH (33 mL) was added K2CO3  and benzyl bromide , and the obtained solution was refluxed for 20 h. After cooling to RT, the reaction mixture was filtered, and the obtained filtrate was evaporated under vacuum. The residue was diluted with H2O and extracted with CHCl3 (50 mL×3), washed with brine, dried over anhydrous Na2SO4, and concentrated. The crude product was purified over SiO2 column (n-Hex. : EtOAc = 7 : 3) to give 4  as a yellow oil.A solution syringaldehyde  in CH1 H NMR  δ: 9.86 , 7.47 , 7.28–7.38 , 7.11 , 5.13 , 3.90 .E)-2-(benzyloxy)-5-(4-(benzyloxy)-3-methoxystyryl)-1,3-dimethoxybenzene (5) in THF (5.5 mL) was stirred at − 78 °C and added t-BuOK solution in THF  over 30 min. The reaction mixture was stirred for 20 min. at the same temperature, and was added 4  in THF (1.0 mL) over 20 min. and the mixture was stirred for 1 h at − 78 °C and for 10 min. at 0 °C. Then, the reaction mixture was stirred for 2 h at RT. The reaction mixture was cooled to 0 °C and diluted with saturated NH4Cl solution and extracted with EtOAc (60 mL×3), washed with saturated NH4Cl solution and H2O, dried over anhydrous Na2SO4, and concentrated. The crude product was purified over SiO2 column (CH2Cl2) to give 5  as a colorless solid.A solution of 1 H NMR  δ: 7.49 , 7.44 , 7.27–7.39 , 7.07 , 6.97 , 6.94 , 6.88 , 6.86 , 6.70 , 5.17 , 5.02 , 3.95 , 3.87 . 13 C NMR  δ: 153.6, 149.8, 148.0, 137.8, 137.0, 136.6, 133.3, 130.8, 128.5, 128.5, 128.1, 127.8, 127.8, 127.2, 127.0, 119.6, 114.0, 109.3, 103.4, 75.1, 71.0, 56.1, 56.0. IR (KBr cm− 1): 3547, 3019, 2399, 1507, 1331, 1214, 1030, 928, 753, 668, 501, 476, 454, 441, 435, 429, 407, 401. EI-MS m/z (%): 482 , 392 (28), 391 (100), 91 (62). HRMS (EI): Calcd for C31H30O5, 482.2093; Found: m/z 482.2092.2.Human lung cancer H23, H292, and A549 cells were purchased from the American Type Culture Collection . H23 and H292 cells were cultured in RPMI medium (Gibco). A549 cells were cultured in DMEM medium (Gibco). The medium was supplemented with 10% FBS (HyClone), 2 mM L-glutamine (Gibco), and 1X Anti-Anti (Gibco). Cells with 70 − 80% confluence were trypsinized with 0.25% trypsin–EDTA (HyClone) and subcultured in the same media. The cells were maintained at 37 °C in an incubator with a humidified atmosphere containing 5% COThe stock solutions of RES and MOS was prepared at concentration of 50 and 12.5mM in DMSO (Sigma) and stored in aliquots at − 20 °C. RES and MOS with designated final concentrations (0 − 200 µM) were diluted with cell culture media for subsequence experiments with a maximal DMSO concentration less than 0.5% DMSO. DMSO was used as the vehicle control.4 cells per well in 96-well plates. The cells were then treated for 24 h with different concentrations (0 − 200 µM) of RES or MOS. After desired incubation, MTT solution (0.5 mg/mL) was added and the cells were incubated in dark for 3 h at 37 °C in the incubator containing 5% CO2. The MTT solution was replaced with DMSO (100 µL/well) to dissolve the purple formazan crystal. The absorbance was measured at 570 nm using a microplate reader . Percentage of cell viability in relation to the non-treated control was calculated from the optical density (OD) ratio of treated to non-treated control cells. IC50 values were calculated using regression analysis from dose-response curves . The cancer selectivity index (SI) was calculated by the following equation: SI = mean IC50 against normal cells/mean IC50 against cancer cells.The effect of RES and MOS on cell viability in lung cancer cells was assessed by using MTT assay. Briefly, lung cancer cell lines  were seeded overnight at a density of 1 × 10Lung cancer cells  were plated in triplicate into 6-well plates at 300 cells/well. Following overnight attachment, cells were treated with various concentrations (0 − 25 µM) of RES or MOS for 24 h. After treatment, the medium containing RES or MOS was replaced with the fresh medium. The colonies were allowed to form for 7 days, and the medium was changed every two days. For colony staining, colonies were washed once with PBS, then fixed by adding fixative ) for 5 min and stained with crystal violet (0.05% (w/v)) in 4% paraformaldehyde for 30 min. The excess crystal violet was washed with distilled water several times and let air dry at RT. The colonies were photographed with a digital camera, and the acquired images were analyzed using the ImageJ software , Bethesda, MD, USA).5 cells/well overnight and synchronized by serum deprivation for 48 h before treatment. The cells were treated with RES or MOS at 0, 1, 2.5, and 5 µM for 24 