Column Chromatography Research Articles

Effect of the 35 nm and 70 nm Size Exclusion Chromatography (SEC) Column and Plasma Storage Time on Separated Extracellular Vesicles

Effect of the 35 nm and 70 nm Size Exclusion Chromatography (SEC) Column and Plasma Storage Time on Separated Extracellular Vesicles

Improvement of functional dyspepsia with Suaeda salsa (L.) Pall via regulating brain-gut peptide and gut microbiota structure.

The traditional Chinese herbal medicine Suaeda salsa (L.) Pall (S. salsa) with a digesting food effect was taken as the research object, and its chemical composition and action mechanism were explored. The chemical constituents of S. salsa were isolated and purified by column chromatography, and their structures were characterized by nuclear magnetic resonance. The food accumulation model in mice was established, and the changes of the aqueous extract of S. salsa in gastric emptying and intestinal propulsion rate, colonic tissue lesions, serum brain-gut peptide hormone, colonic tissue protein expression, and gut microbiota structure were compared. Ten compounds were isolated from S. salsa named as naringenin (1), hesperetin (2), baicalein (3), luteolin (4), isorhamnetin (5), taxifolin (6), isorhamnetin-3-O-β-D-glucoside (7), luteolin-3'-D-glucuronide (8), luteolin-7-O-β-D-glucuronide (9), and quercetin-3-O-β-D-glucuronide (10), respectively. The aqueous extract of S. salsa can improve the pathological changes of the mice colon and intestinal peristalsis by increasing the rate of gastric emptying and intestinal propulsion. By adjusting the levels of 5-HT, CCK, NT, SS, VIP, GT-17, CHE, MTL, and ghrelin, it can upregulate the levels of c-kit, SCF, and GHRL protein, and restore the imbalanced structure of gut microbiota, further achieve the purpose of treating the syndrome of indigestion. The effect is better with the increase of dose. S. salsa has a certain therapeutic effect on mice with the syndrome of indigestion. From the perspective of "brain-gut-gut microbiota", the mechanism of digestion and accumulation of S. salsa was discussed for the first time, which provided an experimental basis for further exploring the material basis of S. salsa.

Determination of Bovine Lactoferrin in Powdered Infant Formula and Adult Nutritionals by Heparin Affinity Extraction and Reverse-Phase High Performance Liquid Chromatography/Ultraviolet Detection (HPLC/UV): Single-Laboratory Validation, First Action 2021.10.

Infant formulas, pediatric and adult nutritional products are being fortified with bovine lactoferrin (bLF) due to its beneficial impacts on immune development and gut health. Lactoferrin supplementation into these products requires an analytical method to accurately quantify the concentrations of bLF to meet global regulatory and quality standards. To develop and validate a lactoferrin method capable of meeting the AOAC Standard Method Performance Requirements (SMPR requirements 2020.005). Powder formula samples are extracted using warm dibasic phosphate buffer, pH 8, then centrifuged at 4 °C to remove insoluble proteins, fat, and other solids. The soluble fraction is further purified on a HiTrap heparin solid-phase extraction column to isolate bLF from interferences. Samples are filtered, then analyzed by LC-UV using a protein BEH C4 analytical column and quantitated via external calibrant. The limit of quantitation (2 mg/100g), repeatability (2.0-4.8% RSD), recovery (92.1-97.7%) and analytical range (∼4-193 mg/100g) all meet the method requirements as stated in SMPR 2020.005 for lactoferrin. The reported single lab validation results demonstrate the ability of this lactoferrin method to meet or exceed the method performance requirements to measure soluble, intact, non-denatured bLF in infant and adult nutritional powder formulas. The use of a heparin affinity column to isolate lactoferrin from bovine milk products combined with a selective analytical chromatographic column provides suitable analyte specificity without requiring proprietary equipment or reagents.

Evaluation of Novel Benzo-annelated 1,4-dihydropyridines as MDR Modulators in Cancer Cells.

