Gel Thickness Research Articles

Preparation of Cassia Bean Gum/Soy Protein Isolate Composite Matrix Emulsion Gel and Its Effect on the Stability of Meat Sausage.

The use of plant-derived emulsified gel systems as fat substitutes for meat products has always been an important direction in the development of healthy foods. In this study, a composite matrix emulsion gel was prepared with soy protein isolate (SPI) and different concentrations of cassia bean gum (CG), and then the selected emulsion gel was applied to meat sausage as a fat substitute to explore its stability. Our results showed that the hardness, chewiness, viscosity, shear stress, and G' and G″ moduli of the emulsion gel increased considerably with the cassia bean gum concentration, the thickness of the emulsion gel increased, and the pore size decreased. The gel strength of the 1.75% CG/SPI emulsion gel was the highest, which was 586.91 g. The elasticity was 0.94 mm, the masticability was 452.94 mJ, and the water-holding capacity (WHC) was 98.45%. Then, the 1.75% CG/SPI emulsion gel obtained via screening was applied as a fat substitute in meat sausage. With an increase in the substitution amount, the cooking loss, emulsification stability, pH, color difference, texture, and antioxidant activity of the meat sausage before and after freezing and thawing increased first and then decreased. The indexes of meat sausage with 50% fat replacement were not considerably different from those of full-fat meat sausage. This study can provide a theoretical basis for the application of plant-derived emulsified gel systems as fat substitutes in meat sausage.

Convenient rapid prototyping microphysiological niche for mimicking liver native basement membrane: Liver sinusoid on a chip

Liver is responsible for the metabolization processes of up to 90 % of compounds and toxins in the body. Therefore liver-on-a-chip systems, as an in vitro promising cell culture platform, have great importance for fundamental science and drug development. In most of the liver-on-a-chip studies, seeding cells on both sides of a porous membrane, which represents the basement membrane, fail to resemble the native characteristics of biochemical, biophysical, and mechanical properties. In this study, polycarbonate (PC) and polyethylene terephthalate (PET) membranes were coated with gelatin to address this issue by accurately mimicking the native basement membrane present in the space of Disse. Various coating methods were used, including doctor blade, gel micro-injection, electrospinning, and spin coating. Spin coating was demonstrated to be the most effective technique owing to the ability to produce thin gel thickness with desirable surface roughness for cell interactions on both sides of the membrane. HepG2 and EA.HY926 cells were seeded on the upper and bottom sides of the gelatin-coated PET membrane and cultured on-chip for 7 days. Cell viability increased from 90 % to 95 %, while apoptotic index decreased. Albumin secretion notably rose between days 1–7 and 4–7, while GST-α secretion decreased from day 1 to day 7. In conclusion, the optimized spin coating process reported here can effectively modify the membranes to better mimic the native basement membrane niche characteristics.

A 96-Well Polyacrylamide Gel for Electrophoresis and Western Blotting.

Western blotting is a stalwart technique for analyzing specific proteins and/or their post-translational modifications. However, it remains challenging to accommodate more than ~10 samples per experiment without substantial departure from trusted, established protocols involving accessible instrumentation. Here, we describe a 96-sample western blot that conforms to standard 96-well plate dimensional constraints and has little operational deviation from standard western blotting. The main differences are that (i) submerged polyacrylamide gel electrophoresis is operated horizontally (similar to agarose gels) as opposed to vertically, and (ii) a 6 mm thick gel is used, with 2 mm most relevant for membrane transfer (vs ~1 mm typical). Results demonstrate both wet and semi-dry transfer are compatible with this gel thickness. The major tradeoff is reduced molecular weight resolution, due primarily to less available migration distance per sample. We demonstrate proof-of-principle using gels loaded with molecular weight ladder, recombinant protein, and cell lysates. We expect the 96-well western blot will increase reproducibility, efficiency (cost and time ~8-fold), and capacity for biological characterization relative to established western blots.

Colloidal magnetic gels for the decontamination of limited access metallic surfaces

Handling nuclear metallic waste is a crucial issue for the nuclear industry, notably by using adapted decontamination processes. Colloidal gels can be applied by spraying for nuclear decontamination of large and plane metallic surfaces, such as walls or floors. However, this implementation mode limits their use for the decontamination of inaccessible or complex geometries surfaces. For that purpose, decontamination magnetic gels have been formulated by incorporating magnetite particles in a pre-synthesized colloidal gel able to decontaminate stainless steel. Such gels can thus be attracted by a magnet, allowing remote application to surfaces with limited access. The presence of magnetite particles does not influence the inherent decontamination properties of the gel, but plays a significant role on the spreading properties by increasing the gel viscosity. Then, a linear relationship between the gel thickness and the decontamination possibilities has been highlighted. Furthermore, as the magnetite particles dissolve in the gel with time, spreading properties, and consequently the decontamination properties, are modified. Finally, a formulation compromise has to be found for a long-term storage of the gels, while maintaining their efficiency.

