Formulation design and characterization of silymarin liposomes for enhanced antitumor activity.

Liposomes, a nanoscale carrier, plays an important role in the delivery of drug, affects the in vivo efficacy of drugs. In this paper, silymarin(SM)-loaded liposomes was optimized using the response surface method (RSM), with entrapment efficiency (EE%) as an index. The formulation was optimized as follow: lecithin (7.8mg/mL), SM/lecithin (1/26) and lecithin/cholesterol (10/1). The optimized SM liposomes had a high EE (96.58 ±3.06%), with a particle size of 290.3 ±10.5nm and a zeta potential of +22.98 ±1.73mV. In vitro release tests revealed that SM was released in a sustained-release manner, primarily via diffusion mechanism. In vitro cytotoxicity studies demonstrated that the prepared SM liposomes had stronger inhibitory effects than the model drug. Overall, these results indicate that this liposome system is suitable for intravenous delivery to enhance the antitumor effects of SM.

The abnormalities of lipid metabolism in children and adolescents with major depressive disorder and relationship with suicidal ideation and attempted suicide.

Background: Major depressive disorder (MDD) is a widespread health issue in many countries, which has an extremely negative impact on the health of children and adolescents in particular. In the context of depression and metabolic disorders, dyslipidemia and metabolism-related problems become more prominent comorbidities. However, they continue to be the main barrier to the successful recovery of the clinical progress. In this study we investigated the rate of dyslipidemia, additional risk factors among Chinese children and adolescents with MDD, and association of the suicidal behavior with lipid levels. Methods: The study took 756 people from the Third People's Hospital of Fuyang between January 2020 and December 2021, aged between 8 and 18, with major depressive disorders diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5). We determined the FBG (fasting blood glucose) and lipid parameters in all subjects and also investigated the history of suicidal ideation, the cases of attempted suicide, and the scores of depressive symptoms. Sociodemographic and clinical data were gathered and analyzed using the SPSS-23.0 version. Results: The prevalence of hypercholesterolemia, hypertriglyceridemia, high LDL-C, and low HDL-C were 5.42 % (41/756), 10.58 % (80/756), 3.84 % (29/756) and 5.42 % (41/756) respectively. For hypercholesterolemia and hypertriglyceridemia, they were positive associated with suicidal ideation and suicide attempts, and the positive correlation is shown between low HDL-C levels and suicide attempts. Nevertheless, non-ideation and inversely suicidal attempts were not discovered among high-LDL-C subjects. Logistic analysis showed that high levels of FBG (OR = 2.86, 95 % CI: 1.31-6.25, P = 0.008) and worse LDL-C (OR = 357.82, 95 % CI: 66.16-1935.10, P < 0.001) are the independent associated factors for hypercholesterolemia. More hospitalizations (OR = 1.89, 95 % CI: 1.07-3.35, P = 0.028), obesity (OR = 2.55, 95 % CI: 1.25-5.18, P = 0.010), high levels of TC (OR = 2.15, 95 % CI: 1.03-4.48, P = 0.042), and higher doses of antidepressants (OR = 1.02, 95 % CI: 1.00-1.04, P = 0.029) were independently associated factors for hypertriglyceridemia, while high levels of HDL-C (OR = 0.11, 95 % CI: 0.04-0.31, P < 0.001) were protective factors. In addition, high levels of TC (OR = 113.94, 95 % CI: 20.01-648.85) were statistically different (P < 0.001) and suggested that the factor was significantly related to high LDL-C. Meanwhile, older age (OR = 1.25, 95 % CI: 1.02-1.52, P = 0.030) and high levels of TG (OR = 3.00, 95 % CI: 1.98-4.55, P < 0.001) were independent factors contributing to low HDL-C. Conclusion: The high prevalence of dyslipidemia in childhood and adolescence among children and adolescents with depressive disorder has become a public health issue. Hypercholesterolemia and hypertriglyceridemia showed a positive correlation with suicidal thoughts and suicidal attempts. Monitoring the incidence of suicidal thoughts and attempts among them would carry some predictor meaning in therapy and for jumping back to health.

The prevalence of underweight and obesity in Chinese children and adolescents with major depressive disorder and relationship with suicidal ideation and attempted suicide.