Multidrug resistance (MDR) is the main problem in anticancer therapy today. Causative transmembrane efflux pumps in cancer cells have been reconsidered as promising anticancer target structures to restore anticancer drug sensitivity by various strategies, including MDR modulators. MDR modulators interfere with the efflux pumps and improve the cellular efficiency of chemotherapeutics. So far, only a few candidates have gone through clinical trials with disappointing results because of low specificity and toxic properties. This study aimed to find Novel MDR modulators to effectively combat multidrug resistance in cancer cells. We synthesized various novel benzo-annelated 1,4-dihydropyridines to evaluate them as MDR modulators towards ABCB1 in cancer cells. Synthesized compounds were purified by column chromatography. The MDR modulation of ABCB1 was determined in cellular efflux assays using the flow cytometry technique and cellular fluorescent measurements by the use of each fluorescent substrate. Compounds were yielded in a two-step reaction with structurally varied components. Further, substituent- dependent effects on the determined MDR inhibiting properties towards ABCB1 were discussed. Cellular studies prove that there is no toxicity or restoration of cancer cell sensitivity towards the used anticancer drug. Novel MDR modulators could be identified with favorable methoxy and ester group functions. Their use in both ABCB1 non-expressing and overexpressing cells proves a selective toxicity-increasing effect of the applied anticancer agent in the ABCB1 overexpressing cells, whereas the toxicity effect of the anticancer drug was almost unchanged in the non-expressing cells. These results qualify our novel compounds as perspective anticancer drugs compared to MDR modulators with nonselective toxicity properties.

Characterisation and critical processes identification for production of herbal preparations using 1H-NMR and chemometrics: A case study of Trichosanthis Pericarpium injection.

Herbal preparations are extensively utilised for the treatment of diseases in Asian countries. However, the variations in origin, climate, and production processes can lead to inconsistencies in the quality of herbal preparations. Existing quality control methods only target a few components in the finished product but ignore the control in the pharmaceutical process. Therefore, this study intends to develop a comprehensive component analysis method for intermediates in the pharmaceutical process to reveal the change patterns of substances and deepen the process understanding. This study aims to develop a rapid and comprehensive process characterisation and critical process identification method for herbal preparations. Six batches of Trichosanthis Pericarpium injection (TPI) intermediates were collected from the production process. Proton nuclear magnetic resonance (1H-NMR) spectra were acquired for qualitative and quantitative analysis of the se intermediates. Subsequently, chemometrics were used to identify critical processes and potential chemical markers. A total of 39 components in intermediates were identified, and the transfer of 25 components during the production process was investigated. Column chromatography was determined as the critical process. Nine components were identified as chemical markers. The application of 1H-NMR facilitated a comprehensive reflection of the chemical composition information of process intermediates, enabling investigations into the transfer of multi-component substances and accurate identification of critical processes and chemical markers.

Antibacterial activity of Dioscorea bulbifera Linn. extract and its active component flavanthrinin against skin-associated bacteria

BackgroundDioscorea bulbifera Linn. has been used for wound care in Thailand. However, a comprehensive evaluation of its antibacterial activity is required. This study aimed to investigate the antibacterial efficacy of D. bulbifera extract against skin-associated bacteria and isolate and characterize its active antibacterial agent, flavanthrinin.MethodsAir-dried bulbils of D. bulbifera were pulverised and extracted with hexane, dichloromethane, ethyl acetate, methanol, ethanol, and distilled water; vacuum filtered; concentrated; freeze-dried; and stored at -20 ºC. Antibacterial activity of the extracts was assessed using microdilution techniques against several skin-associated bacteria. Thin-layer chromatography (TLC) bioautography was used to identify the active compounds in the extract, which were fractionated by column chromatography and purified by preparative TLC. The chemical structures of the purified compounds were analysed using nuclear magnetic resonance (NMR). The cytotoxicity of the extract and its active compounds was evaluated in Vero cells.ResultsThe ethyl acetate extract exhibited distinct inhibition zones against bacteria compared to other extracts. Therefore, the ethyl acetate extract of D. bulbifera in the ethyl acetate layer was used for subsequent analyses. D. bulbifera extract exhibited antibacterial activity, with minimum inhibitory concentrations (MICs) of 0.78–1.56 mg/mL. An active compound, identified through TLC-bioautography, demonstrated enhanced antibacterial activity, with MICs of 0.02–0.78 mg/mL. NMR analysis identified this bioactive compound as flavanthrinin. Both D. bulbifera extract and flavanthrinin-containing fraction demonstrated potent antibacterial activity against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and S. epidermidis. The flavanthrinin containing fraction demonstrated low cytotoxicity against Vero cells, showing CC50 values of 0.41 ± 0.03 mg/mL. These values are lower than the MIC value, indicating that this fraction is safer than the initial ethyl acetate extract.ConclusionsDioscorea bulbifera extract and its bioactive component flavanthrinin demonstrated significant antibacterial activity against the skin-associated bacteria Staphylococci, including MRSA. Flavanthrinin has potential as a complementary therapeutic agent for managing skin infections owing to its potent antibacterial effects and low cytotoxicity.