h. After treatment, cells were stained with PI using the methods previously described [The cell cycle distribution was determined by flow cytometry. A549 cells were plated into 6-well plates at 1 × 104 cells/well overnight and treated with various concentrations at 0, 2.5, and 5 µM of RES or MOS for 2 h. After treatment, the cells were stained with 10 µM DCFH2-DA  for 30 min at 37 °C in the dark. Subsequently, stained cells were photographed under a fluorescent microscope .The intracellular accumulation of reactive oxygen species (ROS) was assessed by fluorescence microscopy. The lung cancer cell lines (A549 and H23) were seeded in 96-well black plates with clear bottom at 1 × 104 cells/well and treated with various concentrations (0 − 10 µM) of RES or MOS for 24 h. For MOS treatment with NAC, the cells were pretreated with 5 mM NAC for 30 min before being exposed to MOS at 2.5 and 5 µM for 24 h. The cells were stained with Hoechst 33342 (10 µg/mL) and PI (1 µg/mL) for 30 min at 37 °C. Then, the cells were visualized under fluorescent microscope  and the percentages of apoptotic cells were determined.Apoptosis was detected by co-staining with Hoechst 33342 and PI. Nuclear morphology was assessed using the DNA dye Hoechst 33342. The lung cancer cell lines  were seeded in 96-well plates at 1 × 10A549 and H23 cells were plated overnight. The cells were treated with MOS at 0, 1, 2.5, and 5 µM for 24 h. Following specific treatments, cells were lysed and prepared for Western blotting as was described previously [3 cells/well in DMEM medium containing 1% FBS for 7 days to form primary spheroids. At day 7, primary spheroids were harvested and resuspended as single cells using 0.25% trypsin–EDTA (HyClone) and seed onto 24-well ultralow attachment plates for 14 days to form secondary spheroids. After 14 days of secondary spheroid development, each secondary spheroid was collected, dissociated into a single spheroid of the same size, and treated with non-cytotoxic concentrations (0 − 2.5 µM) of RES or MOS for 3 days. Phase-contrast images of secondary spheroids were captured  after treatment using a phase-contrast microscope (Nikon ECLIPSE Ts2). At day 3, a single spheroid was co-stained with Hoechst 33342 and PI at 37 °C for 15 min and photographed under a fluorescence microscope (Nikon ECLIPSE Ts2).To generate CSC-rich population, A549 cells were grown in a 24-well ultralow attachment plates at a density of 1.5 × 104 cells per well in 96-well plates overnight. The cells were then treated with 5 µM of RES or MOS and incubated for 24 h. Following fixation with 4% paraformaldehyde for 15 min, cells were permeabilized with 0.2% (v/v) Triton X-100 and blocked with 10% (v/v) FBS for 20 min at RT. The cells were incubated overnight at 4 °C in the presence of p-Akt or CD133 primary antibodies at a dilution of 1:100 in 4% FBS. After incubation, Alexa Fluor 488 or Alexa Fluor 594 conjugated with goat anti-rabbit IgG secondary antibody at a ratio of 1:500 in 4% FBS was added and incubated for 1 h at RT in the dark. Cell nuclei were stained with Hoechst 33342 (10 µg/mL) for 15 min at RT and then photographed under a fluorescent microscope .For staining cell monolayers, A549 and H23 cells were seeded at a density of 1 × 10For staining spheroid (3D), A549 cells were allowed to form primary and secondary spheroids as described before. At day 14 of secondary spheroid development, CSC-rich populations were treated with 1 µM of RES or MOS for 24 h, and then the cells were stained following the same procedure as described previously for cell monolayer staining.The binding of investigated compounds to Akt1 was performed through molecular docking. The crystal structure of Akt1 (PDB code: 5KCV) bound to miransertib, an oral allosteric Akt inhibitor , was dowThe predicted binding modes of targeted compounds from molecular docking studies were used as initial structures for the molecular dynamics (MD) simulation using the Amber18 and AmberTools19 packages. The protein and compound parameters were used for the FF14SB force field  and geneAll experiments were performed at least three times, and all results are expressed as the mean ± standard deviation. Statistical analyses were performed using GraphPad Prism 7.0 . The unpaired t-test was used for the statistical analyses between two groups. P < 0.05 was considered statistically significant.SM_7) in Fig. 1 and 4. The aldehyde of 1 was reduced by sodium borohydride to get an alcohol 2, which was then converted to a bromide followed by the Michaelis–Arbuzov reaction [3, the key reagent for the Horner–Wadsworth–Emmons reaction. The obtained 3 was converted to E-stilbene derivatives 5 by the Horner–Wadsworth–Emmons reaction [1. Finally, 5 was catalytically hydrogenated to yield MOS (SM_7). 