Simultaneous extraction and preconcentration of two dyes with the same color from food samples by dual gel electro-membrane and determination by spectrophotometric method

In this study, the dual gel electromembrane extraction (G-EME) method coupled with UV–Vis spectrophotometry was developed to monitor the trace amount of Tartrazine (Tt) and auramine O (AO) in food samples. These dyes have different acidic and basic properties, but their colors are the same. The main driving forces of the dual G-EME are electro-migration and electroendosmosis (EEO). Tt as acidic and AO as basic analyte from 10 mL aqueous sample solution (pH 5.27) with a voltage of 65 V and a time of 29 min were extracted into 600 µL acidic (pH 9) and basic (pH 4) acceptor solution after passing through the agar membrane. The parameters, including the agar gel concentration (% w/v), the thickness of the agar gel, the pH of the gel, and the acceptor phases were optimized by one factor at a time also the design of the Experiment program was used to analyze and optimize the parameters such as the pH of the donor phase, voltage, time and stirring rate. Under the optimal conditions, the linear range for both dyes Tt and AO was 0.05–2.0 μg mL−1 with R2 > 0.99, the detection limits were 0.0256 and 0.0310 μg mL−1 and % recovery 85.0–111 and 91.7–112 respectively. Finally, the proposed method was successfully applied to the determination of Tt and AO in food samples. This novel approach offers a sensitive and accurate way to monitor the presence of these dyes in food products, which can be crucial for ensuring food safety and quality control.

Dextrin-assisted gel electromembrane extraction of chiral drugs: Improving the extraction efficiency and investigation of enantioselectivity of extraction

The present study investigates the use of dextrins (maltodextrin, β-cyclodextrin, and hydroxypropyl-β-cyclodextrin) to improve the efficiency of the agarose-based gel electromembrane extraction technique for extracting chiral basic drugs (citalopram, hydroxyzine, and cetirizine). Additionally, it examines the enantioselectivity of the extraction process for these drugs. To achieve these, dextrins were incorporated into either the sample solution, the membrane, or the acceptor solution, and then the extraction procedure was performed. Enantiomers were separated and analyzed using a capillary electrophoresis device equipped with a UV detector. The results obtained under the optimal extraction conditions (sample solution pH: 4.0, acceptor solution pH: 2.0, gel membrane pH: 3.0, agarose concentration: 3 % w/v, stirring rate: 1000 rpm, gel thickness: 4.4 mm, extraction voltage: 62.3 V, and extraction time: 32.1 min) indicated that incorporating dextrins into either the sample solution, membrane or the acceptor solution enhances extraction efficiency by 17.3–23.1 %. The most significant increase was observed when hydroxypropyl-β-cyclodextrin was added to the acceptor solution. The findings indicated that the inclusion of hydroxypropyl-β-cyclodextrin in the sample solution resulted in an enantioselective extraction, yielding an enantiomeric excess of 6.42–7.14 %. The proposed method showed a linear range of 5.0–2000 ng/mL for enantiomers of model drugs. The limit of detection and limit of quantification for all enantiomers were found to be < 4.5 ng/mL and <15.0 ng/mL, respectively. Intra- and inter-day RSDs (n = 4) were less than 10.8 %, and the relative errors were less than 3.2 % for all the enantiomers. Finally, the developed method was successfully applied to determine concentrations of enantiomers in a urine sample with relative recoveries of 96.8–99.2 %, indicating good reliability of the developed method.

Application of diffusive gradient in thin films probes to monitor trace levels of labile methylmercury in freshwaters.