Background: The high rates of obesity and suicide have become serious public health problems worldwide, especially in children and adolescents with major depressive disorder (MDD). This research aimed to explore the rates of underweight, overweight or obesity, suicidal ideation and attempted suicide in hospitalized children and adolescents with MDD. Then, we analyzed the correlation between underweight or obesity and suicidal ideation and attempted suicide, and finally obtained the independent influencing factors of underweight or obesity. Methods: A total of 757 subjects in the Third People's Hospital of Fuyang from January 2020 to December 2021 were enrolled in this study. According to the underweight, overweight and obesity screening table for school-age children and adolescents published and implemented by the health industry standard of China, all subjects were divided into different body mass index (BMI) categories. We measured fasting blood glucose (FBG) and lipid levels in all subjects and assessed suicidal ideation, attempted suicide, and the severity of depressive symptoms. The socio-demographic and clinical data were collected and analyzed by SPSS 22.0. Results: The rates of underweight, overweight, obesity, suicidal ideation and attempted suicide were 8.2% (62/757), 15.5% (117/757), 10.4% (79/757), 17.2% (130/757), and 9.9% (75/757), respectively. Correlation analysis indicated that BMIs level was positively correlated with age, age of first hospitalization, total duration of disease, number of hospitalizations, FBG, TG (triglyceride), TC (total cholesterol), LDL (low density lipoprotein), and negatively correlated with HDL (high density lipoprotein). Binary logistic regression analysis showed that male and high level of HDL were risk factors for MDD inpatients with underweight, while high level of TG was a protective factor. Meanwhile, higher levels of FBG, TG and CGI-S were risk factors and suicidal ideation and high dose of antidepressant drugs were protective factors for obesity in children and adolescents with MDD. Conclusion: The prevalence of underweight, obesity, suicidal ideation and attempted suicide were high in children and adolescents with MDD, and severe depressive symptoms are independent risk factors for obesity, while suicidal ideation and high dose of antidepressants may be protective factors for obesity.

Optimizing the preparative capacity of two-dimensional liquid chromatography based on analytes retention behaviors.

In this work, a solute retention optimization method (SRO) was proposed to exploit the purification potential of two-dimensional liquid chromatography (2D-LC). According to our findings, the complementarity of 2D-LC correlates with some specific impurities. In the two methods used in 2D-LC, the retention order of these impurities and target compound is completely opposite. Taking full advantage of the complementarity is crucial to enhance the saturation capacity (wmax) of 2D-LC by SRO. For the purpose of validating the effectiveness of SRO, a reverse-phase liquid chromatography (RPLC) coupled with hydrophilic interaction chromatography (HILIC) was developed to purify p-chlorobenzoic acid from substituted benzenes. By using the overloading effects of analytes as indicators, the wmax of RPLC × HILIC was determined by the bisection method, and finally defined by the extremely high loading volume of 4.9 mL. A touch-peak separation of impurities and the target compound occurred precisely during the secondary separation. The effectiveness of SRO was also verified by the greater purification efficiency of RPLC × HILIC than that of HILIC × RPLC. Subsequently, a RPLC × RPLC method was developed by SRO to prepare the reference materials of caffeine from tea extracts. Only by an analytical C18 column, 15.6 mg of caffeine with the purity of 98.3% was obtained at once with the recovery up to 82.3%. However, without the aid of SRO, the purity rapidly decreased to 62.0%. Compared to other methods, SRO-based 2D-LC offers certain advantages in terms of purity, recovery, and the purification efficiency, suggesting that it is particularly effective in developing preparative 2D-LC facing complex matrices.

Phenolic Compounds and Bioactivities from Pomegranate (Punica granatum L.) Peels.

Pomegranate peels, which are normally processed as the main byproduct of pomegranate juice production, are worthy of being researched and utilized for the aim of economic and environmental benefits. In a phytochemical investigation of the peels of Punica granatum L., 10 phenolic compounds containing a common hexahydroxy diphenol moiety were isolated. Three of them were identified for the first time and named as pomegranatins A-C, and from the other seven known ones, two of them were obtained from pomegranate peels for the first time. Their structures were determined via extensive spectroscopic analysis. Besides, for the sake of preliminarily comprehending their biological activities, in vitro antimicrobial, antioxidant, as well as antitumor assays were detected. In the DPPH antioxidant assay, six compounds presented significant free radical scavenging ability. Two compounds exhibited moderate antimicrobial activities against Candida albicans; one compound could inhibit the proliferation of both C. albicans and Escherichia coli within limits. Four compounds possessed weak antitumor activity toward the Hela cell line without taking into account the bioavailability of ellagitannins. Overall, these results provided further information on the structural diversity of bioactive compounds present in pomegranate peels, as well as on their biological activities.