1 H and 13 C-NMR were identical to that of the natural product [In this study, we explored the anticancer effects of RES and its analog MOS against human lung cancer cells. The chemical structures of RES and MOS are shown in Fig. reaction  with trireaction  with aro product  Fig. 1CCSM_7 in 50) value of 30.34, 5.54, and 7.45 µM for H23, H292, and A549, respectively, whereas the IC50 values of RES were > 200 µM for all lung cancer cell lines  were evaluated. The cells were treated with various doses  of RES or MOS for 24 h, and cell viability was examined with the MTT assay. Following treatment with MOS, the viability of lung cancer cells was significantly decreased in a dose-dependent manner . The cells were treated with various concentrations (0–10 µM) of RES or MOS for 24 h and then subjected to Hoechst 33342/PI double staining to determine apoptotic and necrotic cells by fluorescence microscope. Hoechst 33342 staining was used to evaluate nuclear condensation and DNA fragmentation, both of which are characteristics of apoptosis . PI fluoTaken together, our results indicated that MOS had a greater capacity to suppress cell growth and induce apoptosis in lung cancer cells than RES. Structural activity relationship (SAR) is commonly used in drug design and discovery, and it involves modifying the chemical structure of lead compounds to improve their efficacy, potency, selectivity, and safety  Thus, st2-DA fluorescence probe was used to detect intracellular ROS levels in the cells by fluorescence microscopy. Our results showed that relative fluorescence intensity of DCFH2-DA was significantly increased in both A549 and H23 cells in a dose-dependent manner (2.5–5 µM) at 2 h of MOS treatment  for 3 days. To measure the viability and apoptosis of CSCs, Hoechst 33342/PI double staining was performed on day 3 after treatment with the compounds. The CSC viability was significantly decreased, as indicated by condensed, and fragmented nuclei stained with the DNA dye Hoechst 33342. PI-stained CSC appeared bright red, indicating cell death, and rupture. In the spheroid formation assay, treatment with both RES and MOS reduced the spheroid size and suppressed CSC viability in a dose-dependent manner compared with the untreated control  for 24 h, and the expression levels of CD133, Sox2, p-Akt, Akt, p-GSK-3β, and GSK-3β were determined by Western blot analysis. MOS significantly reduced the expression levels of p-Akt/Akt, p-GSK-3β/GSK-3β, and c-Myc in a dose-dependent manner in A549 cell; however, the p-GSK-3β/GSK-3β ratio was not altered in H23 cells Fig. C and D. We also confirmed the effect of MOS suppressed CSC-like phenotype via Akt signaling compared to PI3K/Akt pathway inhibitor LY294002. Inactivation of PI3K using LY294002 has been demonstrated to suppress the Akt activity through inhibiting the phosphorylation of Akt at both T308 and S473, consequently inducing cell cycle arrest and apoptosis , 50. To To predict the possible binding mode of RES and MOS with Akt, a molecular docking study was performed. RES and MOS were docked into the allosteric binding site of Akt1 using AutoDock Vina. The values of the binding affinities of compounds are presented in Fig. A 100-ns MD simulation of each Akt1/ligand complex was conducted to optimize the docking-predicted binding modes of two natural compounds. To explore the dynamic stability of the two systems, the root mean square deviation (RMSD) values were calculated during 100-ns MD simulations. As shown in Fig. The per-residue binding energy decomposition was performed to obtain details of Akt1–ligand complexes and identify the key residues responsible for ligand binding. Generally, the residue would be considered necessary for binding when the binding energy is more negative than − 2 kcal/mol. Trp80 and Tyr272 have been reported as essential residues for the potent allosteric Akt-1 inhibitor in clinical trials , 51. As 50 of less than 10 µM, implying that MOS might be a promising anticancer agent for the treatment of lung cancer. In addition, MOS exhibits low toxicity to normal cells  can suppress colony formation by more than 90%, similar to other lung cancer cell lines A549 and H292 (Fig. CSCs in cancer were found to resist chemotherapeutic drugs and induce disease recurrence after therapy . CSCs calls Fig. A and B, Several studies have demonstrated that