This study aimed to optimize the methods for sampling and analyzing methylmercury (MeHg) concentrated within diffusive gradients in thin films (DGT) and its application to different water bodies. We explored the elution solution for MeHg, comprised of 1.13 mM thiourea and 0.1M HCl, optimizing its volume to 50 mL. In addition, we found that it is necessary to analyze the entire extraction solution after adjusting its pH, to ensure completion of the ethylation reaction. The DGT samplers were deployed in two distinct aquatic environments (i.e., Okjeong Lake and Nakdong River) for up to 6 weeks, and this study demonstrated to predict the time-weighted average concentration with a diffusion coefficient of 7.65 × 10-6 cm2 s-1 for MeHg in the diffusive gel. To assess the diffusive boundary layer (DBL) effects, the DGT samplers with different agarose diffusive gel thickness were deployed. The mass of MeHg accumulated in the DGT resin at a given time decreased with increasing diffusive gel thickness, because of creating longer diffusion pathways within thicker gels. The labile MeHg concentration estimated by the DGT in Okjeong Lake and Nakdong River are found in the range of 61-111 and 55-105 pg L-1, respectively, which were found to be similar to the grab sampling data. Additionally, this study evaluated depth-dependent MeHg in Okjeong Lake. The vertical profile results showed that the concentration of MeHg at the depth of 2.3 and 15.7 m are about 1.5 and 4.6 times of the DGT installed at 0.3 m of the surface layer, respectively, suggesting potential mercury methylation in deep waters. These findings have practical implications for predicting bioavailability, assessing risks, and formulating strategies for water body management and contamination remediation.

A gel-coated air-liquid-interface culture system with tunable substrate stiffness matching healthy and diseased lung tissues.

Since its invention in the late 1980s, the air-liquid-interface (ALI) culture system has been the standard in vitro model for studying human airway biology and pulmonary diseases. However, in a conventional ALI system, cells are cultured on a porous plastic membrane that is much stiffer than human airway tissues. Here, we develop a gel-ALI culture system by simply coating the plastic membrane with a thin layer of hydrogel with tunable stiffness matching that of healthy and fibrotic airway tissues. We determine the optimum gel thickness that does not impair the transport of nutrients and biomolecules essential to cell growth. We show that the gel-ALI system allows human bronchial epithelial cells (HBECs) to proliferate and differentiate into pseudostratified epithelium. Furthermore, we discover that HBECs migrate significantly faster on hydrogel substrates with stiffness matching that of fibrotic lung tissues, highlighting the importance of mechanical cues in human airway remodeling. The developed gel-ALI system provides a facile approach to studying the effects of mechanical cues in human airway biology and in modeling pulmonary diseases.NEW & NOTEWORTHY In a conventional ALI system, cells are cultured on a plastic membrane that is much stiffer than human airway tissues. We develop a gel-ALI system by coating the plastic membrane with a thin layer of hydrogel with tunable stiffness matching that of healthy and fibrotic airway tissues. We discover that human bronchial epithelial cells migrate significantly faster on hydrogel substrates with pathological stiffness, highlighting the importance of mechanical cues in human airway remodeling.

The role of poroelastic diffusion in the transient wetting behavior of hydrogels.

Wetting and water absorption of hydrogels is often encountered in many applications. We seek to understand how wetting behavior can be affected by the time-dependent swelling of hydrogels. We measured the advancing contact angles of water droplets on hydrogels of varying thicknesses where thicker gels absorbed water more slowly. We also observed that, above a threshold advancing speed, water droplets would collapse into a lower contact angle state on the surface. We hypothesized that this collapse threshold speed is a result of competition between the poroelastic diffusion of water into the gel and the advance of the spreading droplet, the thickness of the surface, and the diffusion of water into the gel. Taking the ratio of the diffusion and advancing timescales results in a Peclet number with gel thickness as a characteristic length scale. Our results show that above a Peclet number of around 40, droplets will collapse on the surface across all gel thicknesses, confirming our hypothesis. This work provides simple insight to understand a complex time-dependent wetting phenomenon for a widely used hydrogel.