Rosarugosides A and D from Rosa rugosa Flower Buds: Their Potential Anti-Skin-Aging Effects in TNF-α-Induced Human Dermal Fibroblasts

This present study investigated the anti-skin-aging properties of Rosa rugosa. Initially, phenolic compounds were isolated from a hot water extract of Rosa rugosa’s flower buds. Through repeated chromatography (column chromatography, MPLC, and prep HPLC), we identified nine phenolic compounds (1–9), including a previously undescribed depside, rosarugoside D (1). The chemical structure of 1 was elucidated via NMR, HR-MS, UV, and hydrolysis. Next, in order to identify bioactive compounds that are effective against TNF-α-induced NHDF cells, we measured intracellular ROS production in samples treated with each of the isolated compounds (1–9). All isolates reduced the level of ROS at a concentration of 10 μM. Particularly, two depsides—rosarugosides A and D (2 and 1)—significantly inhibited ROS expression in TNF-α-induced NHDFs compared to the other phenolic compounds. Subsequently, the production of MMP-1 and procollagen type Ι α1 by these two depsides was examined. Remarkably, rosarugoside A (2) significantly decreased MMP-1 secretion at all concentrations. In contrast, rosarugoside D (1) regulated the expression of procollagen type Ι α1. These findings collectively suggest that Rosa rugosa extracts and their isolated compounds, rosarugosides A (2) and D (1), hold significant potential for protecting against aging and skin damage. Overall, these findings suggest that Rosa rugosa extracts and their isolated compounds, rosarugosides A (2) and D (1), have the potential to prevent and protect against aging and skin damage, although more specific quantitative analysis is needed.

Preliminary Screening for the Anthelmintic Activity of Millettia zechiana Harms and Its Modifying Effect on Albendazole

Helminthic infections affect a greater proportion of the world’s population. This study determined the anthelmintic activity of Millettia zechiana and its modifying effect on albendazole. Powdered leaves of M. zechiana were successively extracted with petroleum ether, ethyl acetate, and ethanol. The anthelmintic potential of the M. zechiana leaf extracts and the modifying effects of the extracts on albendazole were determined on Pheretima posthuma. Phytochemical and gas chromatography-mass spectroscopy (GC-MS) analyses were performed to determine the chemical composition of each extract. The plant extracts of M. zechiana had few or all phytoconstituents such as tannins, saponins, flavonoids, glycosides, terpenoids, phytosterols, and alkaloids present. The IC50 obtained for albendazole, petroleum ether, ethyl acetate, and ethanol extracts for paralysis time were 0.936, 1.722, 1.283, and 1.348 mg/mL, respectively. The IC50 obtained for albendazole and the ethanol extract for death time were 4.638 and 4.988 mg/mL. The ethanol extract at 10 and 5 mg/mL caused death in the worms after 152.5±8.66 minutes and 304.8±7.27 minutes of exposure, respectively. Ethanol, ethyl acetate, and petroleum ether extracts of M. zechiana significantly modified the activity of albendazole at concentrations of 2.5 and 1.25 mg/mL (P<0.0001). The ethanol extract which exhibited the best anthelminthic activity was fractionated through column chromatography, and five (5) fractions were obtained. Fractions 1, 2, 4, and 5 had the best paralytic activities against the worms. Fractions 1 and 2 demonstrated better helminthicidal activity than albendazole, which had an IC50 of 3.915. The GC-MS analysis of the ethanol, ethyl acetate, and petroleum ether extracts showed the presence of 10, 10, and 37 compounds, respectively, with 9-octadecenamide, (Z)-, n-hexadecanoic acid, oleic acid, and some aromatic compounds being the most predominant. The results obtained indicate that M. zechiana leaf extract possesses anthelmintic activity.

A Fully Automated Online Enrichment and Separation System for Highly Reproducible and In-Depth Analysis of Intact Glycopeptide.

