Thin Layer Chromatography Analysis Research Articles

Extraction and Purification of Squalene from Virgin Olive Oil via Catalytic Transesterification and Molecular Distillation

Squalene, a highly valuable compound abundant in various natural sources, shows great potential in pharmaceutical, cosmetic, and nutraceutical applications. This research article outlines a comprehensive methodology for extracting and purifying squalene from virgin olive oil, a rich source of the compound. The extraction process begins with degumming, which involves heating the olive oil to 60-70°C to reduce viscosity, followed by the addition of 2-3% warm water to hydrate phospholipids. Food-grade phosphoric acid is then added to react with the phospholipids, forming precipitates. The mixture is stirred for 20-30 minutes and allowed to rest for an additional 20-30 minutes, enabling impurities to settle. The upper layer of degummed oil is separated via decantation or centrifugation and washed with warm water for pH adjustment. Next, transesterification is performed by mixing 100 ml of virgin olive oil with 25% methanol (w/w) and a catalyst (0.5% sodium methoxide or PTSA), and heating the mixture to 80-90°C under reflux for 1-2 hours. Following transesterification, the solvent and acetone are distilled out, and acetone precipitation is repeated 2-3 times to remove unsaponified matter, which is then filtered and evaporated. The concentrated oil undergoes molecular distillation at 180°C and 0.0033 bar pressure for 1 hour, yielding the distillate and residue for further analysis. Qualitative analysis using Thin Layer Chromatography (TLC) involves Merck TLC plates with silica gel 60 F254 and a hexane: chloroform (9:1) mobile phase. Spots are developed with a 10% HCl solution, confirming the presence of squalene with an RF value of 0.93. Quantitative analysis via High-Performance Thin-Layer Chromatography (HPTLC) employs Merck TLC plates with cyclohexane as the mobile phase and CAMAG at 254 nm and 366 nm wavelengths, revealing a squalene purity of 67% and a recovery rate of 69.8%. The initial purification through transesterification facilitated the conversion of ester groups, yielding squalene-rich fractions, while acetone precipitation effectively removed saponified matter. Molecular distillation further enhanced squalene purity. TLC analysis confirmed the qualitative presence of squalene, and HPTLC provided precise quantitative measurements. The obtained squalene purity of 67% significantly enriches the initial content in virgin olive oil, though further optimization could enhance purity and yield. Complementary techniques like GC-MS or HPLC could validate the purification process. This study presents an efficient, replicable procedure for extracting and purifying squalene from virgin olive oil, with significant implications for pharmaceutical, cosmetic, and nutraceutical industries. The findings support a sustainable and ethical shift towards vegetable-derived squalene, meeting market demands while ensuring high-quality production.

Studies Phytochemical Screening of Leaf Extract of Clerodendrum Infortunatum Linn

The leaves of Clerodendrum infortunatum Linn, a member of the Verbinaceae family, contain compounds with biological activity. The current study set out to evaluate various extraction techniques in order to find the optimal ones for extracting flavonoid components from various solvents. Four different solvents were used to extract the leaves of Clerodendrum infortunatum Linn. To determine the percentage (%) yield of each extract, plant material was extracted using a variety of organic solvents in ascending order of polarity, including Petroleum ether (60–80°), Chloroform, Acetone, and Methanol. Each extract was subjected to a thin layer chromatography analysis to determine the amount of components it contained. Using various extraction techniques and (80%) ethanol, N.R. Fransworth extracted a flavonoid-rich fraction for comparative analysis.

Expression and characterization of a novel microbial GH9 glucanase, IDSGLUC9-4, isolated from sheep rumen.

