The fruits from the Arecaceae family, although being rich in bioactive compounds with potential benefits to health, have been underexplored. Studies on their composition, bioactive compounds, and effects of their consumption on health are also scarce. This review presents the composition of macro- and micronutrients, and bioactive compounds of fruits of the Arecaceae family such as bacaba, patawa, juçara, açaí, buriti, buritirana, and butiá. The potential use and reported effects of its consumption on health are also presented. The knowledge of these underutilized fruits is important to encourage production, commercialization, processing, and consumption. It can also stimulate their full use and improve the economy and social condition of the population where these fruits are found. Furthermore, it may help in future research on the composition, health effects, and new product development. Arecaceae fruits presented in this review are currently used as raw materials for producing beverages, candies, jams, popsicles, ice creams, energy drinks, and edible oils. The reported studies show that they are rich in phenolic compounds, carotenoids, anthocyanins, tocopherols, minerals, vitamins, amino acids, and fatty acids. Moreover, the consumption of these compounds has been associated with anti-inflammatory, antiproliferative, antiobesity, and cardioprotective effects. These fruits have potential to be used in food, pharmaceutical, and cosmetic industries. Despite their potential, some of them, such as buritirana and butiá, have been little explored and limited research has been conducted on their composition, biological effects, and applications. Therefore, more detailed investigations on the composition and mechanism of action based on in vitro and/or in vivo studies are needed for fruits from the Arecaceae family. Geonoma, Syagrus, Bactris, Atallea, Allagoptera, Astrocaryum, and Euterpe are the genera with the highest number of occurrences worldwide  wh wh30] whThe stems of the patawa palm tree have up to 26 m height and diameter from 15 to 45 cm B. The leJuçara, a palm tree native to the Brazilian Atlantic Forest, is found mainly between the states of Bahia and Rio Grande do Sul, and in the riparian forests in the states of Minas Gerais, Goiás, Mato Grosso do Sul, São Paulo, and Paraná. It is also found in the northeast of Argentina and the southeast of Paraguay in tropical forests between sea level and up to 1000 meters altitude C. It is The stem of juçara measures between 20 and 25 meters in height and has from 10 to 15 cm of diameter ,35. The The açaí is native to the Amazon where it occurs mainly in floodplains and in permanently flooded areas. On the other hand, it can also grow in dry lands in low-density forests. The moist soil and the high light intensity, due to the limited vegetation cover in lowland soils, are favorable to the development of hydrophilic vegetation species such as açaí ,43,44. TThe stem of açaí is smooth, thin, generally straight, and gray. It presents between 9 and 15 leaves, has height from 10 to 15 meters, and a diameter of 12 to 18 cm when the tree reaches the adult age D. The frThe açaí, which is widely consumed by the population of northern Brazil, has been reported as a superfruit. In the last decade, the açai showed a growing demand in the national and international market, attracting investments and research. As a result, the Brazilian production of processed açaí pulp grew about 89% between 2010 and 2020, reaching approximately 1.7 million tons and generating about USD 800 million for the Brazilian economy in 2020 ,50. The Buriti is a palm tree found mainly in the biomes Amazon and Cerrado and it is also known as miriti, buriti coconut, buriti palm, and swamp palm. It grows in swamps close to permanent watercourses and on top of mountains, which is an advantage since these areas are not suitable for other activities . The burThe fructification of the buriti happens between December and June, and each plant can produce between 150 and 200 kg of fruit per harvest. The fruits have an average weight of 50 g, longitudinal diameter of 5.25 cm, and a cross-sectional diameter of 3.91 cm E 53,54],5453,54]Buritirana is also known as buriti mirim, buriti bravo, caranã in Brazil, and aguajillo in Colombia and Venezuela. The fruits are globose to oblong–ellipsoid F. The puButia odorata is a palm tree which is native to southern Brazil and east of Uruguay. It usually grows in open areas such as fields, savannas, dunes, and sandbanks in the Pampa biome [The pa biome , and is pa biome . Currentpa biome ,65,66.The butiá palm tree has a single, straight, and inclined stem 3–6 m high, without visible palm hearts at the top. Its leaves (7–32) are pinnate, grey-green, and serrated, and the fruits are pale yellow to reddish-orange, with an average diameter of 1.7 to 4.2 cm . The mes−1), lipids (2.18 to 21.02 g 100 g−1), and energy values  show great variation. The pulps of bacaba and buritirana have the highest content of lipids and energy value . On the other hand, the butiá pulp shows the lowest values for these parameters . The buritirana and açaí pulp show the highest protein content . Açaí, patawa, and bacaba present the highest content for carbohydrates , and the lowest value is observed for juçara palm (5.46 g 100 g−1). The highest fiber content was reported for buritirana (65.46 g 100 g 1), followed by patawa pulp (29.70 g 100 g−1). These fruits are richer in fibers when compared to commercial and popular fruits such as banana (10.50 g 100 g−1), mango (6.71 g 100 g−1), watermelon (8.73 g 100 g−1), and tamarind (13.93 g 100 g−1) [−1, respectively). A diet rich in fiber has been related with health benefits such as blood pressure reduction, improvement in serum lipid profile, and glycemic control [100 g−1) . The ing100 g−1) . Butiá a control .−1, respectively). The RDI of K, which contributes to the reduction in blood pressure and the risk of cardiovascular diseases, is 3510 mg per day for healthy adults [The consumption of minerals is necessary for the proper functioning of the organism, and they are related to energy at the cellular level and macronutrient metabolism . Moreovey adults . The cony adults ,80.−1) and Mg (up to 317 mg 100 g−1). Considering the RDI for a healthy adult for Ca (1300 mg per day) and Mg (400 mg per day), the intake of 100 g of açaí represents 36 and 76% of the RDI for Ca and Mg, respectively. Magnesium and calcium form stable complexes with phospholipids that are part of cell membranes. The action of these minerals, which can act synergistically, depends on its the concentration in the cells [−1), Mn (0.61–45 mg 100 g−1), I (0.28–46.60 mg 100 g−1), and P (6.90–186 mg 100 g−1). Moreover, the highest concentrations of these minerals were found in the same fruits with higher potassium levels. Schulz et al. [Myrcianthes pungens, Myrciaria cauliflora, and E. edulis. The consumption of 100 g of juçara and açaí provides more than 100% of the RDI for Mn (2.3 mg day−1) and iron (8 mg day−1). Furthermore, 100 g of buritirana can afford more than 100% of Mn, and 36% of I. Juçara, açaí, buritirana, and buriti are rich in Ca (up to 462 mg 100 ghe cells . Other m−1) and Cr (0.12 mg 100 g−1). The RDI for these minerals is 0.055 mg per day for Se and 0.03 mg per day for Cr. Chromium is linked to gene expression, energy production, synthesis of lipoproteins or lipids, and regulation of glucose metabolism [The buriti pulp showed a high level of Se .The linoleic acid, an essential PUFA, was found in açaí 48.05%), butiá (32.80%), and juçara (26.10%). The majority of the fruits present a balanced fatty acid composition, with high content in monounsaturated fatty acids (MUFAs) and saturated fatty acids (SFAs). In addition, the high levels of unsaturated fatty acids in its pulp make this raw material susceptible to oxidation reactions which may cause physical and sensory changes . The pat8.05%, buThe oils obtained from bacaba and patawa pulp have high concentrations of SFAs , mainly lauric and stearic acids. These oils can be used in the oleochemical industry and for the development of new lipid-based formulations with diverse industrial applications ,104. Cap−1), followed by juçara, açaí, and patawa . The α-tocopherol was the only tocopherol identified in açaí oil, which presented the highest concentration reported for this isomer (645.00 mg kg−1) [−1) and other refined oils such as soybean (352.00 mg kg−1), sunflower (575.00 mg kg 1), and corn (207.00 mg kg−1) [The highest concentration of tocopherols was observed for buriti (1688.58 mg kgmg kg−1) . This comg kg−1) ,117. Themg kg−1) .−1, respectively). The main phytosterol present was β-sitosterol. Data on the profile of the phytosterols for juçara, açaí, buritirana, and butiá were not found in the literature. It has been reported that the phytosterols can reduce the serum levels of fat-soluble vitamin E (α-tocopherols) and β-carotene, which has pro-vitamin A activity [The patawa oil showed the highest content of phytosterols, followed by bacaba and buriti , when compared to açaí and buriti. The most prevalent amino acids in patawa were leucine, threonine, isoleucine, lysine, and valine. These branched-chain amino acids are related to the protein synthesis, as they stabilize the protein structure through hydrophobic interactions [ractions . Data on−1 protein, respectively). Tryptophan, a precursor of serotonin which is involved in the modulation and regulation of anxiety and mood, is not usually found in foods [Buriti is rich in threonine, leucine, and tryptophan -catechin in bacaba, and cyn 3-O-rutinoside and (−)-epicatechin in patawa were the main compounds assessed. Açaí and juçara have been reported as superfruits, and compounds such as apigenin, cyn 3-O-rutinoside, rutin, myricetin, quercetin, kaempferol, and vanillic acid have been reported in their composition. On the other hand, protocatechuic acid, rutin, chlorogenic acid, (−)-epicatechin, and luteolin are the main compounds found in buriti. For butiá, the main compounds are chlorogenic acid, quercetin, and myricetin. The presence of these compounds in the fruits from the Arecaceae family are presented and discussed in the next sections.−1, respectively). Santos et al. (2015) reported for buriti pulp from the Amazon 118 ± 2 mg GAE 100 g−1 of TPC. On the other hand, Candido et al. (2015) reported that buriti pulp from Cerrado biome had higher values of phenolic compounds when compared to the fruit of the Amazon biome. Royo et al. [−1 in the leaves, roots, and petioles, respectively. It has been also reported that the fruit is rich in carotenoids such as trans-β-carotene (373.00 μg 100 g−1), all-trans-α-carotene (230.00 μg 100 g−1), trans-lutein (198.00 μg 100 g−1) and 9-cis-β-carotene (11.00 μg 100 g−1) [o et al.  reported100 g−1) .O-glucoside 100g−1, respectively)  [−1), (+)-catechin (961.21 μg g−1), (−)-epicatechin (1109.93 μg g−1), and luteolin (1060.90 μg g−1) [Açaí and juçara are rich in bioactive compounds such as cyanidin 3-glycoside and cyanidin 3-rutinoside, lutein, α-carotene, and β-carotene, which are associated with the prevention of inflammatory diseases and cancer ,157. A hctively) . This cactively) . The mai μg g−1)   ,160.