Reversed-phase High-performance Liquid Chromatography Analysis Research Articles

Advancements in RP-HPLC Method Development and Validation: A Comprehensive Review

Reverse-phase high-performance liquid chromatography (RP-HPLC) has emerged as a cornerstone technique in analytical chemistry, offering high sensitivity, selectivity, and efficiency in separating and quantifying a wide range of compounds. This comprehensive review explores recent advancements in RP-HPLC method development and validation, focusing on key strategies, challenges, and emerging trends. The review encompasses a detailed examination of critical parameters such as column selection, mobile phase optimization, detection techniques, and validation protocols. Furthermore, it discusses innovative approaches and technologies that enhance the robustness, speed, and accuracy of RP-HPLC analyses. Through a thorough analysis of current literature and case studies, this review provides valuable insights for researchers and practitioners engaged in method development and validation in the field of chromatographyies.

Comprehensive Analysis of Antioxidant and Phenolic Profiles of Thai Medicinal Plants for Functional Food and Pharmaceutical Development.

Medicinal plants, are resources of traditional medicines, have played a significant role in human culture throughout history. The connect of traditional knowledge with contemporary scientific findings, display the potential of these plants for nutritional and pharmaceutical applications. This study aimed to investigates the antioxidant activity and phenolic compositions of seven medicinal plants (Kaempferia parviflora, Hibiscus sabdariffa, Phyllanthus emblica, Terminalia chebula, Terminalia bellirica, Gynostemma pentaphyllum, and Stevia rebaudiana) from Kalasin Province, Thailand. 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power (FRAP) assays were used to evaluate the the antioxidant activities. The qualitative method was used for the phytochemical screening. Additionally, reverse-phase high-performance liquid chromatography (RP-HPLC) analysis reveals their phenolic compositions. Results revealed that phenols, flavonoids, and terpenoids were found in all medicinal plants studied, with flavonoids and phenols most predominant. Notably, P. emblica and T. bellirica show significant antioxidant properties and high levels of phenolics and flavonoids. P. emblica had the highest phenolic acid concentrations, with ρ-coumaric (79.43µg/g), chlorogenic (45.22µg/g), and ferulic acid (29.58µg/g) being dominant. The research emphasizes the value of these plants in developing functional foods and pharmaceuticals, underlining the importance of conserving biodiversity and traditional knowledge for future health product innovation.

Exploring the phytochemical and biological properties of Salsola foetida: a promising wild plant from Southeastern Algeria

ABSTRACT This study presents an initial exploration of the phytochemical composition and biological properties of Salsola foetida, a wild plant native to southeastern Algeria. The investigation encompassed an analysis of the biochemical content, phytochemical profile, and bioactivity of the plant’s aerial parts. The biomass of S. foetida was found to comprise 51.2 ± 0.4% ash, with elemental analysis revealing the presence of carbon, oxygen, calcium, sodium, chlorine, magnesium, potassium, and sulfur. Carbohydrates were identified as the predominant constituents, surpassing the levels of proteins and lipids. Phytochemical screening, coupled with Fourier Transform Infrared (FT-IR) analysis, confirmed the presence of various secondary metabolites, including alkaloids and phenols. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis further delineated a spectrum of phenolic compounds, with rutin emerging as the predominant compound at 1.59 µg/ml. Quantification revealed a noteworthy phenolic content of 67.1 ± 0.2 µg GE/mg ED. The extract exhibited robust radical scavenging effects, with the radical 50% inhibitory concentration (IC50) being 61.8 ± 0.2 μg/ml, along with protective effects against H2O2-induced hemolysis and the ability to reduce ferric ions. Additionally, the extract demonstrated anti-inflammatory, antibacterial, and antidiabetic activities. Notably, it exhibited considerable sunscreen properties, boasting a sun protected factor (SPF) value of 21 ± 1. Overall, S. foetida presents promising antioxidant activity and a spectrum of potential biological activities, underscoring its significance as a novel antioxidant candidate for applications in animal nutrition, medicine, and natural product research.

RP-HPLC Method Development, Validation Including Stability Indicating and Forced Degradation Studies for the Estimation of Pemetrexed in API and Pharmaceutical Dosage Form

Stability indicating analytical RP-HPLC (Reverse Phase- High performance Liquid Chromatography) method was developed for the analysis of the drug in the presence of its degradation products and subsequent validation of Pemetrexed that was conducted in API and pharmaceutical dosage form. Pemetrexed was subjected to different stress conditions as per International Conference on Harmonization (ICH) guidelines, stress conditions applied were including the effect of acid, base, oxidative, hydrolysis, thermal and photolytic degradation conditions. Chromatographic separation of API (Active Pharmaceutical ingredient) was achieved on Water’s HPLC instrument and ODS C18, 250x4.6 mm, 5µm particle size column with gradient elution of 0.1% ortho phosphoric acid buffer: Acetonitrile 80:20 ratio was taken as the eluent (mobile phase). The buffer pH was maintained at 3, the flow rate was 1.0 ml/min and wavelength were detected at 225 nm. The Retention time (RT) of Pemetrexed was Found at 2.9 minutes, column temperature was kept ambient and the runtime was 6 min. System suitability parameters like theoretical plates, tailing factor, and Asymmetry values were showed at 3391, 1.41, and 1.101 respectively. The percentage drug purity was found to be 99.99%. Validation of the method was proved that linear relationship over the range of 25-150 µg/mL, with linear regression curve (correlation coefficient) r2 value was noticed at 0.999 and LOD (limit of detection), LOQ (limit of quantification) values was established at 0.33µg/mL, 0.99µg/mL respectively. Recovery studies carried out at the concentrations of 50%, 100% and 150% with percentage recovery values of 100.06, 99.8 and 99.87 respectively. The precision of intraday was 0.64% and inter-day was 0.95%. Robustness of flow rate and mobile phase was changed even though the method was Robust. Forced degradation studies were also conducted to check the stability and suitability of this method to resolve the degradation products. Method validation was proved to be accurate, precise, linear, repeatable and robust. Thus, the accepted method can be proceeded for the estimation of Pemetrexed in pharmaceutical dosage form in a daily basis, due to their simplicity, suitability, accuracy, robustness and reproducibility.