A fully automated online enrichment and separation system for intact glycopeptides, named AutoGP, was developed in this study by integrating three different columns in a nano-LC system. Specifically, the peptide mixture from the enzymatic digestion of a complex biological sample was first loaded on a hydrophilic interaction chromatography (HILIC) column. The nonglycopeptides in the sample were washed off the column, and the glycopeptides retained by the HILIC column were eluted to a C18 trap column to achieve an automated glycopeptide enrichment. The enriched glycopeptides were further eluted to a C18 column for separation, and the separated glycopeptides were eventually analyzed by using an orbitrap mass spectrometer (MS). The optimal operating conditions for AutoGP were systemically studied, and the performance of the fully optimized AutoGP was compared with a conventional manual system used for glycopeptide analysis. The experimental evaluation shows that the total number of glycopeptides identified is at least 1.5-fold higher, and the median coefficient of variation for the analyses is at least 50% lower by using AutoGP, as compared to the results acquired by using the manual system. In addition, AutoGP can perform effective analysis even with a 1-μg sample amount, while a 10-μg sample at least will be needed by the manual system, implying an order of magnitude better sensitivity of AutoGP. All the experimental results have consistently proven that AutoGP can be used for much better characterization of intact glycopeptides.

GC–MS based antioxidants characterization in Saussurea heteromalla (D. Don) Hand-Mazz by inhibition of nitric oxide generation in macrophages

For centuries, medicinal plants have served as the cornerstone for traditional health care systems and same practice is still prevalent today. In the Himalayan region, Saussurea heteromalla holds a significant place in traditional medicine and is used to address various health issues. Despite its historical use, little exploration has focused on its potential for scavenging free radicals and reducing inflammation. Hence, our current study aims to investigate the free radical scavenging capabilities of S. heteromalla extracts. The n-hexane extract of entire plant revealed promising activity. This extract underwent extensive extraction on a larger scale. Subsequent purification, employing column chromatography, HPLC–DAD techniques, led to the identification of active compounds, confirmed via GC–MS and the NIST database as 1-O-butyl 2-O-octyl benzene-1,2-dicarboxylate and 2,4-ditert-butylphenol. Assessing the free radical scavenging properties involved utilizing RAW-264.7 macrophages activated by lipopolysaccharides. Notably, the compound 2,4-di-tert-butylphenol exhibited remarkable scavenging abilities, demonstrating over 80% inhibition of Nitric oxide. This study stands as the inaugural report on the isolation of these compounds from S. heteromalla.

Investigating the Anti-adipogenic Potential of Xanthium strumarium Linn. Leaves Fractions: HPTLC-MS Characterization, Enzymatic Analysis, and 3T3-L1 Adipocyte Differentiation Assay in Oxidative Stress-modulated Adipogenesis Cascade

Background: Molecular pathogenesis of obesity is initiated by cellular oxidative stress along with increased adipogenesis. For the search of better alternatives of synthetic molecules in terms of their adverse events, naturally, existing compounds are now a research focus. In this context, Xanthium strumarium ( X. strumarium) Linn. exhibited anti-diabetic activities and proved to have efficacy in neutralizing very near pathological targets to obesity. Purpose: This study is aimed toward bioactivity-guided fraction isolation of methanolic leaves extract, where bioactivity refers to anti-adipogenesis and anti-oxidant efficacy in in vitro enzymatic and three-day transfer, inoculum 3×105 cells (3T3-L1) adipocyte differentiation assay. Materials and method: Collection and drying of the raw leaves followed by cold extraction in methanol and flash chromatography for bioactive fraction isolation. 3T3-L1 cell line was utilized for adipocyte differentiation assay of those and on the other hand in vitro pancreatic lipase, α-glucosidase, and α-amylase enzymatic assays were analyzed. Based on exhibited activities, selected fractions are further characterized by high-performance thin-layer chromatography-mass spectrometry (HPTLC-MS). Results: The most active fractions from these assays underwent characterization via HPTLC-MS. Fractions 2 and 3 exhibited potent activities in all enzymatic assays (α-glucosidase IC50: 2.48 ± 0.015, 2.84 ± 0.030 µg/mL; α-amylase IC50: 1.98 ± 0.050, 1.79 ± 0.045 µg/mL; pancreatic lipase IC50: 3.16 ± 0.030, 3.18 ± 0.040 µg/mL) and displayed significant inhibition of adipocyte differentiation in 3T3-L1 cell line. These fractions further identified through HPTLC-MS, contained compounds like β-sitosterol and quercetin, affirming their potential bioactivity. Conclusion: The study underscores the potential of X. strumarium Linn. fractions as promising natural alternatives for combating obesity-related pathways, highlighting their significance in developing future anti-obesity therapeutics.