−1 and 3.625 μg 100 g−1, respectively. Rosso and Mercadante [−1 for buriti pulp which represents a retinol activity equivalent (RAE) of 7280 RAE 100 g−1. Buriti presents a higher total carotenoid content and RAE when compared to fruits considered rich in this bioactive compound such as dendê (129.03 µg g−1/1535 μRAE 100 g−1), pupunha (197.66 µg g−1/1491 μRAE 100 g−1), physalis (80.89 µg g−1/1108 μRAE 100 g−1), tucumã (62.65 µg g−1/850 μRAE 100 g−1), mamey (62.53 µg g−1/ 688 μRAE 100 g−1), and marimari (37.98 µg g−1/ 605 μRAE 100 g−1) [−1 (300 μRAE 100 g 1) has been reported for açaí. This value is higher when compared to the concentrations reported for tropical fruits such as cajá (120 μRAE 100 g−1) [−1, respectively) [−1) [Buriti has been reported as the richest source of carotenoids found in Brazil ,161,162.rcadante  reported100 g−1) . A conce100 g−1) , mango cctively) , and acely) [−1) .−1) and buriti (226 μg g−1). The values of lutein in these fruits are higher than in vegetables considered rich in lutein, such as caruru (119 μg g−1), mentruz (111 μg g−1), and taioba (104 μg g−1). Moreover, the concentration of lutein in açaí is higher than the concentration reported in nasturtium (450 μg g−1), an edible flower that was considered the richest source of lutein reported in the literature [Lutein, a dehydroxylated carotenoid belonging to the class of yellow-colored xanthophylls, is predominant in açaí  [−1) and Juçara (186 mg 100 g−1) have the highest content of vitamin C. Butiá presents higher amount of vitamin C when compared with mangaba (190 mg 100 g−1), cajá (26.5 mg 100 g−1), murici (148 mg 100 g−1), jaboticaba (238 mg 100 g−1) [−1 of juice), and Christmas orange (84.03 mg AA 100 mL−1 of juice) [Vitamin C is the most important water-soluble antioxidant, acting on scavenging free radicals such as hydroxyl radical, superoxide, singlet oxygen, and hydrogen peroxide . Acerolactively) . Butiá (100 g−1) , pear orf juice) .−1), (−)-epicatechin (211.12 μg g−1), and rutin (161.20 μg g−1). The authors also reported lower concentration of trans-resveratrol, sinapic and ellagic acids, apigenin, and naringenin in the pulp of butiá. These bioactive compounds are related to anticarcinogenic, cytoprotective, antioxidant, cardioprotective, and neuroprotective activity [The in vitro antioxidant activity (AA) observed through 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity (DPPH) and oxygen radical absorption capacity (ORAC) has been reported for the pulp of butiá . The higactivity . The pheactivity ,172.−1) for extracts recovered using ethyl acetate as solvent. The AA for the extracts of buriti was higher when compared to the results reported for extracts of açaí and juçara for DPPH, FRAP, and ORAC . Considering the patawa, the authors indicated that patawa presented AA that was about 41 times higher using ABTS radical when compared to bacaba, juçara, and buriti. Considering that the ABTS radical is soluble in organic and aqueous solvents, this indicates that patawa has antioxidant components with different solubility. In addition, the absorption at a wavelength of 734 nm of the ABTS radical eliminates possible interference from color, resulting from unsatisfactory extraction processes [The AA using the ABTS assay was higher for patawa and açaí , followed by patawa (1869.90 μmol FeSO4 g−1 and 1626.70 μmol TE g−1) and juçara (1745.33 μmol FeSO4 g−1 and 1266.36 μmol TE g−1). The differences observed between the AA assays are related to the differences within the assays, such as the radicals, pH, temperature, time, solvents, and method of extraction [Buriti showed the highest antioxidant capacity in FRAP and ORAC assays , the induction of the lysis was not observed. The authors also report that the blood cells treated with the extracts  were protected when exposed to the peroxide radicals produced by the thermal decomposition of AAPH  dihydrochloride)) [The AA for the pulp, shell, and seed of buriti was evaluated using thiobarbituric acid reactive substances (TBARS) and the oxidative hemolysis inhibition, DPPH, ABTS, and FRAP assays. The shell of buriti showed significantly higher AA when compared to the seed and pulp. Moreover, the bioaccessibility of phenolic compounds for pulp, shell, and seed after simulated in vitro digestion decreased from 553, 1288, and 597 mg Lloride)) . Table 5−1) presented activity against Gram-negative bacteria such as Salmonella Typhimurium, Escherichia coli O157: H7, and Pseudomonas aeruginosa), with zones of inhibition ranging from 59.50 to 86.00 mm using the agar diffusion method.The antimicrobial potential of extracts obtained from butiá has been reported ,189,190.Listeria monocytogenes and Bacilus cereus were not inhibited at the highest concentrations tested (11 mg mL−1). Gram-positive bacteria are more sensitive to antimicrobial compounds from plant origin when compared to Gram-negative bacteria [On the other hand, bacteria . Such bebacteria ,189,193.Escherichia coli inoculated in sliced cheese with a minimal inhibitory concentration (MIC) value of 15 mg mL−1. The logarithmic decrease observed for the number of colony forming units (CFUs) of Escherichia coli in the samples treated with the extract after 72 h was eight times higher when compared to the control . The authors associated the antimicrobial activity of the extract with the main compounds such as Z-10-pentadecenol (80.1%) and palmitic acid (19.4%), identified in the butiá extract [The extracts of butiá obtained with acetone showed activity against  extract .Butiá odorata showed antimicrobial and antibiofilm activity against 26 serovars of Salmonella spp. isolated from food and environment where food was prepared. The MIC of Butiá odorata extract ranged from 10 to 19 mg mL−1. This inhibition may be correlated with the presence of bioactive compounds in the extract such as phenolics, phenols, and flavonoids. The main compounds identified in the extracts were the 5-(hydroxymethyl)-2-furfural (65.17%), followed by the pyranone, 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (8.49%). Polovková and Šimko [Salmonella spp. and Gram-negative and Gram-positive bacteria has been reported [Haubert et al.  reportednd Šimko  reportedreported .Klebsiella pneumonia, Pseudomonas aeruginosa, and Staphylococcus aureus has been reported (Pseudomonas aeruginosa (59%), Klebsiella pneumonia (62%), and Staphylococcus aureus (43%) when compared to the control group without treatment. The inhibition of the bacterial growth was related to the particle size and the phytochemicals, such as quercetin, eugenol, and vanillic acid, present in the oil. Leão et al. [The antimicrobial activity of nonencapsulated buriti oil against reported . The nono et al.  reportedThe inflammatory process is a complex immune response of the organism to heal infections or repair damaged tissue. However, inflammation can produce an uncontrolled response or can be related to the disruption of the homeostasis state of physiological processes. It can lead to chronic systemic damage and inflammatory diseases such as diabetes, asthma, Alzheimer’s, atherosclerosis, cancer, neurodegenerative, and neurological diseases . The freSeveral plants have been used in folk medicine as an alternative to the treatment of chronic inflammation with fewer side effects and low toxicity. Nonsteroidal anti-inflammatories (NSAIDs), which are the traditional anti-inflammatory drugs used, can cause adverse effects such as a decrease in COX-prostaglandin production, gastrointestinal disorders, kidney problems, and severe peptic ulcer. A positive correlation has been reported between ingestion of foods rich in phenolic compounds and a negative modulation of the inflammatory response ,199. TheSilva et al.  studied Xie et al.  evaluate−1) obtained from açaí was investigated using 3T3-L1 adipocytes [−1). The bacaba phenolic extract showed the same behavior, demonstrating that BPE attenuates adipogenesis through downregulation of PPARγ2 and C/EBPα during differentiation’s early to middle stages. This, in turn, decreases the induction of metabolic genes associated with the adipocyte phenotype, such as FABP-4 and adiponectin [The anti-inflammatory and antilipidemic activity of extracts rich in polyphenols , in partnership with the International Agency for Research on Cancer (IARC), reported an increase in cancer from 18.1 million of cases in 2018 to 19 million in 2020, with 10 million deaths. It is expected that there will be 28.4 million new cases of cancer worldwide until the year 2040, which represents an increase of approximately 47% compared to 2020. In countries where the human development index (HDI) is considered low or medium, this expectation of cancer incidence for 2040 is estimated to increase by 96% when compared to 2020 .Poor eating habit is considered the most important factor in the incidence of cancer and other noncommunicable diseases (NCDs) such as neurological, inflammatory, cardiovascular, and endocrine diseases . Epidemi−1. Furthermore, a cell shrinkage and reduction of the cell monolayer area was observed in the MCF-7 breast cancer cells with the treatment at a concentration of 400 μg mL−1. The results indicated that the extracts obtained from bacaba induced apoptosis in MCF-7 cells. The authors suggested that bacaba extracts present chemopreventive potential and correlated such effects with dietary phenolics that are important preventive agents in cancer diseases.Finco et al.  evaluateFuentes et al.  evaluatev/v) obtained from the pulp and peel of butiá against cell lines strains of human intestinal cancer (Caco-2), cervical cancer lineage (HeLa), and human papillomavirus cells (SiHa and C33a). The ethanolic extract presented higher activity against the SiHa and C33a strains with a 50% inhibitory concentration (IC50) of 528 and 411 μg mL−1, respectively. On the other hand, the butiá extract did not reach half of the maximum inhibitory concentration (IC50) for the cell lines strains Caco-2 and HeLa. The treatment with the butiá extract did not affect the cell viability of murine fibroblasts and human keratinocytes. The chemopreventive activity was different between the cancer cell line strains. The variation in the chemopreventive activity among the cancer cells tested indicated different mechanisms of action for the ethanolic extract of butiá pulp. The authors indicated that (+)-catechin (259.00 mg kg−1), (−)-epicatechin (211.00 mg kg−1), and rutin (161 mg kg−1), present in the extract, were the compounds with bioactivity. The cytoprotective, antioxidant, cardioprotective, neuroprotective anticarcinogenic, and chemopreventive activities for these compounds are widely reported in the literature [Boeing et al.  evaluateterature ,222,223.Medeiros et al.  investigp-coumaric, and chlorogenic), flavonoids ((+) catechin, (−)-epicatechin, rutin, kaempferol), phytosterols, tocopherols, and carotenoids. These compounds are responsible for the antioxidant activity and potential health benefits such as antimicrobial, chemopreventive, cardioprotective, anti-inflammatory, and antiobesity effects. The Arecaceae palm tree fruits have potential for use in food, pharmaceutical, biotechnology, and cosmetic industries. Despite their rich nutritional and bioactive compounds composition, butiá (Butia odorata) and buritirana (Mauritiella armata) need more attention, considering the limited studies that have been reported in the literature. This review demonstrates the importance of valorizing underexploited Brazilian native fruits whose products and coproducts are rich in phytochemicals with potential benefits for human health. It also highlights the need for further research on the composition, health effects, processing, and full use of these raw materials. Moreover, the mechanisms and the synergism between the bioactive compounds of these fruits, and their effects, especially in human models, need to be further studied. In addition, government public policies and partnerships with local producers may improve the socioeconomic situation of extractivist populations, and can also encourage the consumption and processing of these fruits, ensuring the preservation of natural resources. This review can contribute to the dissemination of such products whose consumption and applications may help to promote a healthy diet.Arecaceae palm tree fruits have high nutritional value and are rich in bioactive compounds such as phenolic acids (gallic, vanillic,