Biosensor for Rapid Measurement of Lactate in Exhaled Breath Condensate

Lactate concentration plays an important role in disease diagnostics. Higher lactate concentrations are associated with sepsis and septic shock [2], trauma [3], tissue hypoxia due to acute lung injury [4], and respiratory diseases [5]. Lactate level is also widely used in monitoring athletes’ training and fitness as long-term exhausting exercise leads to a rapid increase in lactate level and result in muscle weakening and fatigue [6]. Compared with the traditional blood sample media which is invasive and capillary blood monitoring is not suitable for routine clinical use, the exhaled breath condensate (EBC) is simple and non-invasive method with no potential for adverse effects for lactate analysis.In this presentation, a point-of-care electrochemical biosensor is introduced for the EBC lactate analysis. This disposable and low-cost sensor is aimed to be inserted in an EBC collection device developed by our team that enables real-time monitoring and rapid measurements of EBC lactate level. The sensor measurement is done directly after EBC sample collection, that minimizes the risk of sample degradation and is cheaper than the traditional laboratory techniques such as fluorometry or mass spectrometry.The sensor is modified with poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), Prussian blue (PB) and lactate oxidase (LOD) enzyme. Lactate concentration is measured from amperometric current response generated by the redox reactions on sensor surface. The enzyme gel formulation is optimized based on rotating disk electrode (RDE) experiments and kinetics analysis, investigating the effect of LOD concentration and gel thickness, and was then applied to disposable sensors.Finally, human EBC analysis is conducted on healthy subjects at rest and after 30 min of intense aerobic cycling exercise. Sensor’s results in real EBC lactate measurements demonstrate a good correlation with fluorometry and mass spectrometry calibrations and exhibit high stability and sensitivity in both short-term and long-term use. Mass production of the sensor is done by robotic gel dispensing using a Biodot aspirate dispensing machine.Note: This presentation is based on the corresponding author's recent publication ‘Rapid Measurement of Lactate in the Exhaled Breath Condensate: Biosensor Optimization and In-Human Proof of Concept’ in ACS Sensors (doi.10.1021/acssensors.2c01739) [1]

2D shear wave elastography for the assessment of quadriceps entheses-a methodological study.

To establish the scanning protocol for 2-dimensional shear wave elastography (SWE) on normal entheses by investigating the possible confounding factors that may increase the variability of measured elasticity. 30 normal quadriceps entheses were scanned using SWE to compare the stiffness and coefficient variation by changing the ultrasonic coupling gel thickness, knee position, region of interest size, and scanning plane. No significant difference in median shear wave velocity (SWV) was observed in different coupling gel thicknesses. The median SWV was higher in the knee flexion position than in the extended position (p < 0.001). Increased knee flexion led to stiffer quadriceps enthesis and higher SWV (ρ = 0.8, p < 0.001). The median SWV was higher when the diameter region of interest was 4.0mm than 2.0mm (p = 0.001). The median SWV was higher in the transverse plane than in the longitudinal plane (p < 0.001). Strong correlation was found between SWV and the degree of the shear wave to muscle fiber direction (ρ = 0.8, p < 0.001). The coefficient variation was lower in a gel thickness of 2.5cm, with an extended knee, a region of interest of 2.0mm, and a longitudinal plane (p > 0.05). For interobserver reliability for the proposed protocol, the intraclass correlation coefficients was 0.763. In this study, we determined supine position with the knee extended; using 2.0mm diameter region of interest and image acquisition at the longitudinal plane with thicker layer coupling gel seems most appropriate to reliably image healthy quadriceps entheses with SWE.

Gel-assisted Welding of Epoxy Composite with Ethylene Glycol Mixed Catalyst

Effective and high-strength welding of thermosetting composites is badly in demand and always a challenge in industrial applications. Here, a gel-assisted welding method was proposed for traditional epoxy composites using a gel of 1,5,7-triazabicyclo [4.4.0] dec-5-ene mixed with ethylene glycol (TBD/EG gel) for pretreatment. Firstly, the influence of TBD/EG gel thickness, pretreatment time, pretreatment temperature, and pretreatment after soaking for the welding strength of the epoxy composite was studied. Then, the appropriate TBD/EG gel thickness (2 mm) and TBD/EG gel pretreatment conditions (140 °C for 60 min) were obtained. Compared to the different pretreatment methods for welding, the welding of TBD/EG gel pretreatment is feasible.

Failure analysis of copper substrate crack in high-voltage power supply

Aiming at the crack phenomenon of a copper substrate circuit in a high-voltage power supply, the slice analysis was carried out and showed that the crack part was the dielectric layer of the copper substrate. The finite element analysis is used to study the reason for dielectric layer cracking and discuss the improvement measures from the thickness of thermal conductive gel and the layout of components. The simulations results show that the reason for dielectric layer cracking is that the surface-mounted device (SMD) is too close to the mounting hole when the copper substrate bottom is coated with thermal conductive gel, reducing the thickness of thermal conductive gel or keeping the SMD away from the mounting holes can avoid the dielectric layer cracking, but it is easier to change the location of SMD.

Experimental Investigation on the Penetrability Mechanism of Gel Slug During Well Completion Processes.