Maximization of uricase production in a column bioreactor through RSM based optimization.

Uricase (EC 1.7.3.3) is an oxidoreductase enzyme which is widely exploited for diagnostic and treatment purposes in medicine. This study has been focused on producing recombinant uricase from E. coli BL21 in a bubble column bioreactor (BCB) and finding the optimal conditions for maximum uricase activity. The three most effective variables on uricase activity were selected through Plackett-Burman design from 8 different variables and were further optimized by central composite design of RSM. The selected variables included the inoculum size (%v/v), IPTG (Isopropyl β-d-1-thiogalactopyranoside) concentration (mM) and the initial pH of culture medium. The activity of uricase, final OD600 (Optical Density at 600nm wave-length) and final pH were considered as the responses of this optimization and were modeled. As a result, the activity of 5.84 U.ml-1 and final OD600 of 3.42 were obtained at optimum conditions of 3 %v/v of inoculum size, IPTG concentration of 0.54 mM and pH=6.0. By purifying the obtained enzyme using a Ni-NTA agarose affinity chromatography column, 165±1.5 mg uricase was obtained from a 600 mL cell culture. The results of this study show that bubble column bioreactors can be a highly effective option for large scale uricase production.

Two Novel Angiotensin-Converting Enzyme (ACE) Inhibitory and ACE2 Upregulating Peptides from the Hydrolysate of Pumpkin (Cucurbita moschata) Seed Meal.

Pumpkin (Cucurbita moschata) seed meal (PSM), the major byproduct of pumpkin seed oil industry, was used to prepare angiotensin-converting enzyme (ACE) inhibitory and angiotensin-converting enzyme 2 (ACE2) upregulating peptides. These peptides were isolated and purified from the PSM hydrolysate prepared using Neutrase 5.0 BG by ultrafiltration, Sephadex G-15 column chromatography, and reversed-phase high-performance liquid chromatography. Two peptides with significant ACE inhibition activity were identified as SNHANQLDFHP and PVQVLASAYR with IC50 values of 172.07 and 90.69 μM, respectively. The C-terminal tripeptides of the two peptides contained Pro, Phe, and Tyr, respectively, and PVQVLASAYR also had Val in its N-terminal tripeptide, which was a favorable structure for ACE inhibition. Molecular docking results declared that the two peptides could interact with ACE through hydrogen bonds and hydrophobic interactions. Furthermore, the two peptides performed protective function on EA.hy926 cells by decreasing the secretion of endothelin-1, increasing the release of nitric oxide, and regulating the ACE2 activity. In vitro simulated gastrointestinal digestion showed the two peptides exhibited good stability against gastrointestinal enzyme digestion. In conclusion, PSM is a promising material for preparing antihypertensive peptides.

Radical scavenging dihydroxycinnamic natural compounds from Trianthema pentandra

BackgroundTrianthema pentandra is a promising plant with numerous medical applications. Previous studies have confirmed that the plant is strongly cytotoxic in brine shrimp lethality tests and has antibacterial effects. However, the compounds responsible for these biological activities have not been identified. This study aimed to screen plants in an antiradical assay using 1,1-diphenyl-2-picrylhydrazyl (DPPH). This assay was used to guide the isolation of active compounds, which were identified at the molecular structural level by spectroscopic methods. MethodsThe plant was extracted using ethanol, labelled F1 and fractionated by solvents of different polarities (F2 chloroform, F4 ethyl acetate, F5 aqueous methanol, and F6 n-hexane). The ethyl acetate and aqueous methanol (F4 and F5) fractions were selected for further purification based on their yields and activities. They were run on chloroform-methanol and n-hexane-ethyl acetate gradients, respectively. Fractions resulting from column chromatography were further screened for DPPH antioxidant activity, upon which fraction F5-1-077 had unusually high activity (IC50 = 3.57 µg/ml). Further purification of these fractions through recrystallization and high-performance liquid chromatography (HPLC) led to the isolation of three compounds. Results: Among these compounds, two were identified and characterized using FT-IR, LC-MS, and complete nuclear magnetic resonance (NMR) data, and 2,3-dihydroxycinnamic acid ethyl ester and its analog acid were isolated from the ethyl acetate fraction (F4). The former compound has been proven to have good activity in comparison with standards, ascorbic acid, and butylated hydroxytoluene (BHT), even at a low concentration of 5 µg/ml with an IC50 value of 0.13 µg/ml in the DPPH assay. The molecular docking data supported the potent activity of the compound with binding affinity, ∆G = -6.4 kcal/mol compared to the ascorbic acid (∆G = -5.0 kcal/mol) used as standard. ConclusionThis study serves as a groundbreaking foundation for the investigation of the natural dihydroxycinnamic compounds present in this plant.