These newly synthesized surfactants were screened for blood hemolysis assay and cell toxicity studies using the NIH/3T3 cell line to assess their biocompatibility. Then amphotericin B was loaded into niosomal vesicles, and the drug entrapment efficiency of these surfactants was measured using UV–visible spectroscopy. The morphology of drug-loaded niosomes of synthesized surfactants was investigated using AFM, and their size, polydispersity, and zeta potential were measured with the Zetasizer instrument. Finally, a simulation study was performed to determine the pattern of self-assembly of the synthesized amphiphiles. Both synthesized nonionic surfactants showed good entrapment efficiency of 60.65 ± 2.12% and 68.45 ± 2.12%, respectively. It was also confirmed that both these synthesized nonionic surfactants were safe and biocompatible and showed less blood hemolysis (i.e. 21.13 ± 2.11% and 23.32 ± 2.45%) and higher 3T3 cells’ viability at 150 µg/mL concentration as compared to Tween®-80. The antifungal potential of amphotericin B-loaded niosomes has been evaluated against unicellular multi-fungal species, which showed a promising potential for fungicidal activity. These results are substantiated by constructing a safe vehicle system for drug delivery.Most of the drugs are hydrophobic and have low water solubility, therefore posing issues in their absorption and bioavailability. Nonionic surfactants improve the solubility of hydrophobic drugs by entrapping them in their lipid bilayers. Two nonionic surfactants NODNH-16 and NODNH-18 are synthesized and characterized using different techniques i.e. EI-MS,  Cholesterol and Tween®-80 were acquired from BDH, UK, and Merck, Germany through local suppliers. Sabouraud dextrose agar and Capek’s dox agar , standard fungicidal drugs including miconazole , nystatin , and amphotericin B (Am-B)  were acquired from their vendors.In this research work, HPLC-grade solvents were used. 4 Hydroxybenzaldehyde, nicotinic hydrazide, Dulbecco’s modified Eagle’s medium (DMEM), K2.2.2CO3 and refluxed for 12 h. When the reaction was completed, it was cooled to ambient temperature, concentrated, and products were purified through column chromatography. Then, in a round-bottom flask (50 mL), 70 mg of nicotine hydrazide  and 187.2 mg of 4-(octadecyloxy) benzaldehyde (0.5 mmol) were added in 20 mL ethanol and stirred for 20 min at 80°C then added a few drops of anhydrous acetic acid and the reaction was refluxed for 3 h at the same temperature. The reaction was stopped, cooled down to ambient temperature, and filtered. The white powder of desired product (NODNH-18) was obtained with a 90% yield. In another reaction, the nonionic surfactant NODNH-16 was synthesized by following the same procedure. The white powder of desired product (NODNH-16) was obtained with an 84% yield, as shown in In the initial reaction, 122 mg (1.0 mmol) of 4-hydroxy benzaldehyde was reacted with 499.9 mg  of 1-bromohexadecane in 10 mL acetone in the presence of 345.5 mg  of KYield: for NODNH-16, 84%, m.p. 117.2–119.5 °C and for NODNH-18, yield: 90%, m.p. 118.5–120.8 °C.2.3.λmax was plotted against the respective concentration of nonionic surfactants. Lines were drawn to connect respective concentrations against their absorption and CMC was assigned to the point where discontinuity occurs in the graph’s straight line  of synthesized nonionic surfactants was determined using a UV–visible spectrophotometer . Solutions of various concentrations of the synthesized nonionic surfactants were prepared in ethanol ranging from 0.050 mM to 0.001 mM and then scanned in the UV–visible region (200 to 800 nm) by spectrophotometer. Absorbance at 2.4.2.4.1.The drug-loaded vesicles of synthesized nonionic surfactants NODNH-18 and NODNH-16 were prepared through the hydration of the surfactant thin film method  as reported  was taken in an Eppendorf tube and centrifuged for 15 min at 12,000 rpm using a centrifuge . The drug-loaded vesicles settled to the bottom of the Eppendorf and formed a solid pellet. The procedure was repeated three times to completely remove all of the free drugs from the supernatant  was taken in a dialysis membrane . The membrane was then dipped in a beaker having 50 mL buffer solution (PBS) of respective pH followed by shaking at 50 rpm/min at 37 °C. Then at specific times, aliquots of 2 mL were withdrawn and refilled with fresh buffer solution to prevent drug saturation in the media. The amount of drug release in each dissolution media was determined spectrophotometrically at 405 nm.2.4.5.−1. Samples were prepared and analyzed with the KBr-disk method where KBr powder was used for the preparation of disks.Drug–excipients interactions in the formulation were assessed using FTIR spectroscopy . IR spectra of dried vesicles containing drugs and individual components of the vesicle, i.e. drug, cholesterol, and surfactants NODNH-18 and NODNH-16 were recorded from 400 to 4000 cm2.4.6.3 cubic simulation box and subsequently solvated by the addition of discrete numbers of water molecules. All MD simulations were started with the random distribution of the surfactant molecules in the cubic periodic box which was produced using Gromacs utility ‘genbox’. Energy minimization was done for each molecule as a first step employing the steepest descent algorithm till the maximum force of the system came to <0.001 N/mol. After energy minimization, a constant number of particles (N), volume (V), and temperature (T) and the NPT ensemble  were sequentially carried out for 100 picoseconds each to equilibrate the system at 1 bar pressure and 298 K temperature. Micelles were formed at 30 ns and the period of the last 5 ns of this run was used for analysis. The inspection and visualization of trajectories and arrangement of amphiphiles in bulk water were performed with visual molecular dynamics VMD 1.9.1  simulation was used. All MD simulations were performed with GROMACS 2016.4  and centrifuged in similar conditions. RBCs were dispersed in PBS in a 1:10 ratio  and 4 mL of different concentrations of synthesized surfactants (62.5–1000 mg/mL) and 200 µL of RBCs were mixed. For reference standards, the commercially available Tween®-80 was screened. Samples were incubated at 37 °C for 4 h, then centrifuged to separate hemolyzed blood in the form of supernatant and read at 540 nm to determine free hemoglobin spectrophotometrically. The RBCs suspension was similarly mixed with water and PBS to obtain the 100% and 0% hemoglobin release. The following equation was then used to determine the % hemolysis:The newly synthesized nonionic surfactants NODNH-18 and NODNH-16 were screened for hemolysis assay against human blood. The fresh human blood for this study was obtained from the Center for Bioequivalence Studies and Clinical Research (CBSCR), University of Karachi. To separate red cells from the blood, it was centrifuged for 10 min at 700 Abt, Ab0, and Ab100 indicate the absorbance of the test sample, PBS, and water, respectively  cells were rooted in 96-well plates containing DMEM (Dulbecco’s modified Eagle medium) supplemented with penicillin and streptomycin antibiotics (50 IU/mL each). Cells were incubated in cultured media for 24 h at 37 °C in a 5% CO2 humidified environment. After 24 h, cells were exposed to the same volume of fresh media containing synthesized surfactants NODNH-16 or NODNH-18 in 62.5–1000 μg/mL concentrations. Cells viability was assessed via the well-known 3-c-2,5-diphenyltetrazolium bromide  assay. In short, 20 µL of MTT solution (0.5 mg/mL) was added to each well and incubated for 2 h in the same environmental condition as described for cell culturing. After incubation, the supernatant was removed from each well and 50 µL DMSO was added to each well. The formazan amount formed was determined using microplate readers  at 670 nm. Untreated cells were taken as 100% viable and Tween®-80 was used as the reference standard. Percent cell viability for each sample was calculated using the following formula:To assess the cell line toxicity potential of synthesized nonionic surfactants, NIH/3T3   at 28 °C for 48 h. Multi-cellular and dimorphic plant-associated fungal isolates i.e. Trichoderma longibrachiatum(MW564026), Purpureocillium lilacinum (MW566158), Aspergillus terreus (MW570851), Aspergillus flavus, Paecilomyces variotii, and Fusarium oxysporum, and two yeast species Meyerozyma guilliermondi (MW564205) and Saccharomyces cerevisiae were grown onto sabouraud dextrose and czapek’s dox agar  for 2–7 days at room temperature , and S. cerevisiae grown in Sabouraud dextrose broth  and cells were harvested after centrifugation at 9000 g. Cell suspension equivalent to 0.5 McFarland standard (1.5 × 108 CFU/mL), was prepared in 10 mL sterile phosphate buffer saline solution (PBS). Confluent lawns were prepared onto SDA plates and allowed to dry for 5–10 min. Standard fungicide at a final concentration of 15 µg and the vesicle formulations NODNH-16 and NODNH-18, comprising 60.89% (i.e. 9.13 µg) and 68.63% (i.e. 10.29 µg) encapsulated amphotericin B respectively, was dispensed into the wells (6 mm), bored with help of a sterile cork-borer, and plates were incubated at 28 °C for 24–48 h  was prepared in PBS from five days old cultures. Spores were inoculated with the help of a sterile swab on SDA and incubated at 25 °C for five to seven days. The antifungal activities were performed in triplicates. The results were interpreted and compared with the zones of inhibition in mm(±SD)produced by carrier vesicles and standard drugs as per CLSI (2012) guidelines , nystatin , and amphotericin B  were reconstituted at a concentration of 1500 µg/mL for the assay  was calculated by the following formula.AFI = Zone of inhibition of amphotericin B loaded liposomal vesicles/Zone of inhibition of amphotericin B. AFI was calculated in replicate experiments.AFI values >1 indicate the niosomal vesicles are more active against the tested fungal species than the standard antifungal used.2.7.p values were considered statistically significant.All experiments were performed in triplicate and results were expressed as mean ± SEM. One-way analysis of variance (ANOVA) was used for statistical analysis using Graph Pad Prism software (Version 5). Less than or equal to .05 3.3.1.The CMC of an amphiphilic molecule is the concentration above which the individual monomers of the surfactant or amphiphile start to assemble in micelles thus playing a vital role in the formation of micelles. A balanced hydrophobic and hydrophilic segment of amphiphilic molecules ensures the core/shell structured micelles formation in an aqueous medium. The aggregation behavior of nonionic surfactants is a function of the molecular weight of hydrophilic and hydrophobic segments Griffin, . The det3.2.3.2.1.The morphology of drug-loaded vesicles of newly synthesized nonionic surfactants NODNH-16 and NODNH-18 was assessed using atomic force microscopy (AFM). The drug-loaded vesicles of synthesized surfactants were found spherical as shown in 3.2.2.For proper characterization of a nano-drug delivery system, its capability of entrapping drug amounts is very important. The amphotericin-B entrapment efficiency by NODNH-16 and NODNH-18-based vesicles was determined following the separation of the un-entrapped drug by cold centrifugation. The entrapped drug in the sedimented vesicular pellet was determined by dissolving it in methanol and subsequent reading on UV-spectroscopy at a maximum wavelength of 405 nm. The amount of drug entrapped in newly synthesized surfactant NODNH-18 was 68.63 ± 3.43% and similarly, the drug entrapment efficiency of NODNH-16-based vesicles was 60.89 ± 2.88% . Among o3.2.3.The drug release pattern of drug-loaded vesicular formulation of NODNH-16 and NODNH-18 was invested at three different pH . The drug-loaded vesicular formulation of NODNH-18 showed the slow-release behavior of the drug. It was noted that the highest amount (i.e. 38.67 ± 0.6%) of the drug (amphotericin B) released at pH 1.2 was after 12 h of the study whereas the maximum drug released at pH 4.2 was observed as 42.25 ± 0.5% after 12 h. At pH 7.0, the maximum amount of drug release was found to be 34.41 ± 0.6 at the 11th hour of the study as shown in 3.2.4.