This study aimed to identify and characterize a novel endo-β-glucanase, IDSGLUC9-4, from the rumen metatranscriptome of Hu sheep. A novel endo-β-glucanase, IDSGLUC9-4, was heterologously expressed in Escherichia coli and biochemically characterized. The optimal temperature and pH of recombinant IDSGLUC9-4 were determined. Subsequently, substrate specificity of the enzyme was assessed using mixed-linked glucans including barley β-glucan and Icelandic moss lichenan. Thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) analyses were conducted to determine the products released from polysaccharides and cello-oligosaccharides substrates. The recombinant IDSGLUC9-4 exhibited temperature and pH optima of 40 °C and pH 6.0, respectively. It exclusively hydrolyzed mixed-linked glucans, with significant activity observed for barley β-glucan (109.59 ± 3.61 μmol·mg-1·min-1) and Icelandic moss lichenan (35.35 ± 1.55 μmol·mg-1·min-1). TLC and HPLC analyses revealed that IDSGLUC9-4 primarily released cellobiose, cellotriose, and cellotetraose from polysaccharide substrates. Furthermore, after 48 h of reaction, IDSGLUC9-4 removed most of the glucose, indicating transglycosylation activity alongside its endo-glucanase activity. The recombinant IDSGLUC9-4 was a relatively acid-resistant, mesophilic endo-glucanase (EC 3.2.1.4) that hydrolyzed glucan-like substrates, generating predominantly G3 and G4 oligosaccharides, and which appeared to have glycosylation activity. These findings provided insights into the substrate specificity and product profiles of rumen-derived GH9 glucanases and contributed to the expanding knowledge of cellulolytic enzymes and novel herbivore rumen enzymes in general.

Phytochemical profile and determination of cytotoxicity, acute oral toxicity, genotoxicity, and mutagenicity of aqueous and ethanolic extracts of Pseudobombax marginatum (A. St.-Hil.) A. Robyns

ABSTRACT Pseudobombax marginatum, popularly known as “embiratanha,” is widely used by traditional communities as anti-inflammatory and analgesic agent. This study aimed to determine the phytochemical profile as well as cytotoxicity, acute oral toxicity, genotoxicity, and mutagenicity attributed to exposure to aqueous (AqEx) and ethanolic (EtEx) extracts of embiratanha bark. Phytochemical screening was conducted using thin-layer chromatography (TLC). Cell viability was analyzed using MTT assay with human mammary gland adenocarcinoma (MDA-MB-231) and macrophage (J774A.1) cell lines, exposed to concentrations of 12.5, 25, 50, or 100 µg/ml of either extract. For acute oral toxicity, comet assay and micronucleus (MN) tests, a single dose of 2,000 mg/kg of either extract was administered orally to Wistar rats. TLC analysis identified classes of metabolites in the extracts, including cinnamic acid derivatives, flavonoids, hydrolyzable tannins, condensed tannins, coumarins, and terpenes/steroids. In the cytotoxicity assay, the varying concentrations of extracts derived from embiratanha induced no significant alterations in the viability of MDA-MB-231 cells. The lowest concentration of EtEx significantly increased macrophage J774A.1 viability. However, the higher concentrations of AqEx markedly lowered macrophage J774A.1 viability. Animals exhibited no toxicity in the parameters analyzed in acute oral toxicity, comet assay, and MN tests. Further, EtEx promoted a significant reduction in DNA damage index and DNA damage frequency utilizing the comet assay, while the group treated with AqEx exhibited no marked differences. Thus, data demonstrated that AqEx or EtEx of embiratanha may be considered safe at a dose of 2,000 mg/kg orgally under our experimental conditions tested.

Phytochemical Analysis, Antioxidant Potential and Antibacterial Activities of Different Anatomical Parts of Hypericum cordifolium Choisy.

The genus Hypericum comprises a large number of species. The flower, leaf, stem, and root of the Hypericum species are widely used in traditional medicine in different cultures. Many Hypericum species have been well investigated phytochemically and pharmacologically. However, only a few reports are available on the H. cordifolium native to Nepal. The present study aims to evaluate the phytochemical composition of different extracts, qualitative analysis of methanol extract of the flower and leaf using thin-layer chromatography (TLC), and the antioxidant properties of components by the TLC-DPPH. assay. The phenolic and flavonoid contents were estimated in different extracts of the leaf and stem, and their antioxidant and antibacterial activities were evaluated. In the phytochemical screening, phenolics and flavonoids were present in ethyl acetate, methanol, and 50% aq methanol extracts of both the leaf and stem. In TLC analysis, the methanol extract of flowers showed the presence of 11 compounds and the leaf extract showed the presence of 8 compounds. Both extracts contained chlorogenic acid and mangiferin. Hyperoside and quercetin were present only in the flower extract. In the TLC-DPPH. assay, almost all of the flower extracts and 5 compounds of the leaf extract showed radical scavenging potential. Estimation of phenolics and flavonoids showed that all the leaf extracts showed higher amounts of phenolics and flavonoids than stem extracts. Among leaf extracts, greater amounts of phenolics were detected in 50% aqueous methanol extract (261.25 ± 1.66 GAE/g extract) and greater amounts of flavonoids were detected in methanol extract (232.60 ± 10.52 CE/g extract). Among stem extracts, greater amounts of flavonoids were detected in the methanol extract (155.12 ± 4.30 CE/g extract). In the DPPH radical scavenging assay, the methanol extract of the leaf showed IC50 60.85 ± 2.67 µg/ml and 50% aq. methanol extract of the leaf showed IC50 63.09 ± 2.98 µg/ml. The methanol extract of the stem showed IC50 89.39 ± 3.23 µg/ml, whereas ethyl acetate and 50% aq. methanol extract showed IC50 > 100 µg/ml. In the antibacterial assay, the methanol extract of the leaf showed the inhibition zone of 12-13 mm and the stem extract showed the inhibition zone of 7-11 mm against S. aureus, E. coli, and S. sonnei, whereas both extracts were inactive against S. typhi. The findings of this study support the traditional use of this plant in Nepal for the treatment of diseases associated with bacterial infections. The present study revealed that the underutilized anatomical parts of H. cordifolium could be the source of various bioactive phytochemicals like other Hypericum species.