Anti-inflammatory activities of some Anthemis species used in the treatment of inflammation-related diseases, GC/MS and LC-MS/MS analysis with bioactivity-guided fractionation

Recent studies have focused on Anthemis sp. because of their rich chemical composition and application in the treatment of diseases associated with inflammation. The purpose of this study was to determine the active chemicals responsible for the anti-inflammatory properties of Anthemis cotula, A. tomentosa, A. austriaca, and A. tinctoria var. tinctoria. Therefore, the 5-Lipoxygenase (5-LOX) was used to assess the anti-inflammatory properties of methanol extracts that were extracted from the aerial parts of the mentioned species, with an IC50 value of 23.88 μg/mL, the methanol extract of A. tinctoria var. tinctoria demonstrated anti-inflammatory effects close to the standard (Indomethacin, IC50: 18.05 μg/mL). Compared to the other extracts, the methanol extract of A. tinctoria var. tinctoria demonstrated significantly greater anti-inflammatory activity. A. tinctoria var. tinctoria and A. tomentosa methanol extracts, which were the most active in comparison to other extracts, were fractionated using the bioactivity-guided fractionation method. Their anti-inflammatory properties were evaluated to determine the compounds responsible for the activity. With an IC50 value of 3.44 μg/mL, the ethyl acetate fraction of A. tinctoria var. tinctoria (ATiEA) was found to be the most active of all the fractions. After ATiEA was further fractionated using column chromatography, it was found that ATiEA-II, one of its sub-fractions, was more effective than other sub-fractions against the 5-LOX, with an IC50 value of 2.93 μg/mL. Furthermore, the sub-fractions' total phenol and flavonoid contents were examined to confirm their anti-inflammatory properties. The sub-fraction ATiEA-II exhibited higher total flavonoid content (743,86 μg QE/g dry weight) and total phenol content (4659.36 μg GA/g dry weight) than other sub-fractions, which was in line with its anti-inflammatory activity. GC/MS and LC-MS/MS were used to clarify the effective compounds of the sub-fraction ATiEA-II. Experiments on the anti-inflammatory properties of different Anthemis sp. have demonstrated the good anti-inflammatory potential of this species. Specifically, ATiEA-II, a sub-fraction of A. tinctoria var. tinctoria, may contain a novel anti-inflammatory drug molecule.

Ultrasonication assisted percolation extraction, isolation, identification and in silico studies of secondary metabolites from Caesalpinia bonduc (L.) Roxb. seeds with special reference to the target proteins involved in Benign Prostatic Hyperplasia

Medicinal plant-derived phytochemicals provide a vast pool of diverse bioactive compounds for novel drug development. Caesalpinia bonduc L. Roxb. is a traditional medicinal plant that is reported for diverse pharmacological properties. This study aims to compare conventional (maceration) and novel extraction techniques (ultrasonication assisted maceration extraction) to evaluate their efficiency, yield and bioactive potential of hydroalcoholic extracts from C. bonduc seeds. Furthermore, the study also aimed to isolate the bioactive compounds from the hydroalcoholic seed extract obtained from ultrasonication assisted maceration and in-silico studies of isolated compounds against the protein targets involved in Benign Prostatic Hyperplasia (BPH). For comparing the influence of extraction methods, the seeds were extracted using a novel and conventional techniques. Chemical profiling, antioxidant activities, cytotoxic and apoptotic studies were conducted using obtained extracts. The extract yielded from the novel technique recorded the highest yield, significant antioxidant activity, cytotoxicity and chemical content when compared to the conventional technique. From the hydroalcoholic extract, three major bioactive compounds, namely ethyl glucoside, methyl lineolate and linoleic acid, were isolated using column chromatography. The structures of the compounds were elucidated on the basis of extensive NMR (1H, 13C, DEPT, 2D NMR- HMBC and HSQC) and HR-ESI-MS analysis. In-silico anti-BPH activity of isolated compounds clearly reveals that the compounds were targeting 5α‒Reductase and α1-Adrenoceptor linked to the BPH progressions. Based on this investigation, it is demonstrated that isolated molecules or active molecule-enriched fractions of C. bonduc should be further researched to confirm the anti-BPH activity through in-depth preclinical and clinical research for the development of products. Ethyl glucoside and methyl lineolate were isolated for the first time from C. bonduc.