−1 for OH, at 2926 cm−1 for NH2, at 1712 cm−1 for CH2, at 1626 cm−1 for carbonyl group, at 1550 cm−1 for alkene and the bands at 1249.4 cm−1 represent C–O stretch in the ester group. The absorption peak at 3416 cm−1 and 1669.7 cm−1 indicates the characteristic peaks for -OH and CH = CH groups respectively in the cholesterol. The FTIR spectra of the drug carrier (compound) show a peak at 3268 cm−1 which is responsible for H-N, at 2918.6 cm−1 for CH3, at 2850.4 cm−1 for CH2, 1651 cm−1 for carbonyl group of amides, at 1608.0 cm−1 for H-N bending vibrations as shown in −1 in the FTIR spectrum of formulation indicating that the characteristic peak of OH of the pure drug was slightly shifted toward a lower frequency, the peak of the H-N amine group appeared at 3261.0 cm−1 which indicated the frequency shifted slightly toward high frequency, and the CH3 peak at 2918 cm−1, CH2 at 2850 cm−1, carbonyl group of amide at 1651 cm−1, H-N at 1295 cm−1 and C-N at 1247 cm−1 appeared nearly in the same region at 2920.1 cm−1, 2851.6 cm−1, 1648.2 cm−1, 1300.1 cm−1, and 1249.4 cm−1 respectively. The main characteristic peaks of the pure drug were present in the formulation spectrum indicating the presence of the drug in an intact form in the formulation. A small change i.e. higher or lower frequency shift was noted which indicated the physical interaction of the drug and carrier , cholesterol, synthesized surfactant carriers, and a dried vesicular formulation containing all ingredients were performed. The FTIR of the drug show their characteristic peak at 3387 cm3.2.5.Numerous studies and experimental techniques have been performed to elucidate and characterize vesicles’ structures, their dynamic behavior, and thermodynamic properties as they are gaining considerable applications in diverse technological and fundamental fields  followed by C. albicans ATCC 10231 21 (±0.87), C. galeberata ATCC 20001 19 (±0.92), M. guilliermondi 16.7 (±0.51), and T. longibrachi 17 (±1.2). However, NODNH-18 revealed an increase in ZOI against the S. cerevisiae 30 (±0.81) followed by C. galeberata ATCC 20001 22(±0.31), T. longibrachi 20 (±1.1), C. albicans ATCC 10231 18(±0.2), A. terreus 18.1(±0.79), and P. lilacinum 18 (±0.08), respectively (variotii . The higectively . Furtherectively . It is nectively .C. albicans ATCC 10231, C. galeberata ATCC 20001, and M. guilliermondi (-resistant C. galeberata ATCC 20001 as compared to NODNH-16. Furthermore, the AFI for the NODNH-16 and NODNH-18 also compliments the fungicidal activities against multi-cellular fungal species, presented in T. longibrachiatum, P. lilacinum, and A. Terreus (AFI of the carrier vehicle NODNH-16 exhibits an enhanced efficacy of amphotericin B against human pathogens, i.e.  Terreus .4.The nonionic surfactants have got the great attention of pharmaceutical scientists in recent years due to their greater biocompatible nature. Nonionic surfactants form closed bilayer vesicles or micellar structures upon their contact with an aqueous environment. Due to their remarkable capacity and properties such as biocompatibility, cost-effectiveness, and stability, these vesicles are regarded as excellent carrier for the delivery of drugs. Two newly synthesized nicotinic hydrazine-based nonionic surfactants have been screened out here for CMC, biocompatibility, and their potential for drug encapsulation and release was evaluated. Both the synthesized nonionic surfactants NODNH-16 and NODNH-18 have a great ability to entrap the greater amount of model hydrophobic drug amphotericin B. Drug-loaded vesicles of the synthesized surfactants were of small size with less polydispersity index value. From the results, it can be concluded that both newly synthesized nonionic surfactants are promising candidates to be used for vesicular drug delivery of less water-soluble drugs like Amphotericin B and showed a significant potential for fungicidal activity.


Echinococcus granulosus (E. granulosus). This study aimed to understand the clinical characteristics of human CE in Ningxia Hui Autonomous Region (NHAR) located in northwest China and to investigate the antibody profiles against the recombinant E. granulosus antigen P29 (rEg.P29) in plasma of CE patients.Cystic echinococcosis (CE) is a neglected parasitic zoonotic disease caused by the larval stage of the tapeworm Eg.P29 and hydatid cyst fluid (HCF) were assessed by indirect ELISA.A total of 37 human CE patients, along with 37 healthy donors enrolled in this study and demographic and clinical data were analyzed, including age, gender, laboratory data, symptoms, and cysts description. Plasma levels of cytokines, total IgG, and total IgE were determined by sandwich ELISA kits. Specific antibodies against rEg.P29 in CE patients was significantly higher than in healthy donors, and the dominant IgG subclass was IgG4. Further analysis of different patient groups revealed that rEg.P29-specific IgG and IgG4 were only elevated in CE patients with CE2 type cysts.The results revealed that females have a higher percentage of CE patients than males. The incidence of CE reached a peak in the 41–50 years-old group. The liver was the most frequent location, accounting for 91.9%. Based on the CT images, cysts of 34 patients who had liver involvement, were classified as 1 (2.9%) CE1, 12 (35.3%) CE2, 5 (14.7%) CE3a, 1 (2.9%) CE3b, and 15 (44.2%) CE5. Twenty-nine (78.4%) patients had a single cyst and 8 (21.6%) had at least two cysts. The most frequently reported symptom was upper abdominal pain. The plasma level of IL-6 and total IgE were significantly increased in CE patients compared with healthy donors. Additionally, IgG response to rEg.P29 can be used as an assisted method for imaging techniques to identify cystic activity and determine the best therapeutic approach for CE.This study systematically investigated the clinical characteristics of patients with CE and may provide a reference basis for the diagnosis and treatment of CE in NHAR. Furthermore, tests of specific IgG and IgG4 against r E. granulosus, and it is one of the 17 neglected tropical diseases recognized by the World Health Organization (WHO) [E. granulosus completes its life cycle in two hosts, a definitive carnivore host and an intermediate herbivore host. Human is an accidental intermediate host and becomes infected by ingesting food or water contaminated with the parasite’s eggs. After ingestion, the eggs hatch in the small intestine and become activated six-hooked oncospheres which penetrate through the mucosa and migrate through the circulatory system into internal organs where the oncospheres develop into hydatid cysts [Cystic echinococcosis (CE), also known as hydatid disease, is a complex disease in humans caused by the larval stage of the tapeworm id cysts , 3. The id cysts . CE is eid cysts . The estid cysts . The morid cysts .E. granulosus is the coexistence of both cellular and humoral responses. However, definite immune responses in CE patients are still complex and controversial. It is generally accepted that patients with CE have a mixed Th1/Th2 response. The Th1-dominated immunity in CE is related to protective immunity and well-response to chemotherapy. However, Th2 is associated with susceptibility to the disease and no response to chemotherapy [Based on the ultrasonographic features, the hydatid cysts are differentiated into five types using the WHO-IWGE standard: CE1, CE2, CE3 , CE4, and CE5. The hydatid cyst type is used to assess their stage of development. Type CE1 and CE2 cysts are considered active cysts and usually fertile with viable protoscoleces; type CE3a and CE3b cysts are in a transitional stage; type CE4 and CE5 cysts are inactive and degenerative cysts . The lonotherapy , 11.E. granulosus P29 protein is absent in hydatid cyst fluid, but it is restricted to the metacestode stage of the parasite and the germinal layer of the cyst [E. granulosus P29 protein in humans. Two studies reported that P29 protein is a potential serological marker for the follow-up of human CE, especially in young patients [E. granulosus secondary infection in mice [Several antigenic proteins in the hydatid cyst fluid have been reported to play key roles in immunoregulation mechanisms in patients with CE. the cyst . There apatients , 14. Our in mice . The P29 in mice . HoweverE. granulosus P29 protein in human CE patients. All assays were also performed using hydatid cyst fluid antigen (HCF), and the results were compared.CE is highly endemic in the Ningxia Hui Autonomous Region (NHAR), located in northwest China . In the CE patients participating in this study were enrolled in the two largest hospitals in the Ningxia Hui Autonomous Region (NHAR) in China from May 2020 to March 2022. The demographic and clinical data of the patients, including age, sex, length of hospitalization, medical history of hydatidosis, laboratory data, symptoms, and cyst description were collected. The CE patients involved in this study were subjected to surgical operations and confirmed by histopathology. A hydatid cyst was shown in Fig. Ten milliliters venous blood was drawn into heparin-anticoagulated tubes and plasma was obtained after centrifugation at 2500 rpm for 5 min at room temperature. Plasma samples were stored at – 80 ℃ until further analysis.E. granulosus IgG antibody detection according to the manufacturer’s instructions. Sample Index (SI) was calculated and interpreted for each plasma according to the manufacture’s instructions; ELISA was considered positive for SI > 1.1, negative for SI < 0.9, and equivocal for 0.9 < SI < 1.1.The serological test was performed using a commercial enzyme-linked immunosorbent assay (ELISA) kit  for Cytokines including IFN-γ, IL-2, IL-4, TNF-α, IL-10, and IL-6 were determined by sandwich ELISA kits  according to the manufacturer’s instructions. The optical density (OD) values