Distinguishing between Tectona grandis and acacia auriculiformis woods

The demand for Teak (Tectona grandis Linn F.) wood in India is high due to its durability and aesthetic value, and the country is highly dependent on imports to fulfil the demand. Thus, people try to trade Acacia auriculiformis A. Cunn wood commonly known as auri or acacia in place of teak wood as they have similar appearance. To address this issue, a simple and effective method was developed using Thin Layer Chromatography (TLC) analysis and UV–visible spectroscopy to distinguish between teak and auri timber and their finished products. For the study, we collected wood specimens from the Wood Properties and Processing division of IWST and conducted TLC analysis and UV–visible spectroscopy to distinguish the wood of both species. The identification of distinct peaks in UV–visible and separate spots with different retention factors (RF) in TLC provide a reliable means of differentiation. This study aims to present a solution to the problem of distinguishing between teak and auri, woods particularly in finished products, and the findings of the study may be useful for enhancing the customer satisfaction who are paying of teak and getting auri wood. Thus, also preventing the unethical trade of A. auriculiformis as T. grandis wood.

Exploring the Role of Active Photosynthetic Pigments in Tomato (Solanum lycopersicum) Crop Growth Process

Photosynthesis is a necessary process for plants during which light energy is absorbed via pigments present in leaves for essential processes and needs of the particular plant. This work identified and studied the active photosynthetic pigments in tomato crop (Solanum lycopersicum) at each growth stage and grown in an open field under Sahelian conditions. Phytochemical screening, UV-visible and Fourier transform infrared spectroscopy, and thin layer chromatography analyses were done on tomatoes leaves samples. The results, first, revealed the presence of phytoconstituents such as triterpenoids and carotenoids. Then, UV spectra combined with FTIR and thin layer chromatography showed that chlorophylls (a and b), xanthophyll (lutein) and carotene (mostly β) are being synthesized at different proportions during all growth stages. Besides, the photosynthetic pigments showed more activity during the flowering stage than at the other growth stages. The various action spectra showed intense absorption within the intervals 400 – 510 nm and 560 – 710 nm which seem to be vital radiation ranges for photosynthesis. Knowing these optimum plant’s needs can help in a choice of better outdoor and indoor cultivation conditions that can ensure necessary radiations for optimum plant growth.

Genome Analysis of Multiple Polysaccharide-Degrading Bacterium Microbulbifer thermotolerans HB226069: Determination of Alginate Lyase Activity.