Punicalagin promotes autophagic degradation of human papillomavirus E6 and E7 proteins in cervical cancer through the ROS-JNK-BCL2 pathway.

Punicalagin, which is derived from pomegranate peel, is reported to exert growth-inhibitory effects against various cancers. However, the underlying mechanisms have not been elucidated. Human papillomavirus (HPV), a major oncovirus, utilizes the host autophagic machinery to support its replication. Here, punicalagin markedly downregulated the levels of the major HPV oncoproteins E6 and E7 in cervical cancer cells through the autophagy-lysosome system. Additionally, punicalagin activated the reactive oxygen species (ROS)-JNK pathway and promoted the phosphorylation of BCL2, which led to the dissociation of BCL2 from BECN1 and the induction of autophagy. Treatment with autophagy and JNK inhibitors or ROS scavengers mitigated the punicalagin-induced degradation of E6 and E7. Moreover, the knockout of ATG5 using the clustered regularly interspaced palindrome repeat/Cas 9 system mitigated the punicalagin-induced downregulation of E6/E7. This indicated that punicalagin-induced degradation of E6 and E7 was dependent on autophagy. The results of in vivo studies demonstrated that punicalagin efficiently inhibits cervical cancer growth. In conclusion, this study elucidated a mechanism of punicalagin-induced autophagic degradation of E6 and E7. It will enable the future applications of punicalagin as a therapeutic for HPV-induced cervical cancer.

Dual-tautomerism separation method based on asymmetric transformation: Gram-scale preparation of high-purity punicalagin from pomegranate peel wastes.

In this study, a special orthogonal separation method, named as dual-tautomerism separation (DTS), was developed for the purification of tautomeric compounds from complex matrixes. In DTS, isomers of these compounds are individually collected and asymmetrically transformed to the mixtures of isomers. After separating the mixture with an identical method, high-purity compounds can be prepared from the newly generated isomers but impurities remain in another one. To validate the effectiveness, a DTS was developed to prepare punicalagin in gram-scale from pomegranate peel waste. Isomerization kinetic and thermodynamic of punicalagin were accurately assayed by dynamic HPLC built on low-temperature or/and loop-based stop-flow two-dimensional liquid chromatography. After the isomerization based on it, 9.3 g of pomegranate peel extract was firstly separated on C18 column, and Fα and Fß around α-punicalagin and ß-punicalagin were obtained. Then, the proportion of α-punicalagin in Fα and Fß was optimized to 52.7% and 32.0% based on isomerization kinetics and thermodynamic. With the aid of low-temperature injection, Fα and Fß were loaded and secondly purified. After waste recycling, totally 3.0 g of punicalagin with the purify of 99.5% was obtained within two days, which would strongly support the resource utilization of pomegranate peel waste. Because only an individual method was employed in the two-step purification, the separation in DTS was fully compatible.

Simultaneous determination of loading capacity and selectivity in preparative off-line two-dimensional separation: An application for purification of corilagin from Pomegranate flower extracts.

This work describes the development of capacity orthogonal chromatography (COC), a new technique for simultaneously determining the loading capacity and orthogonality during the construction of two-dimensional (2D) separations. Three steps were required for the construction of a COC based on the correlation between the selectivity factor (α) and both orthogonality and loading capacity. (1) α values of the impurities-target compound were used to normalize the retention of the impurities around the target compound. (2) α values were input into four quadrants of a coordinate system to identify correlations between orthogonality and loading capacity. α values of the impurities must be greater in the first dimension than the second dimension, with iterated analyses performed until an αmax is obtained for the two purification methods. (3) Touch-peak separation using the first-dimensional αmax was performed and the target compound was collected. The co-eluted impurities are further separated in the second dimension. To test the efficiency of this technique, a COC using two methods on a standard C18 column was developed to purify corilagin from pomegranate flower extract. Despite its low abundance, 288 mg of corilagin was obtained by COC and further purified by LH-20 gel chromatography to obtain the compound with an 80.0% recovery and 98.4% purity. Compared to COC, the purity of corilagin independently obtained using the same purification methods and identical loading capacity was poor (60.1% and 61.6%). These results indicate that COC is a useful tool for extending loading capacity in the development of preparative 2D separations.