were transformed into concentrations (pg/ml) according to the cytokine-specific standard curves. The lower limits of cytokine detection are 4.7 pg/ml for IFN-γ and IL-6, and 7.8 pg/ml for TNF-α, IL-2, IL-4, and IL-10. Total IgG from plasma samples was assessed using a commercial enzyme-linked immunosorbent assay (ELISA) kit . Total IgE in human plasma was determined using the IgE Human Uncoated ELISA kit , and calculated as absolute concentrations according to the standard curve.E. coli BL21 pLysS in our laboratory before [g at 4 ℃ for 30 min. The supernatant was used as HCF crude antigen. Protein concentration was measured with a BCA Protein Assay Kit  and stored at − 80℃.Recombinant plasmid P29/pET28a was constructed and converted into y before . HydatidEg.P29 IgG, IgM, IgA, and IgE in plasma samples of patients with CE and healthy donors were assessed by indirect ELISA. Briefly, ninety-six-well polystyrene plates  were coated with 100 μl/well of rEg.P29 antigen at 10 μg/ml and HCF at 0.6 μg/ml in coating buffer  overnight at 4 ℃. Following five washes with PBS containing 0.05% (v/v) Tween-20 (PBS-T), 200 μl of the blocking buffer  were added in each well, and plates were incubated at 37 ℃ for 2 h. After washing the plates five times with PBST, 100 μl of plasma samples were added at 1:500 dilution (1:100 for IgE testing) in blocking buffer and incubated at 37 ℃ for 1 h. Following five times washes, plates were incubated with 100 μl of peroxidase-conjugated goat anti-human IgG (dilution 1:5000 in blocking buffer), goat anti-human IgM (dilution 1:8000 in blocking buffer), goat anti-human IgA (dilution 1:2000 in blocking buffer), goat anti-human IgE (dilution 1:1000 in blocking buffer) and incubated at 37 ℃for 1 h. Subsequently, 100 μl of TMB substrate buffer  was added to each well and incubated at 37 ℃ for 10 min in the dark. Finally, the reaction was terminated with 50 μl stop buffer , and the absorbance of each well was read at 450 nm and referenced to 630 nm by a microplate reader. All samples were tested in triplicate. HRP conjugated antibodies against IgG, IgM, IgA, and IgE were acquired in abcam, USA.Specific anti-rEg.P29 and HCF coated plates were incubated with 100 μl of 1:100 diluted samples in blocking buffer. Horseradish peroxidase (HRP) labeled anti-human IgG1, IgG2, IgG3, and IgG4 conjugates  in blocking buffer were added to each well. HRP conjugated antibodies against IgG subclasses were acquired in Invitrogen, USA.To determine IgG subclasses, rp-value < 0.05 was considered to be statistically significant.Category variables were expressed as absolute frequencies and percentages, whereas continuous parameters were expressed as mean and standard deviations. A Mann–Whitney U test was used to compare the differences between the two groups. A Kruskal–Wallis H rank-sum test was applied to compare continuous variables for multiple groups. Statistical analyses were performed using GraphPad Prism software version 8 ; a two-tailed Demographical information of CE patients and healthy donors are shown in Table In 30 (81.1%) CE patients, the disease was limited to the liver, and the right lobe was the most frequent location. Based on the computed tomography (CT) results of 34 patients with liver echinococcosis, the cysts were classified into different types, and Fig. Among all 37 patients, 8 (21.6%) were asymptomatic; among them, 3 and 5 patients found cysts during postoperative annual ultrasound examination and physical examination, respectively. For symptomatic patients, the most frequently reported symptom was upper abdominal pain, which was observed in 24 (64.9%) patients. Other symptoms were as follows: nausea, vomiting in 8 patients, lower back pain in 2, loss of weight in 3, and respiratory symptoms in 1.The serology test was scored positive in 27 patients, equivocal in 3 patients, and negative in 7 patients, respectively. All healthy donors are negative for the serological test. Laboratory hematological analysis showed 5 patients had an eosinophil increase, 13 patients had a monocyte increase, 3 patients had erythropenia, and 5 cases had hemoglobinia. Decreased levels of TP, ALB, and A/G ratio were observed in 13, 17, and 11 patients, respectively. An increase in alanine aminotransferase (ALT) level in 7 patients, aspartate transaminase (AST) in 8 patients, γ-glutamyl transferase (GGT) in 7 patients. Notably, AST/ALT ratio was increased in 16 patients.p < 0.01). TNF-α exceeded the first standard of the ELISA in only 5 patients and 8 healthy donors. However, there was no significant difference in plasma levels of TNF-α between the CE (51.12 ± 182.6 pg/ml) and HD (34.85 ± 93.26 pg/ml) groups. Levels of IFN-γ, IL-2, and IL-4 in all enrolled subjects were very low and below detection limits. Similarly, the concentration of IL-10 was only detected in one patient. No significant difference in total IgG levels was found between the CE (0.94 ± 0.62 g/L) and HD (0.96 ± 0.45 g/L) groups , while no statistically significant differences were observed in IgM, IgA, and IgE , but there was no significant difference in IgM and IgE   and CE5 (p < 0.05) groups than in the HD group. The levels of IgA against HCF were significantly higher in all CE2 (p < 0.001), CE3 (p < 0.01), and CE5 (p < 0.01) groups than in the HD group  Fig. A. Anti-HEg.P29 and HCF, IgG subclasses were tested in CE patients and healthy donors. The specific IgG4 subclass against rEg.P29 was significantly higher in the plasma of CE patients in comparison to HDs (p < 0.001), while no differences were observed between the two groups in IgG1, IgG2, and IgG3 . HCF-specific IgG2 subclass was barely detectable in the two groups. Furthermore, we compare the specific IgG subclasses to rEg.P29 and HCF in different patient groups. The results showed that IgG4 against rEg.P29 was only significantly higher in the CE2 group than in the HD group (p < 0.001), but there was no significant difference between other patient groups and the HD group , CE3 (p < 0.05), and CE5 (p < 0.05) groups showed significantly higher IgG4 reactivity to HCF compared with the HD group , the remaining patients with at least 2 cysts. Similarly, in another study in China, 75.96% and 24.04% of hepatic CE patients had a single cyst and multiple cysts [In our study, females with CE had a higher occurrence than males. Many previous studies reported similar results , 27, 28.le cysts ; in Mongle cysts ; in Tunile cysts . This inle cysts , 39. PatIn the analysis of symptoms of CE patients in the present study, upper abdominal pain was the most common symptom in 64.9% of the patients, which is consistent with findings of previous studies . Other sE. granulosus infections may lead to liver damage and inflammation, we further assessed the alteration of the routine blood tests, liver function tests, cytokines, and total antibodies in the plasma sample of CE patients. In the current study, elevation in eosinophil was present in 13.5% of cases, which was also consistent with a study conducted by Ebrahimipour et al. [Since r et al. . We alsoE. granulosus cysts, with different antibody isotypes . However, immune regulation mechanisms and the strategies of immune evasion in humans are still not clear [E. granulosus P29 protein is a 29 kDa antigen that is absent in hydatid cyst fluid but localized to the protoscoleces and the germinal layer of the cyst [E. granulosus P29 protein in humans is quite limited.Due to the CE cysts and host cohabiting for a long time, the immune response between the parasite and the host is complex and contradictory. Previous studies revealed that most patients develop a significant antibody response to ot clear . E. granthe cyst . To dateEg.P29 was significantly higher in CE patients than in healthy donors, and the dominant IgG subclass is IgG4. Consistent with our findings, other studies reported that the IgG4 antibodies may play a prominent role in the responses to E. granulosus infection. Antigens-specific IgG4 may serve as a link between AgB and Th2 cell activation and could be used for serological diagnosis of CE [Eg.P29 elicits a Th2 cell-mediated immune response to induce IgG4 production. To date, an accepted view is that antibody responses in CE patients are correlated with the clinical stage and cysts activity. When the patients were divided into different groups based on the cyst types, we found levels of IgG and IgG4 against rEg.P29 were significantly higher only in patients with CE2 type cysts compared with healthy donors. The results in our study were comparable to the previous understanding that IgG4 and IgG1 were mainly increased in patients with active and transitional cysts [Eg.P29-specific antibodies in the plasma of patients with CE3 and CE5 types were not statistically significant compared with healthy controls. This may be due to the high activity of the CE2 type cyst, which can secrete a large amount of antigenic material and induce a strong immune response, while the immunity of the body is weakened by the gradual decrease in the activity of the cysts from CE3 to CE5. Meanwhile, we assessed the antibody responses to HCF in CE patients. We found IgG and IgA against HCF were higher in CE patients, and the dominant IgG subclasses were IgG1, IgG3, and IgG4. Unlike our results, Aceti et al. reported a predominance of IgG1, IgG2, and IgG3 antibodies among sera from hydatid patients [In the present study, the level of IgG against ris of CE , 46. It al cysts , 48. Thepatients . These dEg.P29 was increased in the CE2 group compared with the HD group, but there were no significant differences of the CE3 and CE5 groups with the HD group. These findings suggest that tests based on IgG and IgG4 against rEg.P29 together with imaging techniques, can be used for the identification of cystic activity, leading to the best therapeutic approach for CE. This study was conducted using a limited number of patients with unequal numbers of patients in different stages. Larger and further research is required to confirm our results.To our best understanding, this is one of few systematic study to investigate the clinical characteristics of patients with CE in NHAR. These results may provide a reference basis for the diagnosis and treatment of CE in the region. Our further study found that specific IgG and IgG4 against r