Polysaccharide-degrading bacteria are key participants in the global carbon cycle and algal biomass recycling. Herein, a polysaccharide lyase-producing strain HB226069 was isolated from Sargassum sp. from Qingge Port, Hainan, China. Results of the phylogenetic of the 16S rRNA gene and genotypic analysis indicated that the isolate should be classified as Microbulbifer thermotolerans. The whole genome is a 4,021,337bp circular chromosome with a G+C content of 56.5%. Analysis of the predicted genes indicated that strain HB226069 encoded 161 carbohydrate-active enzymes (CAZymes), and abundant putative enzymes involved in polysaccharide degradation were predicted, including alginate lyase, fucosidase, agarase, xylanase, cellulase, pectate lyase, amylase, and chitinase. Three of the putative polysaccharide lyases from PL7 and PL17 families were involved in alginate degradation. The alginate lyases of strain HB226069 showed the maximum activity of 117.4 U/mL at 50°C, pH 7.0, and 0.05M FeCl3, while exhibiting the best stability at 30°C and pH 7.0. The Thin Layer Chromatography (TLC) and Electrospray Ionization Mass Spectrometry (ESI-MS) analyses indicated that the alginate oligosaccharides (AOSs) degraded by the partially purified alginate lyases contained oligosaccharides of DP2-DP5 and monosaccharide while reacting for 36h. The complete genome of M. thermotolerans HB226069 enriches our understanding of the mechanism of polysaccharide lyase production and supports its potential application in polysaccharide degradation.

Targeted preparation of six rare ginsenosides using two β-glucosidases from Bifidobacterium adolescentis

In this study, six rare protopanaxadiol (PPD)-type ginsenosides, namely, F2, CK, C-Mc1, C-Mc, CO, and CY, were prepared using enzymatic hydrolysis. Firstly, major (PPD)-type ginsenosides, including Rb1, Rb2, Rc, and Rd, were biotransformed by employing single and combined β-glucosidases BaBgl1A and BaBgl3A. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analyses revealed that ginsenosides F2, CO, and C-Mc1 could be produced by BaBgl1A, while rare ginsenosides CK, CY, and C-Mc could be produced by the combined use of BaBgl1A and BaBgl3A. Furthermore, optimal preparation conditions for these rare ginsenosides were elucidated, with yields exceeding 76.34%. Additionally, large quantities of rare ginsenosides were prepared to determine their structures. Characteristic absorption peaks in 13C NMR spectra and ion peaks in MALDI-TOF-MS suggested that the six high-purity rare ginsenosides (i.e., F2, CO and C-Mc1, CK, CY, and C-Mc) could be produced at high-efficiency. The findings of this study offer insights into novel enzymolysis methods for the targeted preparation of rare PPD-type ginsenosides, thus widening the range of raw materials that could be directed to in-depth biological activity research.

Determination of Physicochemical Characteristics, Phytochemical Profile and Antioxidant Activity of Various Clover Honeys.

This paper reports on some physicochemical and phytochemical characteristics (i. e. pH, electrical conductivity, colour, moisture content, total phenolic content, sugar profile) and in vitro antioxidant activity of honeys harvested from five legume species, red clover (Trifolium pratense), balansa clover (T. michelianum), Persian clover (T. resupinatum), purple clover (T. purpureum) and sanfoin, also known as holy clover (Onobrychis viciifolia), that were grown in enclosed shade houses to ensure that the honeys' characteristics are reflective of a truly monofloral honey. Glucose and fructose, determined via High-Performance Thin-Layer Chromatography (HPTLC) analysis, were found as the main sugars in all investigated honeys with the ratio of fructose to glucose ranging from 1 : 1.2 to 1 : 1.6. The honeys' pH values ranged from 3.9 to 4.6 which met Codes Alimentarius (CA) requirements. The moisture content was found to be between 17.6 and 22.2 % which in some cases was slightly higher than CA requirements (≤20 %). The honeys' colour values, prior and after filtration, were between 825.5-1149.5 mAU and 532.4-824.8 mAU respectively, illustrating golden yellow to deep yellow hues. The total phenolic content (TPC) of the honeys was determined using a modified Folin-Ciocalteu assay. Their antioxidant activity was captured by the Ferric Reducing-Antioxidant Power (FRAP) assay as well as HPTLC analysis coupled with 2,2-diphenyl-1-picrylhydrazyl (DPPH) derivatisation. The highest total phenolic content was found in red clover honey (45.4 mg GAE/100 g) whereas purple clover honey showed the highest level of activity in the FRAP assay (7.3 mmol Fe2+/kg). HPTLC-DPPH analysis of the honeys' organic extracts demonstrated the presence of various bioactive compounds that contribute to their overall antioxidant activity. This study developed a methodology for producing monofloral clover honeys in a space limited, enclosed production system, which allowed to collate important baseline data for these honeys that can serve as the foundation for their potential future development into commercial honeys, including honeys that can be used for medicinal purposes.