various anti-cancer agents induce cancer cell apoptosis through ROS generation . It has MOS was demonstrated to have a superior activity to suppress CSCs than RES at the same concentration, indicated by the reduction of CSC marker, pluripotent transcription factor, and stem cell-like phenotypes Figs.  and 6. LGSK-3β was recognized as a downstream effector of Akt and has been indicated as a cellular regulator of growth, drug resistance, metastasis, and CSC maintenance . Akt canCompounds possessing the ability to inhibit Akt are under investigation in a clinical setting . The majIn this study, we have unraveled the anticancer activity of RES and its modified analog MOS against CSC of lung cancer cells. MOS was more potent than its parental compound in terms of cytotoxicity and CSC suppression. This study demonstrated that MOS suppresses lung CSCs via the Akt/GSK-3β/c-Myc pathway. Furthermore, molecular docking, and MD simulation revealed the possibility that MOS interacts with the Akt molecule. The binding affinity to the allosteric site of the Akt protein of MOS was greater than that of RES. Our findings highlight the therapeutic potential of MOS in the treatment of lung CSCs and support further research toward the clinical development of this compound.Below is the link to the electronic supplementary material.Additional file 1


Spodoptera litura, two separated testes fuse into a single testis during the larva-to-pupa transformation. We identified five α and five β integrin subunits that were closely associated with testicular fusion. Integrin α1 and α2 belong to the position-specific 1 (PS1) and PS2 groups, respectively. Integrin α3, αPS1/αPS2, and αPS3 were clustered into the PS3 group. Integrin β1 belonged to the insect β group, and β2, β3, and β5 were clustered in the βν group. Among these integrins, α1, α2, α3, αPS1/PS2, αPS3, β1, and β4 subunits were highly expressed when the testes fused. However, their expression levels were much lower before and after the fusion of the testis. The qRT-PCR and immunohistochemistry analyses indicated that integrin β1 mRNA and the protein were highly expressed in the peritoneal sheath of the testis, particularly when the testes fused. These results indicate that integrins might participate in S. litura testicular fusion.Integrin members are cell adhesion receptors that bind to extracellular matrix (ECM) proteins to regulate cell–cell adhesion and cell-ECM adhesion. This process is essential for tissue development and organogenesis. The fusion of two testes is a physiological phenomenon that is required for sperm production and effective reproduction in many Lepidoptera. However, the molecular mechanism of testicular fusion is unclear. In  Integrins are present in all metazoan animals, and they are cell adhesion receptors formed by the non-covalent association of α and β subunits. Each subunit consists of a type I transmembrane glycoprotein, a large extracellular domain, and a short cytoplasmic tail ,2. The cIn mammals, 18α and 8β subunits have been reported and assembled into 24 distinct integrin heterodimers with different ligand binding characteristics and different tissue distributions . As specDrosophila, it has been shown that integrin mediating cell-ECM adhesion can play important roles in muscle-tendon attachment at myotendinous junctions of the muscle tissue during larval development [Drosophila’s early oogenesis, integrin can medicate cell–basement membrane interactions to regulate epithelial cell division and differentiation [Integrins have been shown to perform vital reproductive activities, including gamete formation, fertilization, and implantation ,21. In melopment . During ntiation .Bombyx mori, αPS3 is specifically expressed in granulosa cells [B. mori hemocytes and regulated the development of plasmatocytes [Spodoptera exigua, the reduction in β1 integrin by RNAi can cause a marked loss of cell–cell contacts and a remarkable midgut epithelium cell death when the Cry toxin infects the pests [Helicoverpa armigera, β integrin could be seen as a hemocytic receptor of C-type lectin during the encapsulation reaction analogous to granuloma formation in vertebrates [Ostrinia furnacalis, β1 integrin was expressed in the hemocytes and regulated the spreading of plasmatocytes [Integrin expression in Lepidoptera demonstrates considerable tissue specialization. For example, in sa cells . These eatocytes . In Spodhe pests . In Helitebrates . In Ostratocytes . These sD. melanogaster [B. mori [S. litura (S. litura) (Lepidoptera: Noctuidae) is a major