Enterococcus durans and Enterococcus faecalis had the highest results in removing acrylamide, with 33 and 30% removal, respectively. Those two LAB were further examined for their binding abilities under optimized conditions of pH (4.5–6.5), temperature (32°C - 42°C), time (14–22 h), and NaCl (0–3% w/v) using BBD. pH was the main factor influenced the acrylamide removal compared to other factors. E. durans and E. faecalis exhibited acrylamide removal of 44 and 53%, respectively, after the in vitro digestion. Zeta potential results indicated that the changes in the charges were not the main cause of acrylamide removal. Transmission electron microscopes (TEM) results indicated that the cell walls of the bacteria increased when cultured in media supplemented with acrylamide.Acrylamide is an unsaturated amide that forms in heated, starchy food products. This study was conducted to (1) examine the ability of 38 LAB to remove acrylamide; (2) optimize acrylamide removal of selected LAB under various conditions  using the Box–Behnken design (BBD); (3) the behavior of the selected LAB under the simulated gastrointestinal conditions; and (4) investigate the mechanism of adsorption. Out of the 38 LAB,  In 2002, the Swedish National Food Authority and Stockholm University announced that acrylamide could form in food heated above 120°C  were also tested for their ability to bind acrylamide after its formation in food. . LAB areLb. bulgaricus and Lb. rhamnosus to fired potatoes and fermented bread, respectively, removed 85% of the dietary A.A. (in-vitro simulated digestion system using the INFOGEST2.0 model and (4) characterization of the cell wall of the chosen strains, using scanning electron microscopy in combination with energy-dispersive X-ray spectroscopy (SEM-EDS), zeta potential, transmission electron microscopy (TEM), and Fourier transforms infrared spectroscopy (FTIR) to interpret the mechanism of adsorption.After the publication of research by ary A.A. . With thEnterococcus; (2) Pediococcus; and (3) Streptococcus. A loopful of the bacterial stock was inoculated into 10 ml of a broth of de Man, Rogosa, and Sharpe (MRS)  and then incubated at 37°C for 20 h to activate the isolates.Thirty-eight strains of LAB strain that our laboratory had already isolated from food products  were exaWe prepared an acrylamide stock solution by dissolving 50 mg of powdered acrylamide  in a 50-ml volumetric flask of deionized water, giving a concentration of 1 mg/ml. This was used in the screening stage to prepare two working solutions, diluting the stock solution to 50 μg/ml and 100 μg/ml in 10 ml of MRS broth. These working concentrations were selected based on the AA concentrations used in various food system reported in the previous studies .An aliquot of 1% of activated culture was inoculated into two samples of 10 ml of sterilized MRS broth supplemented with acrylamide, one at 50 μg/ml and the other at 100 μg/ml. The samples were then incubated at 37°C for 20 h. Subsequently, a pour-plate method was used to perform bacterial enumeration with MRS agar, and anaerobic incubation was at 37°C for 20 h. In preparation for acrylamide analysis by LC–MS–MS, bacterial cells were removed from the samples by centrifugation . Acrylamide analysis was conducted on the supernatants collected, with each sample being analyzed in duplicate. Each group of samples was matched with controls of MRS broth, MRS with bacteria, and MRS spiked with acrylamide without bacteria .BBD was employed for the optimization of the four variables, each at three levels: pH of 4.5, 5.5, and 6.5, using 1.0 mol/l HCl or 1.0 mol/l NaOH; the temperature of 32°C, 37°C, and 42°C, using anaerobic incubators; incubation period of 14, 18 and 22 h; and NaCl of 0%, 1.5 and 3% w/v. Experiment design and statistical analysis were performed using Minitab v.21. Twenty-seven experimental runs with three repetitive central points were used to investigate the four independent variables. Two different LAB strains were investigated under aerobic and anaerobic conditions, with each experiment conducted in triplicate, as shown in p-values to evaluate the significance .Optical density at 600 nm was used to measure the microbial population using a spectrophotometer and 24-well plates, using an Epoch 2 Microplate Spectrophotometer from BioTeck, Santa Clara, California, United States. After determining the bacterial populations, 1.5-ml tubes were used to collect the samples and centrifuge at 10,000 × in-vitro gastrointestinal INFOGEST2.0 protocol . A 1-ml aliquot of the bacterial pellet suspension and aliquot of AA were subjected to in-vitro digestion named INFOGEST2.0 model. The sequence of (1) oral phase , 0.3 M CaCl2, 2 min, 37°C), (2) gastric phase , pH 3.0, 0.3 M CaCl2, 120 min, 37°C) omitting the rabbit gastric extract, followed by (3) intestinal phase  pH 7.0, 0.3 M CaCl2, 120 min, 37°C) was followed. The samples were continuously shaken at 120 rpm during whole in vitro digestion process (4 h and 2 min). During INFOGEST2.0 treatment, samples were collected for the bacterial count and acrylamide analysis. The bacterial count was measured by serial dilution immediately after taking the samples. For acrylamide analysis, the samples were frozen under −20°C for later analysis as described in section 2.6.The protocol  was appl18 . The 1% acetic acid mobile phase in deionized water with a 0.2 ml/min flow rate was employed. An injection volume of 20 μl and a column temperature of 35°C were applied. For quantification, an external acrylamide standard curve of 0, 5, 10, 25, 50, 75, 100, 125 and 150 𝜇g/ml was constructed.To determine the amount of acrylamide in the aqueous fraction, the Agilent 1,290 Infinity LC system was used. It was equipped with the MS/MS detector , Column Hypercarb C9 CFU/ml of the second sub-culture, an aliquot of the activated cultures from the second sub-culture was added at 1% v/v to 10 ml of fresh MRS broth containing 10 μg/ml acrylamide. This was incubated at 37°C with 0% NaCl and pH 6.5 for 18 h. Following incubation, samples were centrifuged at 5,000 x g for 10 min at 4°C. Supernatants were extracted, and bacterial cells were collected in 0.1 M, pH 7.0 phosphate buffer in 1.5-ml tubes and retained under −20°C until analysis.The two LAB strains were activated twice in MRS broth for 24 h at 37°C. To obtain 10The LAB cells’ zeta potential was measured using a micro electrophoretic apparatus, Zeta Plus . The experiment was conducted at ~23°C (room temperature), with the pH adjusted using 0.1 M NaOH and 0.1 M HCl .−1 -FTIR spectroscopy, for which we used a Spectrum Two I.R., combined with a Universal ATR (UATR) unit . Freeze-dried bacterial cell samples were directly positioned on a Diamond/ZnSe crystal plate (Perkin-Elmer). The IR spectral range was 4,000–400 cm−1 .E. durans and E. faecalis were evaluated by Quanta 250 ESEM.  glutaraldehyde containing 1% osmium tetroxide. The radius, height, and elemental composition of 50 ESEM. . From eaLAB cells were characterized by employing a Tecnai G2 TEM operating at 200 kV. The prepared samples were cut into 50–60 nm sections of thickness using an Ultramicrotome  was used to calculate the mean values and standard deviations of the results obtained by screening the samples. BBD was employed, with the responses analyzed using the Minitab v.21.E. durans (accession no. KY795325) (33%) and E. faecalis U2 (accession no. MF067501) (30%) had the highest % acrylamide removal.Enterococcus has not yet been approved as Generally Recognized as Safe by the U.S. Food and Drug Administration  for E. durans and F-value = 0.89 and p-value of (p > 0.587) for E. faecalis. The coefficient of determination values R2 was 0.631 for all models of E. durans, suggesting that the developed models have the goodness of fit that could explain >63% of the total variation. The adjusted R2 was 20.03% for E. durans. E. faecalis had lower results, with a goodness of fit that could explain up to 50.95% of the total variation. The p-values for the lack of fit for the models were not significant for both strains: p > 0.266, p > 0.272 for E. durans and E. faecalis, respectively.E. durans, p > 0.05), except for the relation with temperature alone (p < 0.016). The level of acrylamide was reduced by more than 40% under those conditions, as shown in E. faecalis, there were no significant relationships between pH, incubation temperature, salt, and incubation time, nor their interactions and acrylamide levels, as shown in E. faecalis indicates higher acrylamide removal than E. durans under the same conditions as in E. faecalis performed better under pH 6.5, temperature 32–34°C, incubation time 18 h, and salt 0.0.For E. durans acted better in a warm, acidic environment that lacks NaCl, unlike E. faecalis, which can bind high levels of acrylamide in warm salty surroundings with pH of 6.5. The longer the strains were incubated with the acrylamide, the higher the binding results.By analyzing E. faecalis and E. durans, respectively. This indicates that acrylamide removal is species-dependent under the simulated gastrointestinal system. The results indicate that LAB can remove acrylamide formed during the cooking processes in food during in vitro digestion. The effect of the matrix on acrylamide removal during in vitro digestion was also investigated. Our results showed that the media matrix (the in vitro digestion solutions) had a minor effect (< 1.1%) on acrylamide removal.The remaining acrylamide in the gastrointestinal fluids are presented in E. durans and E. faecalis suspensions were − 11.37 and − 26.27 mV, respectively. Both strains showed poor coagulation results and high adsorption rates, as presented in E. durans and E. faecalis might not relate to their binding abilities.The average zeta potentials of E. durans and E. faecalis are presented in E. durans and E. faecalis showed distinct peaks at the following bands. The strong absorption band was around 1800 cm-1 which may arise due to the stretching vibrations of C=O. The medium absorption bands located at 2860 and 2,900 cm −1 corresponds to C-O stretch. The band at 3002 cm − 1 indicates presence of amine group , nitrogen (N), and carbon (C), which might explain their ability to adsorb acrylamide.E. durans and E. faecalis are displayed in TEM images of After the announcement that acrylamide posed a risk to human health and could be formed in cooked starchy food, researchers studied several approaches to its removal and elimination. In comparison to many approaches that have been suggested, such as changing raw materials, adding new ingredients to recipes, or changing the processing method, LAB presented positive results in removing acrylamide without affecting the sensory characteristics of food .E. durans and E. faecalis had the ability to reduce the levels of acrylamide in MRS medium by up to 33 and 30%, respectively. In a previous study, Streptococcus lutetiensis and Lactiplantibacillus plantarum removed up to 39 and 26% of acrylamide, respectively  NaCl 0.0; (2) incubation temperature 32–34°C; (3) pH 4.5–5.5 and (4) incubation time 18–22 h. Similarly, Lactobacillus acidophilus had the highest removal under 10 ppm Bbenzo[a]pyrene concentration, pH 5, cell density 109 CFU/ml, and incubation period of 24 h , Al-Ain, UAE.