Rapid analysis and classification of blue ballpoint pen inks using high-performance thin-layer chromatography and digital color analysis

Despite the rise of digitalization, the scrutiny of paper documents with stamps and signatures remains crucial, constituting 55–60 % of document examination court cases. Developing accessible and robust ink analysis techniques is an ongoing challenge for forensic scientists. To tackle issues related to the identification and classification of dye components in blue ballpoint pens, we have proposed an advanced approach that integrates high-performance thin-layer chromatography (HPTLC), digital color analysis (DCA), and hierarchical cluster analysis (HCA) for a simplified yet effective procedure.The method encompasses multiple development of TLC plates. Initial separation employs methanol:ethanol (4:1, v/v), followed by a second separation using methanol:propan-2-ol:formic acid (1:1:0.1, v/v/v) to achieve cationic dye separation. This TLC development enhances separation selectivity, contributing to a more precise clustering of pen inks. Employing advanced DCA as a processing method for HPTLC, the optimal descriptor for ink classification is identified as the difference between red (R) and green (G) color components. The proposed clustering algorithm achieved 8 distinct ink clusters, showing improved discriminative capability and a reduced variable space compared to well-established or state-of-the-art methods.The approach's robustness was demonstrated across different readout devices, including smartphones and scanners. Reproducibility tests yielded consistent clustering results, confirming the method's reliability. Moreover, the method proved effective in clustering pen ink extracts from paper substrates, showcasing its potential for real sample analysis.This HPTLC-DCA approach presents a simple, cost-effective, and high-precision solution for clustering blue pen inks, significantly enhancing accessibility in high-throughput forensic applications.

Argemone ochroleuca Phytochemicals and Allelopathic Effect of Their Extracts on Germination of Soybean

Soybean is a high-value food source, and the invasive weeds Mexican prickly poppy (Argemone ochroleuca) could release allelochemicals that inhibit the growth of this crop. The impact of A. ochroleuca on the germination and growth of soybean is not well documented. Therefore, the aim of this study was to evaluate the TLC profiles of different extracts of A. ochroleuca and assess the effects of extracts on the germination of soybean seeds. Shoots and roots of A. ochroleuca were weighed and 100 g of each was separately extracted with 1000 mL deionized water, hexane or acetone. Ten concentrations of water extracts ranging from 10 to 100 mL per 100 mL of deionized water and three concentrations of acetone and hexane extracts ranging from 2.5 to 7.5 g/L were separately used for seed germination bioassays. Thin-layer chromatography (TLC) analysis was used to compare the chemical profiles in the shoot and root water, and in the hexane and acetone extracts of A. ochroleuca. The highest reduction was recorded from the water extract, at 100%. The TLC profiling of A. ochroleuca addressed different classes of compounds, including alkaloids, phenolic acids and flavanoids. There is, however, a need to identify the most active phytochemicals in the suppression of germination.

In vitro Evaluation of Skin Related Enzyme Inhibitory Effect and Emulgel Formulation Development Studies of Onobrychis Argyrea subsp. Argyrea with Phytochemical Analysis.

Species of Onobrychis have been used to treat skin disorders such as wounds and cuts in folk medicine and Onobrychis argyrea subsp. argyrea (OA) commonly known as 'silvery sainfoin', is a member of this genus. In this study, it was aimed to investigate the skin-related biological activities and phytochemical characterization of OA. Moreover, an emulgel formulation was developed from the main methanolic extract of the plant (OAM). Initially, to identifiy of the active fractions, aerial parts of the plant material was extracted with methanol and fractionated by n-hexane, chloroform, ethyl acetate and n-butanol, respectively. Antioxidant activity was determined by CUPRAC, TOAC, FRAP and DPPH assays. Thereafter, the inhibition potential of OAM, novel formulation and all fractions was measured against elastase, collagenase, tyrosinase and hyaluronidase enzymes. OAM was analyzed and characterized by LC/MS-MS. The major bioactive flavonoids which are rutin and isoquercetin were measured and compared as qualitative and quantitative via high performance thin layer chromatography (HPTLC) analysis in OAM and fractions. The results showed that extracts of OA can be a potential cosmeceutical agent for skin related problems.