Two-dimensional chiral metal-organic framework nanosheets L-hyp-Ni/Fe@SiO2 composite for HPLC separation

In this study, we have synthesised a chiral L-hyp-Ni/Fe@SiO2 composite as a chiral stationary phase (CSP) for high-performance liquid chromatography (HPLC) for the first time. This was achieved by coating two-dimensional (2D) chiral metal-organic framework nanosheets (MONs) L-hyp-Ni/Fe onto the surface of activated SiO2 microspheres using the “wrapped in net” method. The separation efficiency of the L-hyp-Ni/Fe chromatographic column was systematically evaluated in normal-phase HPLC (NP-HPLC) and reversed-phase HPLC (RP-HPLC) configurations, employing various racemates as analytes. The findings revealed that 16 chiral compounds were separated using NP-HPLC, and five were separated using RP-HPLC, encompassing alcohols, amines, ketones, esters, alkanes, ethers, amino acids and sulfoxides. Notably, the resolution (Rs) of nine chiral compounds exceeded 1.5, indicating baseline separation. Furthermore, the resolution performance of the L-hyp-Ni/Fe@SiO2-packed column was compared with that of Chiralpak AD-H. It was observed that certain enantiomers, which either could not be resolved or were inadequately separated on the Chiralpak AD-H column, attained separation on the 2D chiral MONs column. These findings suggest a complementary relationship between the two columns in racemate separation, with their combined application facilitating the resolution of a broader spectrum of chiral compounds. In addition, baseline separation was achieved for five positional isomers on the L-hyp-Ni/Fe@SiO2-packed column. The effects of the analyte mass and column temperature on the resolution were also examined. Moreover, during HPLC analysis, the L-hyp-Ni/Fe columns demonstrated commendable repeatability, stability and reproducibility in enantiomer separation. This research not only advances the utilisation of 2D chiral MONs as CSPs but also expands their applications in the separation sciences.

Structural characterization of the polysaccharide from the black crystal region of Inonotus obliquus and its effect on AsPC-1 and SW1990 pancreatic cancer cell apoptosis

In this study, one water soluble polysaccharide (IOP1–1) with a weight average molecular weight of 6886 Da was obtained from the black crystal region of Inonotus obliquus by hot water extraction, DEAE-52 cellulose extraction and Sephadex-100 column chromatography purification. Structural analysis indicated that IOP1–1 was a glucan with a main chain composed of α-Glcp-(1 → 4)-α-Glcp-(1 → 4)-β-Glcp-(1 → 4)-β-Glcp-(1 → 4)-α-Glcp-(1 → 6)-β-Glcp-(1 → 4)-α-Glcp-(1 → 3)-β-Glcp-(1→. The CCK-8 assay results showed that IOP1–1 inhibited AsPC-1 and SW1990 pancreatic cancer cell proliferation in a concentration-dependent manner. Flow cytometric analysis revealed that IOP1–1 induced cell cycle arrest in AsPC-1 and SW1990 cells. Hoechst 33342 staining and Annexin V-FITC/PI double staining analysis showed that IOP1–1 could induce apoptosis in AsPC-1 and SW1990 cells. Furthermore, western blot analysis confirmed that IOP1–1 could induce apoptosis in AsPC-1 and SW1990 pancreatic cancer cells through three pathways: the mitochondrial pathway, the death receptor pathway, and endoplasmic reticulum stress. According to these research data, IOP1–1 may be utilized as an adjuvant treatment to anticancer medications, opening up new application prospects and opportunities.