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Aidi injection (ADI), a traditional Chinese medicine antitumor injection, is usually combined with doxorubicin (DOX) for the treatment of malignant tumours. The cardiotoxicity of DOX is ameliorated by ADI in the clinic. However, the relevant mechanism is unknown.To investigate the effects of ADI on DOX-induced cardiotoxicity and its mechanism.i.p., 0.03 mg/10 g) was administered in the presence or absence of ADI  for two weeks. Heart pathology and levels of AST, LDH, CK, CK-MB and BNP were assessed. H9c2 cells were treated with DOX in the presence or absence of ADI . Cell viability, caspase-3 activity, nuclear morphology, and CBR1 expression were then evaluated. DOX and doxorubicinol (DOXol) concentrations in heart, liver, kidneys, serum, and cells were analysed by UPLC-MS/MS.ICR mice were randomly divided into six groups: control, ADI-L, ADI-H, DOX, DOX + ADI-L and DOX + ADI-H. DOX  improved the cell viability, and IC50 increased from 68.51 μM  to 83.47, 176.9, and 310.8 μM, reduced caspase-3 activity by 39.17, 43.96, and 61.82%, respectively. High-dose ADI inhibited the expression of CBR1 protein by 32.3%, reduced DOXol levels in heart, serum and H9c2 cells by 59.8, 72.5 and 48.99%, respectively.ADI reduces DOX-induced cardiotoxicity by inhibiting CBR1 expression, which provides a scientific basis for the rational use of ADI. Mylabris phalerata Pallas (Meloidae), Astragalus membranaceus (Fisch.) Bge. (Fabaceae), Panax ginseng C. A. Mey.  and Acanthopanax senticosus (Rupr. et Maxim.) Harms . It has exhibited good efficacy, safety and cost-effectiveness in clinical treatment , a traditional Chinese medicine injection, was approved by the China Food and Drug Administration (CFDA) in 1996 for the treatment of malignant tumours, including primary liver cancer, lung cancer, rectal cancer, malignant lymphoma and gynecological malignancies. The ingredients of ADI are extracts from 236, ADI,P. ginseng and A. membranaceus protect against cardiotoxicity caused by DOX  was supplied by Guizhou Ebay Pharmaceutical Co., Ltd. . DOX (purity > 98%) and the internal standard (IS), azithromycin (purity > 98%), were purchased from Dalian Meilun Biotech Co., Ltd. . DOXol was obtained from Glpbio . Hoechst 33342 was purchased from Beyotime Institute of Biotechnology . Aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatinine kinase (CK), CK-MB isoenzyme and caspase-3 activity assay kits were provided by Nanjing Jiancheng Bioengineering Institute . A mouse BNP (brain natriuretic peptide) ELISA kit was purchased from Shanghai MLBIO Biotechnology Co., Ltd. . BCA assay kit, CBR1 and GAPDH antibody were purchased from Abcam . High performance liquid chromatography (HPLC)-grade acetonitrile, methanol and formic acid were supplied by Merck Company Inc. . Distilled water was obtained from Watsons Group Co., Ltd. . All other chemicals and reagents used were of chromatographic or analytical grade.in vivo efficacy of ADI was evaluated using male ICR mice (18–22 g) from Chongqing Tengxin Biotechnology Co., Ltd.  2012-0008). Each cage contained 10 mice that were acclimatised for one week before the experiment. The animal room was well ventilated with sufficient light, room temperature of 18–25 °C and relative humidity of 50–70%. Feeding and management were carried out in strict accordance with the requirements and rules for treatment of laboratory animals.All experimental procedures were approved by the Institutional Animal Ethics Committee and the Experiment Animal Centre of Guizhou Medical University . The n = 6, day 0): (1) saline 0.2 mL/10 g/day (i.p.); (2) low-dose ADI ; (3) high-dose ADI ; (4) DOX 0.03 mg/10 g ; (5) DOX 0.03 mg/10 g  + ADI-L 0.1 mL/10 g/day (i.p.); and (6) DOX 0.03 mg/10 g  + ADI-H 0.2 mL/10 g/day (i.p.). ADI or saline was given 1 h before DOX. Experiment grouping and treatments were performed according to i.p., 0.03 mg/10 g). All mice were euthanized by isoflurane overdose 2 h after DOX administration. The blood, heart, liver and kidneys were collected and stored at −20 °C for quantification of DOX and DOXol. Specimens from each heart were used for histological examination. Serum samples were used to determine the levels of AST, LDH, CK, CK-MB and BNP.Male ICR mice were weighed and randomly divided into six groups  were stained with hematoxylin-eosin (H&E). Images of the stained sections were obtained using a light microscope  with 100Myocardial cellular damage was evaluated by measuring AST, LDH, CK and CK-MB activity in serum using commercially available assay kits. The serum levels of BNP were determined using the commercial kit, according to the manufacturer’s instructions.g. The upper organic phase was transferred into a tube and evaporated to dryness under a gentle stream of nitrogen. The residue was dissolved in the initial mobile phase (500 μL) and centrifuged at 18,000g for 10 min. Subsequently, a sample (2 μL) was injected into the UPLC-MS/MS consisting of an Acquity H-Class UPLC system coupled to a Xevo TQ-S Micro Tandem Mass Spectrometer . Chromatographic separation was performed on an Acquity UPLC BEH C18 column . The column and autosampler tray temperatures were at 45 and 25 °C, respectively. The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B). A linear gradient at a flow rate of 0.30 mL/min was run at 10–90% A over 0–3.5 min, 90% A over 3.5–4.0 min, 90–10% A over 4.0–4.5 min, and 10% A over 4.5–5.0 min. The MS was operated in positive electrospray ionisation mode. The MS parameters were as follows: source temperature 150 °C, desolvation temperature 500 °C, nitrogen gas flow 1000 L/h and capillary voltage 1.5 kV. Quantification was performed using selective reaction monitoring mode: m/z 544.7 → 361.0 (DOX); m/z 545.6 → 362.9 (DOXol); m/z 749.7 → 591.4 (IS). The cone voltages for DOX, DOXol, and IS were 25, 30, and 30 V, respectively. Collision energies for DOX, DOXol, and IS were all 25 eV.Briefly, simple protein precipitation with methanol was performed  and cultured in Dulbecco’s modified Eagle medium supplemented with 10% foetal bovine serum, 100 IU/mL penicillin and 100 μg/mL streptomycin at 37 °C under 5% CO50) were calculated from survival curves.The effects of DOX, DOXol and ADI on cell viability were determined by the MTS assay  after incubation of the cells with various concentrations of DOX  in the absence or presence of ADI  at 37 °C for 24 h. The absorbance was measured at 470 nm and used to calculate the relative ratio of cell viability. The concentrations required to inhibit growth by 50%  in the absence or presence of DOX (5 μM) at 37 °C for 24 h. Activity of caspase-3, a key enzyme marker of apoptosis, was measured using a commercial assay kit according to the manufacturer's protocol. After DOX (5 μM) and ADI  treatment, H9c2 cells were incubated with 10 μg/mL Hoechst 33342 for 10 min after fixing with 4% formaldehyde. Nuclear morphology was observed on a Nikon TE2000 microscope (Japan). Representative images were captured by NIS-Elements F (4.60.00 version) software.g for 5 min , cells were suspended in purified water (1 mL) and broken by repeated freeze-thawing. After determination of the protein concentration using a BCA Protein Assay Kit, the concentrations of DOX and DOXol were then determined by UPLC-MS/MS. All experiments were conducted six times.H9c2 cells were seeded in 24-well plates. When confluence reached 90%, the cells were incubated with DOX (5 μM) and ADI  for 1, 2, 4, 8, 12 and 18 h. Subsequently, the medium was removed and cells were washed twice with phosphate-buffered saline. After centrifugation at 500H9c2 cells were incubated with ADI (10%) or an equivalent volume of PBS for 18 h. Subsequently, protein samples were extracted from cellular lysate. Protein concentration was quantified by BCA assay, then measured by absorption in a spectrophotometer. After that, CBR1 protein was assessed by western blot as described .Data are expressed as the mean ± standard deviation. Statistical analysis included the two-tailed Student’s As shown in i.p.) did not affect the plasma levels of CK, CK-MB, AST or LDH. Treatment with DOX  significantly increased the plasma levels of CK, CK-MB, AST and LDH to 2.31, 2.57, 2.47 and 1.23 times that of the control group, respectively. High-dose ADI (0.2 mL/10 g) decreased the DOX-induced up-regulation of these four enzymes better than low-dose ADI (0.1 mL/10 g). In the 0.2 mL/10 g ADI + DOX group, the levels of myocardial enzymes returned almost to their normal levels  concentration-dependently reduced the survival of H9c2 cells . CombineP < 0.001), while ADI  alone had no significant effect. When DOX was combined with ADI , the caspase-3 activity decreased by 39.17, 43.96 and 61.82%, respectively.As shown in Hoechst 33342 staining was also performed to investigate morphological changes of nuclei subjected to DOX-induced apoptosis . The nucH9c2 cells were incubated with DOX (5 μM) in the absence or presence of 1, 4, or 10% ADI for 1, 2, 4, 8, 12 and 18 h. DOX and DOXol concentrations in cells were quantified by UPLC-MS/MS. As shown in CBR1 contributes mainly to the conversion of DOX to DOXol (Lal et al. Malignant tumours present a serious health threat and have now become the number one cause of death in humans. Anthracyclines are some of the most effective chemotherapeutics for the treatment of malignant tumours, and are widely used clinically to treat hematological malignancies and solid tumours (Martins-Teixeira and Carvalho In recent years, advances in theoretical research on Chinese medicine have led to significant progress in the treatment of malignant tumours, and Chinese medicine has become an important part of their treatment in China (Xiang et al. Therefore, in the present study, we have used a mouse model of DOX-induced myocardial injury to investigate the effects and mechanism of ADI. The dosage of DOX was screened in a preliminary experiment using 2, 3 and 4 mg/kg. The 2 mg/kg dose was too low, with no obvious myocardial toxicity observed. About 30% of the mice were intolerant to 4 mg/kg DOX and died during the experiment. Consequently, a dose of 3 mg/kg was selected for the myocardial injury model. The results from scanning electron microscopy after 15 days of repeated DOX administration showed that the cardiomyocytes of the mice were damaged. The mitochondrial morphology in the model group was abnormal, with the mitochondria appearing swollen. The serum levels of CK, CK-MB, AST and LDH in the model group increased significantly, indicating that the cardiomyocytes were seriously damaged. The levels of BNP, a well-recognized marker of heart failure (Kuwahara et al. In the clinical treatment of hepatocellular carcinoma, ADI is dosed simultaneously with chemotherapy drugs for a period of 10–30 days. Therefore, the mice were dosed with ADI for 15 consecutive days in this experiment. After 15 days of ADI treatment, H&E staining showed that ADI reduced DOX-induced myocardial damage. After treatment with ADI, the serum levels of CK, CK-MB, AST and LDH in the model animals were decreased. ADI also reduced the serum level of BNP in model mice. The results showed that ADI effectively alleviated the myocardial toxicity caused by DOX in animals. In cell experiments, compared with DOX alone, the combined use of ADI and DOX increased the survival of H9c2 cells, reduced caspase-3 activity and improved nuclear morphology in a dose-dependent manner. In summary, the results from the animal model and cell experiments showed that ADI can reduce the myocardial toxicity of the chemotherapy drug, DOX.It is generally believed that the pharmacological effects of drugs are positively correlated with the drug concentration in plasma or target tissues, and also positively correlated with the intracellular drug concentration. We therefore used UPLC-MS/MS to determine the drug concentrations in tissues, organs and myocardial cells. The method used to determine DOX and DOXol in plasma and tissues was an established method (Lu et al. P. ginseng and A. membranaceus have been reported to reduce DOX-induced myocardial toxicity. For example, ginsenoside Rg1 prevents DOX-induced cardiotoxicity by inhibiting autophagy and endoplasmic reticulum stress in mice (Xu et al. Astragalus polysaccharides restore autophagy flux and improve cardiomyocyte function in DOX-induced cardiotoxicity (Cao et al. Astragalus polysaccharide inhibits DOX-induced cardiotoxicity by regulating PI3k/Akt and p38MAPK pathways (Cao et al. Various components in ADI reduced DOXol concentrations in myocardial tissue and cardiomyocytes by inhibiting CBR1 protein expression, thereby reducing DOX-induced cardiotoxicity. This research provides a scientific basis for the clinical use of ADI and DOX.