Chemical profiling of spermidines in goji berry by strong cation exchange solid-phase extraction (SCX-SPE) combined with ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS/MS).

Lycium barbarum fruit (Goji berry) have been used as a traditional Chinese medicine (TCM) with its outstanding biological and pharmacological activities. Spermidine alkaloids are a major class of bioactive constituents in goji berry, nevertheless, detailed information related to its identification remains scarce. In this study, chemical profiling of spermidines in goji berry was carried out by ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Four structure types of standards were used to study the comprehensive fragmentation rules of spermidines. Different types of spermidines were identified by distinctive MS/MS fragment ions. Noticeably, it was first proposed that the co-existence of fragment ions at m/z 220 and 222 was the key characteristic for distinguishing spermidine isomers. According to the structural feature of spermidines, a quick, convenient, highly selective strong cation exchange solid-phase extraction (SCX-SPE) combined with RP-LC procedure was developed for selective enrichment and the MS detection compatibility. A total of 41 out of 58 spermidines were tentatively characterized using the established method, of which 26 were reported for the first time from goji berry. This study provides guidelines and references for the identification of spermidines in natural products.

Cost-effective imprinting to minimize consumption of template in room-temperature ionic liquid for fast purification of chlorogenic acid from the extract of E. ulmoides leaves.

One of the main challenges in large-scale applications of molecularly imprinted polymers (MIPs) is the significant amount of template needed in polymer preparation. A new strategy based on room-temperature ionic liquids (RTILs) was suggested to solve this problem by reducing the amount of template in the polymerization recipe. The MIP was synthesized with a mixture of dimethyl sulfoxide and RTIL (1-butyl-3-methylimidazolium tetrafluoroborate) as porogen, in which chlorogenic acid (CGA) was used as template, 4-vinylpyridine (4-VP) as functional monomer, and ethylene glycol dimethacrylate (EDMA) as cross-linker. The influence of polymerization variables, including CGA concentrations, and the ratio of 4-VP to EDMA on imprinting effect were investigated comprehensively. Moreover, the properties involving the column permeability, the number of binding sites, and the polymer morphology of the CGA-MIP monoliths were studied thoroughly. The MIP monolith had an excellent column permeability (1.53 × 10-13 m2) and allowed an ultra-fast on-line SPE, which dramatically shortens the separation time (< 10 min) and improves the separation efficiency. At high flow velocity (5.0 mL min-1), 50 µL of the extract from Eucommia ulmoides leaves can be loaded directly on the CGA-MIP monoliths and CGA with high purity can be obtained with a recovery of 89.01 ± 0.05%. As a conclusion, the resulting RTIL-induced approach of preparing MIP may be an effective tool in fabricating MIP in a low-cost way. Graphical abstract ᅟ.

Qualitative Analysis of Polyphenols in Macroporous Resin Pretreated Pomegranate Husk Extract by HPLC-QTOF-MS.

INTRODUCTION: Pomegranate (Punica granatum L.) husk is a traditional herbal medicine abundant in phenolic compounds and plays some roles in the treatment of oxidative stress, bacterial and viral infection, diabetes mellitus, and acute and chronic inflammation. OBJECTIVE: Identification and determination of polyphenols in macroporous resin pretreated pomegranate husk extract by high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF-MS). METHODS: The total polyphenols of pomegranate husk were prepared by ethanol extraction followed by pretreatment with HPD-300 macroporous resin. The polyphenolic compounds were qualitatively analysed by HPLC-QTOF-MS in negative electrospray ionisation (ESI) mode at different collision energy (CE) values. RESULTS: A total of 50 polyphenols were detected in the extract of pomegranate husk, including 35 hydrolysable tannins and 15 flavonoids with distinct retention time, fragmentation behaviours and characteristics, and the accurate mass-to-charge ratios at low, moderate and high CE values. Of these, we identified nine compounds for the first time in the pomegranate husk, including hexahydroxydiphenoyl-valoneoyl-glucoside (HHDP-valoneyl-glucoside), galloyl-O-punicalin, rutin, hyperoside, quercimeritrin, kaempferol-7-O-rhahmano-glucoside, luteolin-3'-O-arabinoside, luteolin-3'-O-glucoside, and luteolin-4'-O-glucoside. To validate the specificity and accuracy of mass spectrometry in the detection of polyphenols, as compared to the fragmentation pathways of granatin B in detail, including the HHDP-valoneyl- glucoside was first identified from pomegranate husk. CONCLUSION: The study provides evidence for the quality control and development of novel drugs based on polyphenols from the pomegranate husk. Copyright © 2017 John Wiley & Sons, Ltd.