Reservoir formation damage is an essential problem which troubled oil and gas well production, a smart packer is a promising technology to maintain sustainable development for the oil and gas fields. Currently, the "gel valve" technology has been proved to be feasible with gel slug to seal casing and descend the completion pipe string, while the systemic performance of ideal gel is still not clear. In the underbalanced completion stage with the gel valve, the downward completion string needs to penetrate the gel slug to form an oil and gas passage in the wellbore. The penetration of rod string to gel is a dynamic process. The gel-casing structure often shows a time-dependent mechanical response, which is different from the static response. In the process of penetration, the interaction force between the rod and gel is not only related to the properties of the interface between gel and string but also affected by the moving speed, rod diameter, and thickness of the gel. The dynamic penetration experiment was carried out to determine the penetrating force varied with depth. The research showed that the force curve was mainly composed of three parts, the rising curve of elastic deformation, decline curve for the surface worn out, and another curve of the rod wear into. By changing the rod diameter, gel thickness, and penetration speed, the change rules of forces in each stage were further analyzed, which would provide a scientific basis for a well completion design with a gel valve.

Gelation and Orientation Dynamics Induced by Contact of Protein Solution with Transglutaminase Solution.

Gel growth induced by contact of polymer solutions with crosslinker solutions yields an emerging class of anisotropic materials with many potential applications. Here, we report the case of a study on the dynamics in forming anisotropic gels using this approach with an enzyme as a trigger of gelation and gelatin as the polymer. Unlike the previously studied cases of gelation, the isotropic gelation was followed by gel polymer orientation after a lag time. The isotropic gelation dynamics did not depend on concentrations of the polymer turning into gel and of the enzyme inducing gelation, whereas, for the anisotropic gelation, the square of the gel thickness was a linear function of the elapsed time, and the slope increased with polymer concentration. The gelation dynamics of the present system was explained by a combination of diffusion-limited gelation followed by free-energy-limited orientation of polymer molecules.

Relationship between Rate-Limiting Process and Scaling Law in Gel Growth Induced by Liquid-Liquid Contact.

Gelation through the liquid-liquid contact between a polymer solution and a gelator solution has been attempted with various combinations of gelator and polymer solutions. In many combinations, the gel growth dynamics is expressed as X∼t, where X is the gel thickness and t is the elapsed time, and the scaling law holds for the relationship between X and t. In the blood plasma gelation, however, the crossover of the growth behavior from X∼t in the early stage to X∼t in the late stage was observed. It was found that the crossover behavior is caused by a change in the rate-limiting process of growth from the free-energy-limited process to the diffusion-limited process. How, then, would the crossover phenomenon be described in terms of the scaling law? We found that the scaling law does not hold in the early stage owing to the characteristic length attributable to the free energy difference between the sol-gel phases, but it does in the late stage. We also discussed the analysis method for the crossover in terms of the scaling law.

Wettability of mg-ha/Chitosan-based membrane surfaces: blood vs. autologous platelet liquid (APL).

The physical and physical chemistry is able to influence the interaction of the scaffolds and bone substitutes with the body fluid and blood. The aim of the present investigation was to evaluate the wettability properties of an Mg-HA Chitosan-based Gel with blood vs. autologous platelet gel. A total of 6 study groups were evaluated according to the Mg-HA Chitosan-based Gel thickness (1, 2 and 3 mm) and the fluids (blood vs. autologous platelet gel). The biomaterial wettability was conducted through the sessile drop technique. The study findings showed a significant difference in contact angle between the APL and blood groups (p<0.05). The MG-Ha Chitosan-based membrane thicknesses seem to produce no significant effects on contact angles measurement for all groups (p>0.05). In the present investigation, a similar MG/Ha gel membranes wettability was reported between APL and blood groups. In addition, a high hydrophilicity of MG/Ha gel membranes was reported with a potential advantage in terms of a more effective osteogenic capability in the clinical practice.

Geometry of hydrogel rectal spacer placement and risk of MRI-identified rectal wall infiltration.