In the concentration range of 1–2000 ng/ml, a good linear relationship of voxtalisib was successfully established by the UPLC-MS/MS technology, and the lower limit of quantification (LLOQ) of the analyte was identified as 1 ng/ml. Intra-day and inter-day precisions for voxtalisib were 7.5–18.7% and 13.0–16.6%, respectively, and the accuracies were in the ranges of −14.0–2.0% and −7.2–3.1%, respectively. The matrix effect, extraction recovery, carryover and stability of the analyte were all in compliance with the acceptance criteria of bioassays recommended by FDA. Finally, the pharmacokinetic profile of the analyte had been availably studied by the UPLC-MS/MS bio-analytical method after rats were treated by intragastric administration with voxtalisib (5 mg/kg). The results indicated that the UPLC-MS/MS technology can effectively and quickly quantify the analyte, and this method can also be used for the pharmacokinetic study of voxtalisib, which can provide reference for the optimization of clinical drug management in the later period.Voxtalisib, is a specific, effective, and reversible dual inhibitor, which inhibits both pan-class I phosphoinositide 3-kinase (PI3K) and mechanistic target of rapamycin (mTOR). To date, voxtalisib has been studied in trials for melanoma, lymphoma, glioblastoma, breast cancer, and other cancers. In this study, a highly sensitive and rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technology was applied to the quantitative methodology and pharmacokinetic analysis of voxtalisib in rat plasma. After protein precipitation of the analyte by acetonitrile, the chromatographic separation was performed by gradient elution on an Acquity BEH C18 column  with acetonitrile (solvent A) and 0.1% formic acid (solvent B) as the mobile phase. In the positive ion mode, the mass transfer detection of the analyte and IS was  In addition, voxtalisib blocked the adhesion, proliferation, and in vitro survival of chronic lymphocytic leukemia cells and effectively inhibits T-cell-mediated cytokines that support the production of chronic lymphocytic leukemia  were collected from the Experimental Animal Research Center of The First Affiliated Hospital of Wenzhou Medical University , and were given fresh water and plenty of food every day. In the formal pharmacokinetic study, each rat was given 5 mg/kg voxtalisib in the 0.5% carboxymethylcellulose sodium (CMC-Na) by single intragastric administration after fasting for 12 h. Then, blood samples (approximately 0.3 ml) were acquired at 0.333, 0.667, 1, 1.5, 2, 3, 4, 6, 8, 12, 24 and 48 h, respectively, and placed into polyethylene tubes containing heparin. The samples were immediately centrifuged at 13,000 × g for 10 min at 4°C for separation. The centrifuged supernatants were then transferred into new 0.5 ml polythene tubes and then stored at −80°C for further analysis. In this experiment, the plasma concentration of voxtalisib in rats was measured by UPLC-MS/MS technology, and the main pharmacokinetic parameters of voxtalisib in non-compartmental model were calculated using Drugs and Statistics (DAS) 3.0 software .Chromatographic separation of voxtalisib and IS were performed using a Waters Acquity ultra-performance liquid chromatography (UPLC) system  equipped with an Acquity BEH C18 column , and it was operated under the condition of 40°C column temperature. The mobile phases of gradient elution were acetonitrile (solvent A) and 0.1% formic acid (solvent B), respectively. At the flow rate of 0.30 ml/min, the elution process was 90% B from 0 to 0.5 min; 90–10% B from 0.5 to 1.0 min; 10% B from 1.0 to 1.4 min; 10–90% B from 1.4 to 1.5 min, and finally at 1.5–2.0 min, 90% B was maintained for equilibration. The whole process of analysis was 2.0 min, the temperature of the auto-sampler was set at 10°C, and the injection volume was 1.0 μL for each running process.m/z 270.91 > 242.98 and m/z 572.30 > 246.10, respectively  with an electrospray ionization (ESI) source and a positive ion mode detection by MS/MS. Using selective response monitoring (SRM), the ion transitions of voxtalisib and IS were ectively . OptimizVoxtalisib and IS were dissolved in methanol as the stock solutions, and the concentration of each stock solution was 1 mg/ml. Then, the stock solution was gradient diluted with methanol to obtain the calibration and quality control (QC) samples. The concentrations of the working solutions for calibration curve were 10, 20, 50, 100, 500, 1000, 2000, 5000, 10000, 20000 ng/ml, and the IS concentration was 100 ng/ml. Fresh calibration standards with final concentrations of 1, 2, 5, 10, 50, 100, 200, 500, 1000, 2000 ng/ml were prepared by adding the corresponding concentration of voxtalisib working solutions (10 μL) to the blank rat plasma (90 μL). Then, the same method was used to prepare QC samples with different concentrations  and the lower limit of quantification . Finally, all the stock and working solutions were stored at −80°C until further analysis.The plasma samples were treated with a simple and rapid protein precipitation method. Firstly, adding 10 μL IS working solution to 100 μL plasma sample; Secondly, precipitate the plasma protein by adding 300 μL acetonitrile; Thirdly, the plasma samples in each tube were fully swirled for 2.0 min and centrifuged for 10 min at 13000 × g under 4°C. Finally, we took 100 μL centrifuged supernatant into the special detection bottle of UPLC-MS/MS system, and then used UPLC-MS/MS technology for analysis. In order to ensure the stability of the sample, it is recommended to test immediately. If immediate detection is not available, it can be stored at 4°C in the short-term, and should be stored at −80°C in the long-term.According to the principle of bio-assay validation of FDA, a novel bio-assay method based on UPLC-MS/MS technology was established, including the specificity, carryover, calibration curve, LLOQ, precision, accuracy, matrix effect, recovery, and stability, as similar with those validated in our previous papers .The specificity of UPLC-MS/MS analytical method was determined by analyzing plasma samples from three different batches: blank plasma without analyte and IS, blank plasma spiked with the analyte at LLOQ and IS, as well as rat plasma samples. Then, the retention times of the analyte and IS in the SRM chromatogram were observed to determine whether there were endogenous interference. Carryover was assessed by injecting plasma samples at upper limit of quantification (ULOQ) followed by blank injections.x2) to obtain the calibration curve. The sensitivity of the UPLC-MS/MS bio-analytical method was evaluated by analyzing six replicates of spiked LLOQ samples.The linear regression analysis of voxtalisib/IS peak area ratio (Y) and voxtalisib concentration (X) was performed by the least square regression analysis with a weighted factor  were determined on the same day and on three different days. The intra and inter-day precisions of the method were calculated, which is usually shown as relative standard deviation (RSD%), and the accuracy is commonly represented by relative error (RE%).n = 6), the peak areas of voxtalisib added to the post-extracted blank plasma were compared with those of QC sample with corresponding concentration obtained by diluting standard solution with methanol to calculate matrix effect.At low, medium and high concentrations .Voxtalisib was added into the blank plasma before and after extraction, respectively, and the extraction recovery was evaluated by comparing the peak area of the two, also at three different levels  under four different placement conditions, including short-term stability (3 h at room temperature), long-term stability (3 weeks at −80°C), post-preparation stability (4 h at 10°C in an auto-sampler), and stability through complete three freeze-thaw cycles.We evaluated the stability of this bioassay by measuring rat plasma concentrations of QC samples of voxtalisib at three different concentrations  and water buffers (such as formic acid and ammonium acetate) on different types of analytical columns  were evaluated. After comparing different types of analytical columns in the experiment, the Acquity BEH C18 column  provided good peak shapes, short chromatographic retention times and high chromatographic responses for both the analyte and IS, and the separation effect was better. In addition, acetonitrile and 0.1% formic acid aqueous solution was the best mobile phase, and the analyte and IS obtained better signals and responses.In pharmacokinetic study, we chose simple, time-saving and economical organic protein precipitation method to prepare plasma samples. We precipitated plasma proteins with acetonitrile. The results showed that acetonitrile had higher efficiency of protein precipitation.According to SRM chromatograms under three different conditions in r2 = 0.999). As shown in In this experiment, the linear regression equation of calibration standard curve in the concentration range of 1–2000 ng/ml was y = 0.0231301x + 0.00967189  of 977.74 ± 250.40 ng/ml at 2.45 ± 1.42 h . Moreover, the half-life (t1/2) of its elimination in rats was 10.47 ± 5.00 h.According to max = 301 ± 101 ng/ml, Tmax = 1.53 h, t1/2 = 3.94 ± 0.79 h on the first day after giving patients 50 mg/kg once daily (n = 6), compared to 49 patients in the clinical trial. Consequently, the pharmacokinetics of voxtalisib need to be further studied. Furthermore, this pharmacokinetics of voxtalisib in patients with solid tumors did not provide enough data for repeating the approach in other laboratories , so our method effectively achieved the need of high sample throughput for biological analysis (In a phase I dose-escalation study on the safety and pharmacokinetics of voxtalisib tablets in patients with solid tumors, Cce daily . The exianalysis .In summary, in this experiment, we determined the specificity, carryover, precision, accuracy, extraction recovery, matrix effect, and stability of voxtalisib in rat plasma. This UPLC-MS/MS assay can effectively and quickly quantify the analyte, and this method can also be used for the pharmacokinetic study of voxtalisib in rats, which can provide reference for the optimization of clinical drug management in the later period.