Meta-topolin mediated improved micropropagation, phytochemical profiling, and assessment of genetic fidelity in Chlorophytum borivilianum Sant. et Fernand., an endangered medicinal herb

A highly effective method has been developed to examine the impact of different combination of meta-topolin and various cytokinins on Chlorophytum borivilianum Sant. et Fernand. using apical bud explants. The effects of benzyl adenine purine (BAP), kinetin (Kin), meta-topolin (mT), and thidiazuron (TDZ) were explored separately and in different concentrations (ranging from 0.5 to 3.0 mg L−1) on shoot proliferation, as well as the influence of auxins like Indole -3- acetic acid (IAA), α-Naphthalene acetic acid (NAA) and Indole-3-butyric acid (IBA) on rooting. When grown on MS medium supplemented with mT, highest growth rates were observed followed by BAP. Within the permutations tested, 2.0 mg L−1 of mT and 1.0 mg L−1 of BAP produced the best results with 96.3% success rate and average of 19.2 ± 1.92 shoots measuring 6.72 ± 0.49 cm in length after 8 weeks of culture. Among the various auxins used for in vitro root formation, 95.5% of multiple shoots effectively established roots on MS medium supplemented with 1.0 mg L−1 of IBA resulting in an average of 14.0 ± 1.39 root numbers and 3.36 ± 0.27 cm in length after 4 weeks of culture. Furthmore, 94% of the acclimated plants not only survived but also thrived when grown in a mixture of loam, sand and fym (in 1:2:1 ratio) with an average weight of 16 g. High-performance thin layer chromatography (HPTLC) analysis revealed the presence of diosgenin and stigmasterol in both in vitro regenerants and mother plant, although there were variations in their content. To ensure genetic fidelity among regenerants, ISSR and SCOT markers were used, but no somatic differences were detected. Therefore, the established procedure seems to be easy and consistent for large scale development of genetically identical clones with a greater content of bioactive compounds.

Response surface optimization, purification, characterization and short-chain chitooligosaccharides production from an acidic, thermostable chitinase from Thermomyces dupontii

Chitin, recovered in huge amounts from coastal waste, may biocatalytically valorized for utilization in food and biotech sectors. Conventional chemical-based conversion makes use of significant volumes of hazardous acid and alkali. Alternatively, enzymes offer better process control and generation of homogeneous products. Process variables were derived to achieve augmented levels of chitinase (3.8809 Ul−1 h−1) productivity from a novel thermophilic fungal strain Thermomyces dupontii, ITCC 9104 following incubation (96 h, 45 °C). An acidic thermostable chitinase TdChiT having molecular mass of 60 kDa has been purified. Optimal TdChiT activity has been demonstrated at 70 °C and pH 5. Notably decreased activity over a broad range of temperature and pH was observed following deglycosylation. Half-life, activation energy, Gibbs free energy, enthalpy and entropy for denaturation of TdChiT at its optimum temperature were 197.40 min, 105.48 kJ mol−1, 100.59 kJ mol−1, 102.64 kJ mol−1 and 5.95 J mol−1 K−1. TdChiT has specificity towards colloidal chitin and (GlcNAc)2–4. Metal ions viz. Mn2+, Ca2+ and Co2+ and nonionic surfactants notably enhanced chitinase activity. Thin layer chromatography analysis has revealed effective hydrolysis of colloidal chitin and (GlcNAc)2–4. TdChiT may potentially be employed for design of better, eco-friendly and less resource-intensive industrial procedures for upcycling of crustacean waste into value-added organonitrogens.

Extraction, Isolation and Neuropharmacological Potential of Phyllanthus acidus L.

Because of their broad availability, low cost, low danger, and few side effects, medicinal herbs are vital in traditional medication all over the world. Traditional therapeutic herb Phyllanthus acidus L. is well-known for its calming possessions on the nervous system. Its roots are known for their activity against bacteria, inflammation, immunity stimulation, and erogenous properties, which contribute to potential health benefits. This study investigates the neuropharmacological effects of P. acidus L. in enhancing memory, targeting cognitive disorders and learning deficits. Analysis of the ethanolic extract’s phytochemistry of P. acidus L. revealed a rich composition of phytoconstituents. UV analysis and thin layer chromatography (TLC) bioassay demonstrated significant acetylcholinesterase (AChE) inhibition by the extract. TLC and high-performance thin-layer chromatography (HPTLC) analyses of the extract indicated distinct and well-separated spots, suggesting the presence of active compounds. Additionally, the extract was assessed for its total phenolic, flavonoid, and ascorbic acid content, revealing antioxidant properties through ex-vivo lipid peroxidation assays. The study concludes that the ethanolic extract of P. acidus L. exhibits memory-enhancing, antidepressant, and neuroprotective effects attributed to its AChE inhibition, modulation of neurotransmitters like dopamine and serotonin, and antioxidant activity. This suggests its potential utility in Alzheimer’s dementia treatment, though further research is necessary to elucidate pharmacology with mechanisms of action and applications