agricultural pest of many crops in the tropical and subtropical areas of Asia [S. litura testis produce spermatozoa and provides nutrition for spermatozoa development [S. litura, the two kidney-like testes are separated during the larval stages. However, during their transition from final instar larva to pupa the two separated testes fuse to form a single testis [However, there are still fewer integrin subunits identified in insects. Seven integrin subunits including αPS1, αPS2, αPS3, αPS4, αPS5, βPS, and βν were identified in nogaster  and six [B. mori . S. litu of Asia . The S. elopment . Testicuelopment ,36,37. Ie testis ,36,37,38e testis . HoweverS. litura and analyzed their expression in the testis. The results confirmed the specific expression and localization of integrin subunits in the fusion of testis in S. litura and provided new insights into the molecular mechanism of testicular fusion in lepidopteran insects.In this study, we identified cell adhesion receptor integrins of S. litura larvae were provided by the Entomology Institute of SUN YAT-SEN University  and fed on an artificial diet, including soybean powder [n powder , wheat bn powder . These ln powder .S. litura genome [http://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/) (accessed on 14 March 2022). The amino acid sequences of integrins from Homo sapiens, Drosophila melanogaster, Bombyx mori, Tribolium castaneum, Manduca sexta, Pseudoplusia includens, et al., were downloaded from NCBI  as reference sequences. The integrin sequences were obtained by querying the reference sequences to BLAST against the S. litura genome with the E-value threshold of 10−6 [S. litura were further verified by domain prediction using three online forecasting tools: SMART , Pfam  and PROSITE .The integrin α and β subunits were predicted and analyzed, based on the published a genome , using t of 10−6 ,43. The H. sapiens, D. melanogaster, B. mori, S. litura, and other species were aligned using the CLUSTAL W program [The amino acid sequences of integrins from  program . The phy program  with 100 program . The gen® Select Master Mix (ABI). The reactions included a 10 μL of 2 × SYBR® Select Master Mix (ABI) and 0.6 μL of specific forward and reverse primers (the concentration of each primer was 10 μM). The reactions were performed under the following conditions: 50 °C for 2 min, 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The housekeeping gene gapdh  of S. litura was used as a reference gene [−ΔΔCt [The total RNA was extracted using the Trizol Reagent Kit  according to the manufacturer’s instructions. Two μg RNA from each sample was used for cDNA synthesis. The genomic DNA was digested using the RNase-free DNase I . The first-strand cDNA was synthesized using M-MLV Reverse Transcriptase  and oligo d(T) primers  using the manufacturer’s instructions. qRT-PCR reactions were carried out using the QuantStudio™ 6 Flex Real-Time system  with SYBRnce gene . The rele [−ΔΔCt . The priThe testes from 12 individuals were collected for RNA extraction. The process for the separation of the peritoneal sheath and sperm cells was described previously . The relSlIntegrin β1 was generated by immunized rabbits in the Zoonbio Biotechnology Company . The ratio of the primary antibody against SlIntegrin β1 (1:1000) was incubated at 4 °C overnight, and this membrane was washed three times with 0.1% Triton X-100 in phosphate-buffered saline . The HRP-conjugated goat anti-rabbit IgG (1:5000) was used as the second antibody and incubated at 37 °C for 1 h. Then, this membrane was washed three times with 0.1% Triton X-100 in PBS and twice with PBS alone. The rabbit antibody β-tubulin  was used as a control reference. Using Image J (V1.8.0.112) (downloaded from https://imagej.nih.gov/ij/) (accessed on 1 May 2022), the relative expression of the Integrin β1 protein was estimated based on the integrated density ratio of Integrin β1 to β-tubulin in Western blot results.The total proteins from 20 testes were collected and extracted in a RIPA lysis buffer . A total of 25 μg of proteins from each sample were separated on 10% SDS-PAGE gels, and these proteins were then transferred onto PVDF membranes  using a Trans-Blot Cell . The primary antibody against SlIntegrin antibody  was incubated for 1 h at 37 °C. The slides were washed three times at room temperature, 5 min at a time. The secondary antibody Goat anti-Rabbit IgG Alexa Fluor™ 594  (1:200) and DAPI  (1:2000) were incubated for 30 min at 37 °C. The slides were examined using a confocal microscope .The testes from