312 Background: The use of rectal spacers in the management of localized prostate cancer treated with definitive radiotherapy has become ubiquitous in recent years. However, pre-treatment MRIs often identify varying degrees of hydrogel involvement within the rectal wall. In the present study, we evaluate the geometry of spacer placement and its association with radiological rectal wall infiltration. Methods: We identified all patients who underwent hydrogel rectal spacer placement in preparation for 5-fraction prostate SBRT from 1/2020 to 9/2021. Two specialty trained body radiologists evaluated all MRIs independently. Scans were evaluated for the following spacer parameters: spacer thickness, prostate-rectal distance, symmetry, and degree of rectal wall infiltration. Prostate-rectal distance was measured at the level of the prostatic apex, midgland, and base. Symmetry of the rectal spacer was measured using right or left lateralization from midgland. Degree of rectal wall invasion was categorized as follows: none, muscularis, submucosal, and intraluminal. Results: A total of 336 patients underwent MRI following hydrogel rectal spacer placement from 1/2020 to 9/2021. Patients were excluded from MRI if they had AICD/pacemaker, foreign body, or patient refusal. In those patients with any rectal wall invasion, gel thickness as measured at the base (11 vs. 10 mm, p = 0.02), midgland (14 vs. 10 mm, p &lt; 0.001), and apex (12 vs. 8 mm, P &lt; 0.001) was significantly larger than those patients without invasion. This translated into significantly larger distances between the posterior aspect of the prostate and anterior aspect of the rectum at the level of the apex (12 vs. 8 mm, p &lt; 0.001) and midgland (13 vs. 11, p &lt; 0.001), but not at the base (14 vs. 14 mm, p = 0.5). There was no association seen with asymmetrical spacer placement and rectal wall invasion (p = 0.7). Subgroup analysis of patients with more extensive invasion into the muscularis or submucosa confirmed significantly larger gel thickness at all prostate levels, as well as a larger prostate-rectal distance at the level of the apex and midgland. Significant associations remained consistent with both independent radiological evaluations. Conclusions: Hydrogel spacer rectal wall infiltration was associated with increased axial gel thickness, specifically at the level of the prostatic midgland and apex. Rectal wall infiltration was not associated with lateralization of gel. Rectal infiltration may be a result of surplus hydrogel placed particularly in the region where the potential space between the prostate and the rectum is limited.

Evaluation of the optimal dose for maximizing the anti-adhesion performance of a self-degradable dextran-based material

LYDEX, which is a hydrogel composed of aldehyde-functionalized dextran and succinic anhydride-treated ε-poly-L-lysine, was previously developed by our group as a self-degradable adhesive with an excellent functionality. As a result, this hydrogel has been investigated for a wide range of medical applications, including hemostatic agents, sealants, wound dressings for endoscopes, and anti-adhesion materials. Previously, the unique potential of LYDEX to control the in vivo degradation rate depending on its composition was verified for use in anti-adhesion applications. Although the gel thickness also plays a significant role in determining the degradation rate, this issue was not sufficiently investigated in previous studies. Thus, in the current study, the optimal LYDEX dose (film thickness) for maximizing the anti-adhesion material performance was verified using a rabbit colon model and it was confirmed that the gel film thickness affects the degradation period and anti-adhesion effect. The obtained results suggest the potential for designing LYDEX materials suitable for application at a wider range of in vivo treatment sites, which could be extremely valuable for the development and use of novel anti-adhesion materials with adequate hemostatic and sealant performances.

Isolation of Isoflavones from Iraqi Trifolium pretense

Objective: Trifolium pretense considered one of the most important medicinal plant which belongs to Fabaceae family, this plant commonly known as Red clover, its native region is Europe, Western Asia, and northwest Africa, but planted and naturalised in many other regions. The main compoundes found in Trifolium pretense are isoflavones which is determinrd by genistein, daidzein, biochanin A and formononetin. These compounds structurally related to estradiol-17 beta. Mangement of cancer is the main medicinal uses of Trifolium pretense in addition to decrease cholesterol and LDL and had anti- inflammatory activity, antioxidant and its effect on the Skin, Appendages, and Mucosal Status in Postmenopausal Women. This study was designed to isolate the main isoflavones from Trifolium pretense. Methods: The plant was collecting and washed by tap water to remove dirt and dust and rinsed with distilled water then dried at room temperature. Then, the plant was extracted by ethanol in a soxhlet apparatus and the extract partitioned with ethyl acetate by separatory funnel. The ethyl acetate layer was subjected to standard methods for active constituents identification, and purified by column chromatography. The collected fractions were analysed by TLC comparing with standard isoflavones (genistein, daidzein, biochanin A and formononetin). Preparative TLC was performed using 0.5 mm thickness of silica gel; the isolated bands were conducted with AR grade acetone. Iisolated isoflavones were characterized by 1H and 13-CNMR. Results: Isolated isoflavones (genistein, daidzein, biochanin A and formononetin) were identified by melting points which were identical with to that which were reported in the literature. Moreover, the ¹H-NMR and 13C-NMR analysis were used to identify the target compounds in CDCl3 solvent, and the values of chemical shifts have been discussed according to the literature of analogous compounds.