Comparative negation of amphiphile production using nutrition factors: Amyloids versus biosurfactants

Microbial amphiphiles play an important role in environmental activities such as microbial signaling, bioremediation, and biofilm formation. Microorganisms rely on their unique characteristics of interfaces to carry out critical biological functions, which are helped by amphipathic biomolecules known as amphiphiles. Bacillus amyloids aid in cell adhesion and biofilm formation. Pseudomonas sp. are essential in biofilm development and are a vital survival strategy for many bacteria. Furthermore, Pseudomonas and Bacillus are well-known for their ability to produce biosurfactants with a range of applications, including bioremediation and removing biological pollutants from different environments. The study employed 31 different media types and a range of analytical techniques to assess the presence of amyloid proteins and the absence of biosurfactants in Bacillus licheniformis K125 (GQ850525.1) and Pseudomonas fluorescens CHA0. The presence of amyloid proteins was confirmed through Congo red and thioflavin T staining. The carefully constructed medium also efficiently inhibited the synthesis of biosurfactants by these bacteria. Additionally, surface tension measurements, emulsification index, thin-layer chromatography, and high-performance thin-layer chromatography analyses indicated the absence of biosurfactants in the tested media.

Engineering a recombinant chitinase from the marine bacterium Bacillus aryabhattai with targeted activity on insoluble crystalline chitin for chitin oligomer production

Chitin, an abundant polysaccharide in India, is primary by-product of the seafood industry. Efficiently converting chitin into valuable products is crucial. Chitinase, transforms chitin into chitin oligomers, holds significant industrial potential. However, the crystalline and insoluble nature of chitin makes the conversion process challenging. In this study, a recombinant chitinase from marine bacteria Bacillus aryabhattai was developed. This enzyme exhibits activity against insoluble chitin substrates, chitin powder and flakes. The chitinase gene was cloned into the pET 23a plasmid and transformed into E. coli Rosetta pLysS. IPTG induction was employed to express chitinase, and purification using Ni-NTA affinity chromatography. Optimal chitinase activity against colloidal chitin was observed in Tris buffer at pH 8, temperature 55°C, with the presence of 400 mM sodium chloride. Enzyme kinetics studies revealed a Vmax of 2000 μmole min−1 and a Km of 4.6 mg mL−1. The highest chitinase activity against insoluble chitin powder and flakes reached 875 U mg−1 and 625 U mg−1, respectively. The chitinase demonstrated inhibition of Candida albicans, Fusarium solani, and Penicillium chrysogenum growth. Thin Layer Chromatography (TLC) and LC-MS analysis confirmed the production of chitin oligomers, chitin trimer, tetramer, pentamer, and hexamer, from chitin powder and flakes using recombinant chitinase.

In Vitro Evaluation of Extracts From Ixora Species for a Potential Phytosomal Formulation.