L6D4, L6D6, and PD1 were fixed in 4% paraformaldehyde in PBS. The testes were dehydrated through a series of increasing concentrations of ethanol, which were cleared with xylene, and embedded in preheated melted paraffin at 60 °C, as described previously . The parn = 3). A p-value < 0.05 or <0.01 indicated a significant difference.Gene expression data were analyzed using GraphPad Prism 8 Software , and significant differences were determined by ANOVA. The values provided are the mean ± SEM (standard error of the mean) , B. mori integrin α1 (78.1%), and D. melanogaster integrin αPS1 (41.2%). S. litura Integrin α2 belonged to the PS2 group and was closely related to P. includens integrin α2 (79.4%), M. sexta integrin α2 (67.0%), and B. mori integrin α2 (57.8%). S. litura integrin α3, αPS1, αPS2, and αPS3 were clustered in the PS3 group, which contained D. melanogaster integrin αPS3, αPS4, and αPS5. S. litura integrin α3 had a 60.9% identity to P. includens integrin α3, S. litura αPS1 had a 32.1% identity to B. mori αPS2 (32.1%), and S. litura αPS2 had a 31.1% identity to B. mori αPS3 , and S. litura β2, β3, and β5 were clustered in the insect βν group. S. litura β4, B. mori β4, and Danaus plexippus β3 were clustered together in a single branch, which was different from the other groups. Interestingly, S. litura integrins αPS1/PS2 and αPS3 were located in the same scaffold of the S. litura genome, and the transcription orientation in the genome was the same, while integrin β2, β3, and β4 were also in the same scaffold; however, the transcriptional direction of β3 and β4 was inverse to that of integrin β2  and Vertebrate B , and three insect β groups . Integriegrin β2 .To study the expression patterns of the integrins identified in the testes, we collected the testes that were described in methods 2.1 for mRNA expression analysis by qRT-PCR. Integrin α1, α2, α3, αPS1/PS2, αPS3, β1, and β4 were highly expressed in L6D6 larvae when the testes were fusing and lower expressed in L6D0, L6D2, L6D4 larvae and PD1, PD3 pupae . IntegriPrevious transcriptome analysis demonstrated the differential expression of integrin β1 during the testis fusion process . To examS. litura genome sequences with the reported integrin reference sequences from B. mori, D.melanogaster, and mammals. The sequences of the subunits αPS1 and αPS2 of S. litura were identical  and analyzed in the context of the reproductive cycle, and it was found that the expression of ITGB1b was highly expressed during spermatogenesis, whereas the expression of other ECM-associated molecules was induced mainly in the post-spawning phase, suggesting that integrins were involved in the genesis of the reproductive system [S. litura epithelial cell morphogenesis during the testicular fusion process, which is key to the construction of developing tissues and organs. For example, neural tube closure involves a process of epithelial fusion, whereby initially adjacent neural folds are adhered by cellular protrusions and then form a more stable union, which is followed by the remodeling of epithelial structures to form the neural tube [Drosophila heart development, integrin-mediated cell adhesion and migration were essential for the formation of cardiac chambers, especially for cardioblast polarization [Integrin receptors connect to the cytoskeleton and bind to the ECM to mechanically link cellular components within and outside of the cells. We investigated the expression patterns of integrins during testicular fusion because it has not yet been established what function they play in gonadal morphogenesis in r fusion . The gene system . Thereforal tube . This taral tube . In thisral tube . α6β4, aral tube . During rization ,72. Thismmp2 and mmp3) and ECM proteins, including collagens, proteoglycans, and glycoproteins, were highly expressed in testicular ECM when testicular fusion occurred. During gonadal morphogenesis, integrin plays a variety of critical roles in cell migration, differentiation, and connection between the germ and somatic or somatic and somatic cells [S. litura.We previously reported  that theic cells . It is yS. litura. The molecular mechanisms involved in this process require further study.In summary, this study demonstrated that all integrin α subunits and integrin β1, β4 had a higher expression during testicular fusion. Integrin β1 mRNA and the protein were highly expressed in the peritoneal sheath of the testes when the testes were in the process of fusion. Integrin β1, and other integrins, might be directly involved in the testicular fusion of