Isolation, characterization, identification and quantification of 6-F oxyphenisatin dipropionate, a novel illegal additive, from a fruit-flavored jelly.

This study is aimed to screen, identify and detect illegal additives from healthcare products which claim or imply to have weight-loss effects. Ultra-high performance liquid chromatography-quadruple-time-of-flight mass spectroscopy (UPLC-Q-TOF/MS) was employed to perform non-targeted screening of illegal additives from a total of 26 batches of healthcare products with weight-loss effects. A novel oxyphenisatin dipropionate analog was discovered in a fruit-flavored jelly that was not clearly labeled as containing added drugs. After being separated and purified by silica gel column chromatography, the analog was unambiguously characterized by one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopies. The molecular structure of the analog was finally identified by comparing the spectra of the analog with those of suspected candidates prepared by de novo synthesis strategy. Thereafter, a sensitive and precise reversed phase ultra performance liquid chromatography coupled with photodiode array (UPLC-PDA) detection method was developed and verified for the determination of the analog in 15 batches of real samples. In the MS/MS spectra, the signal intensity of mass/charge ratios (m/z, 242 and 214) of the novel analog fragments was highly similar to that of mass/charge ratios (m/z, 224 and 196) of oxyphenisatin dipropionate fragments. Additionally, the 1D NMR spectrum of the analog was completely consistent with that of one of the suspected candidates prepared by the de novo synthesis strategy. Based on the above analysis, the structure of the analog was determined as 3,3-bis[4'-(propionyloxy)phenyl]-6-fluoro-2-oxoindoline, which was briefly named 6-F oxyphenisatin dipropionate. A developed quantitative method showed good linearity (R2>0.999) in a concentration range of 1.0-100μg/mL. The limits of detection (LOD) and quantification (LOQ) for the analog was 3mg/kg and 10mg/kg, respectively. The average recoveries of the analog from spiked three different matrix samples in low (1 time of LOQ), medium (2 times of LOQ), and high (10 times of LOQ) concentrations were varied from 93.9% to 107.8% with a precision of 0.03-1.56%. Results of quantitative analysis in 15 batches of healthcare products revealed that the content of 6-F oxyphenisatin dipropionate in a fruit-flavored jelly and a solid beverage was 118mg/kg and 330mg/kg, respectively. In terms of its structure, 6-F oxyphenisatin dipropionate replaces hydrogen atom by the fluorine atom at position 6 on the indolinone fragment in oxyphenisatin dipropionate. To our best knowledge, 6-F oxyphenisatin dipropionate has never been detected as an illegal additive in foods. Such illegal addition of the analog to foods is more concealing, thus the supervision and testing departments should attach great importance to its application in food markets.

Antidiabetic and antihyperlipidemic effects of crude fractions from Chlorophytum borivilianum root methanolic extract on streptozotocin induced diabetic rats and phytochemical investigation by LCMS analysis.

To evaluate the in vitro and in vivo pharmacological efficacy of the plant Chlorophytum borivilianum in diabetes and hyperlipidemia and to confine and describe the synthetic constituents from the roots that are in charge of the action. The present study was carried out to investigate the ethno-medical use of Chlorophytum borivilianum root methanolic extract as a potential anti-diabetic and antihyperlipidemic agent in STZ-induced diabetic rats. Extract was tested for in vitro and in vivo biological activities. Soxhlet extraction was carried out using methanol as a solvent, and TLC and column chromatography were used for fractionation. Liquid Chromatography and Mass Spectroscopic study confirmed the structures of isolated compounds. Chlorophytum borivilianum root methanolic extract showed the presence of phytoconstituents as Dihydrocapsaicin, Reserpine, Deserpidine, Biliverdin-IX-α, and Cassiamin C having a therapeutic effect. Dihydrocapsaicin was identified at RT 7.572 and the Chlorophytum borivilianum root chloroform methanolic extract fraction noticeably depleted increased blood glucose levels and had positive effects on altered lipid profile after administering a dose of 150 mg/kg orally compared with oral hypoglycemic drug metformin. All the results are dose-dependent. Active chloroform-methanol fraction from methanol extract showed the presence of anti-diabetic compound, Dihydrocapsaicin. The chloroform-methanol fraction from the methanolic extract of Chlorophytum borivilianum root can inhibit the parameters linked to diabetes and hyperlipidemia.