"Two-dimensional" molecularly imprinted solid-phase extraction coupled with crystallization and high performance liquid chromatography for fast semi-preparative purification of tannins from pomegranate husk extract.

In this study, "two dimensional" molecularly imprinted solid-phase extraction (2D-MIP-SPE) of semi-preparative grade was constructed to fast purify ellagitannins in pomegranate husk extract with the help of crystallization and reverse-phase liquid chromatgoraphy (RPLC). Ellagic acid and punicalagin imprinted polymers were synthesized in batch mode and two semi-preparative MIP-SPE columns were individually packed. After investigaing "functional complementation", 2D-MIP-SPE was constructed using ellagic acid MIP and punicalagin MIP-SPE as the first and second dimension, respectively. Then, pomegranate husk extract was fast divided into four fractions individually enriching in ellagic acid, granatin A, punicalagin and ellagic acid glucoside by 2D-MIP-SPE. With the aid of crystallization and RPLC, ellagic acid (13.5mg) and punicalagin (53.4mg) were fast obtained in 30min. Ellagic acid glucoside was purified to the purity near 100% with a recovery of 86.1%. Granatin A (92%) was directly obtained by 2D-MIP-SPE with the recovery of 81.8%. All above indicated that 2D-MIP-SPE was highly efficient in natural product purification. The concept of "functional complementation" was expected to be a useful tool in the construction of 2D-MIP-SPE.

Cost-effective imprinting combining macromolecular crowding and a dummy template for the fast purification of punicalagin from pomegranate husk extract.

The combination of molecular crowding and virtual imprinting was employed to develop a cost-effective method to prepare molecularly imprinted polymers. By using linear polymer polystyrene as a macromolecular crowding agent, an imprinted polymer recognizable to punicalagin had been successfully synthesized with punicalin as the dummy template. The resulting punicalin-imprinted polymer presented a remarkable selectivity to punicalagin with an imprinting factor of 3.17 even at extremely low consumption of the template (template/monomer ratio of 1:782). In contrast, the imprinted polymer synthesized without crowding agent, did not show any imprinting effect at so low template amount. The imprinted polymers made by combination of molecular crowding and virtual imprinting can be utilized for the fast separation of punicalagin from pomegranate husk extract after optimizing the protocol of solid-phase extraction with the recovery of 85.3 ± 1.2%.

Effect of minimizing amount of template by addition of macromolecular crowding agent on preparation of molecularly imprinted monolith.

One of the main challenges in the preparation of molecularly imprinted polymers (MIPs) is the substantial initial amount of template needed because of the requirement of high load capacities for most applications. A new strategy of macromolecular crowding was suggested to solve this problem by reducing the amount of template in the polymerization recipe. In a ternary porogenic system of polystyrene (PS) (crowding agent), tetrahydrofuran, and toluene, an imprinted monolithic column with high porosity and good permeability was synthesized using a mixture of ellagic acid (template), acrylamide, and ethylene glycol dimethacrylate. The effect of polymerization factors, including monomer-template molar ratio and the molecular weight and concentration of PS, on the imprinting effect of the resulting MIP monoliths was systematically investigated. At a high ratio of monomer-template (120:1), the greatest imprinting factor of 32.4 was obtained on the MIP monolith with the aid of macromolecular crowding agent. The PS-based imprinted monolith had imprinting even at the extremely high ratio of functional monomer to template of 1510:1. Furthermore, an off-line solid-phase extraction based on the ground MIP was conducted, and the purification recovery of ellagic acid from pomegranate-rind extract was up to 80 %. In conclusion, this approach based on macromolecular crowding is simple, and is especially valuable for those applications of MIP preparation for which a rare template is used.