Background Ixora species are perennial shrubs and flowering plants belonging to the family Rubiaceae. The leaf and flower parts of Ixora coccinea (I. coccinea)andIxora alba (I. alba) were aimed at isolating their active fractions. The present study was to determine in vitro antitumor activity against malignant melanoma cell lines for phytosome formulation. Materials and methods Two species, I. coccinea (red flowers and leaves) and I. alba (white flowers and leaves), were selected, and this study focused on determining the active fraction by comparing the in vitro antimicrobial and antioxidant potentials of petroleum ether, chloroform, ethyl acetate, and hydroalcoholic (ethanol:water, 70:30 v/v) extracts. The identified potent extract was subjected to in vitro anticancer activity in malignant melanoma cell lines. Results A phytochemical study revealed phytosterols, flavonoids, proteins, amino acids, alkaloids, carbohydrates, phenols, tannins, and diterpenes. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to evaluate the antioxidant effect of I. coccinea and I. alba leaf and flower extracts. In the DPPH assay, I. coccinea flower hydroalcoholic extract (ICFHA) had an IC50 value of 248.99 µg/mL, and I. coccinea leaf hydroalcoholic extract (ICLHA) had an IC50 value of 268.87 µg/mL. These two extracts had a lower value with a higher antioxidant effect. In the total antioxidant assay, I. coccinea leaf ethyl acetate extract (ICLEA) and I. coccinea leaf chloroform extract (ICLCE) have 77.4 ± 0.05 and 68.9 ± 0.03 mg of ascorbic acid equivalent per gm of extract, respectively. These two extracts exhibited a high antioxidant effect. The antimicrobial potential was evaluated using selected bacterial and fungal strains using the agar-well diffusion method. Petroleum ether and chloroform extracts of I. coccinea and I. alba leaves and flowers did not possess antimicrobial activity with any of the bacterial or fungal strains. An ethyl acetate extract and a hydroalcoholic extract of I. coccinea leaves and flowers showed antimicrobial activity against Enterococcus faecalis, Candida albicans, and Staphylococcus aureus. An ethyl acetate extract of I. coccinea flower and a hydroalcoholic extract of I. alba leaf showed a significant zone of inhibition when compared with standard chloramphenicol for all three selected strains, which may be due to the presence of active phytoconstituents. ICLHA showed a MIC of ≤300 µg/mL for Enterococcus faecalisandStaphylococcus aureusand ≤400 µg/mL for Candida albicans microbial strains. The high total flavonoid content was reported in ICLEA at 771.31 µg/mL and in I. coccinea flower ethyl acetate extract (ICFEA) at 694.69 µg/mL. High-performance thin layer chromatography (HPTLC) analysis showed a high quercetin (QCE) content in the ICLEA extract. To prove the in vitro skin anticancer activity, an MTT assay was performed for the ICLEA extract in a malignant melanoma cell line, and the IC50 value was reported as 7.96 µg/mL. Conclusion I. coccinea leaf ethyl acetate extract revealed a significant total flavonoid content in analysis through the aluminum chloride method, and the presence of a high QCE content was confirmed by HPTLC analysis. The in vitro skin anticancer activity of ICLEA was confirmed by the MTT assay; therefore, it was concluded that the ICLEA extract was a potent fraction and was selected to develop a phytosome.

Composition of biologically active substances of common cranberry fruits (Oxycoccus palustris Pers.) in various methods of preservation

Introduction. The study of common cranberry fruits is promising due to the wide variety of biologically active substances included in their composition, a wide range of therapeutic properties, as well as the ability of cranberry fruits for long-term storage and resistance to harsh environmental conditions. Fresh fruits contain the maximum amount of biologically active substances and, accordingly, have the most pronounced pharmacological effect. To preserve the composition and content of biologically active substances, preservation is used in pharmacy. Most often they resort to drying. Freezing can be considered a new and promising method for preserving the quality of medicinal plant materials. Purpose of the study. Study of the effect of freezing and drying on the qualitative composition and quantitative content of biologically active substances in common cranberry fruits. Material and methods. The object of the study was fresh, frozen, and dried common cranberry fruits collected in the Pskov and Novgorod regions in 2022 during the fruiting period. The study of the composition of biologically active substances (BAS) and quantitative assessment of the content were carried out according to pharmacopoeial methods, using analysis using thin layer chromatography (TLC), spectrophotometry and redox titration methods. Results. Using TLC analysis, organic acids (ascorbic, malic, citric, tartaric and succinic), flavonoids (rutin, quercetin), tannins (gallic acid) and anthocyanins were identified in the fruits. There were no differences in the qualitative composition of dried and frozen fruits. The content of organic acids, tannins, flavonoids and anthocyanins in cranberry fruits using different preservation methods was determined. It has been established that when fruits are dried, the content of all studied groups of biologically active substances decreases, on average by 40–60% of the initial content in fresh raw materials. Ascorbic acid is destroyed to a greater extent (90–92%). Exposure to low temperatures leads to a slight decrease in biologically active substances in common cranberry fruits (7–14%). Conclusion. The influence of preservation methods on the composition and content of biologically active substances in common cranberry fruits has been studied. The possibility of using frozen cranberry fruits as an alternative to fresh and dried raw materials in the manufacture of drugs in allopathy and homeopathy has been shown.