Predictive Quantitative Read-Across Structure-Property Relationship Modeling of the Retention Time (Log tR) of Pesticide Residues Present in Foods and Vegetables.

The retention time (log tR) of pesticidal compounds in a reverse-phase high-performance liquid chromatography (HPLC) analysis has a direct relationship with lipophilicity, which could be related to the ecotoxicity potential of the compounds. The novel quantitative read-across structure-property relationship (q-RASPR) modeling approach uses similarity-based descriptors for predictive model generation. These models have been shown to enhance external predictivity in previous studies for several end points. The current study describes the development of a q-RASPR model using experimental retention time data (log tR) in the HPLC experiments of 823 environmentally significant pesticide residues collected from a large compound database. To model the retention time (log tR) end point, 0D-2D descriptors have been used along with the read-across-derived similarity descriptors. The developed partial least squares (PLS) model was rigorously validated by various internal and external validation metrics as recommended by the Organization for Economic Co-operation and Development (OECD). The final q-RASPR model is proven to be a good fit, robust, and externally predictive (ntrain = 618, R2 = 0.82, Q2LOO = 0.81, ntest = 205, and Q2F1 = 0.84) that literally outperforms the external predictivity of the previously reported quantitative structure-property relationship (QSPR) model. From the insights of modeled descriptors, lipophilicity is found to be the most important chemical property, which positively correlates with the retention time (log tR). Various other characteristics, such as the number of multiple bonds (nBM), graph density (GD), etc., have a substantial and inversely proportionate relationship with the retention time end point. The software tools utilized in this study are user-friendly, and most of them are free, which makes our methodology quite cost-effective when compared to experimentation. In a nutshell, to obtain better external predictivity, interpretability, and transferability, q-RASPR is an efficient technique that has the potential to be employed as a good alternative approach for retention time prediction and ecotoxicity potential identification.

Study on the identification and quantification of sodium benzoate in different brands of jelly by high performance liquid chromatography

A reversed-phase high-performance liquid chromatography analysis was carried out for rapid determination and quantification of sodium benzoate in different jelly products. Sodium benzoate is allowed food additive by universal laws in processing in restrictive amounts, but its content must be declared and must not exceed the established limits by legislation. An experimental study for the level of sodium benzoate in different brands of jelly available in the markets, stores and shops in the Tangail region in Bangladesh was determined by high-performance liquid chromatography. Chromatographic separation was achieved with isocratic solvent system of sodium acetate and acetic acid buffer (pH = 4.0): acetonitrile as 70:30 with a flow rate of 0.8 mL/min; chromatograms were recorded at 254 nm. The injection volume was 20 µL. The concentrations of sodium benzoate in all the samples were calculated by the external standard method with calibration of correlation coefficient 0.985 from the standards calibration curves. The limit of detection and quantification for sodium benzoate was 0.0003 mg/100 mL and 0.0009 mg/100 mL respectively. Quantification of the selected brand jellies revealed that the level of the used sodium benzoate was within the FDA standard range. But by comparing with the Bangladesh Standard and Testing Institute (BSTI), Brand-3 jelly samples were found to exceed the current legal limits.

The Development and Validation of Simultaneous Quantitative Analysis Reversed-phase High-performance Liquid Chromatography for Sitagliptin Phosphate Monohydrate and Dapagliflozin Propanediol Monohydrate Fixed-dose Combination Dual-layered Tablet

Background: Sitagliptin phosphate monohydrate-dapagliflozin propanediol hydrate fixeddose combination (FDC) dual-layered tablet is used for type 2 diabetes treatment. Simultaneous quantitative analysis can shorten the analysis time of sitagliptin phosphate monohydrate-dapagliflozin propanediol monohydrate FDC dual-layered tablets and increase their efficiency. Objective: This study aimed to develop the simultaneous quantitative analysis for sitagliptin phosphate monohydrate-dapagliflozin propanediol monohydrate FDC dual-layered tablet, a type 2 diabetes treatment. Methods: Simultaneous quantitative analysis using the rapid and selective reversed-phase highperformance liquid chromatography (RP-HPLC) method was developed and validated using method validation. RP-HPLC analysis was conducted using an ultraviolent absorption spectrophotometer and a Zorbax C18 column (4.6 x 150 mm, 5 μm). The flow rate and injection volume were set to 1.5 mL min-1 and 20 μL, respectively. The wavelength was set at 205 nm. Results: The retention times of sitagliptin phosphate monohydrate and dapagliflozin propanediol monohydrate were 2.28 mins and 10.65 mins, respectively. The relative standard deviations of the system suitability for validation of simultaneous quantitative analysis were 0.03% for sitagliptin phosphate monohydrate and dapagliflozin propanediol monohydrate. The chromatogram confirmed that there was no peak interference between the two main components and between the main component and the excipients. In addition, It revealed a favorable linearity with correlation coefficients of 0.9999 in the concentration range of 20-120% compared to the standard solution. Conclusion: The developed simultaneous quantitative analysis shortened the analysis time and high efficiency of the sitagliptin phosphate monohydrate-dapagliflozin propanediol monohydrate FDC bilayer tablet. The validity of the analytical method was verified through accuracy and precision, detection and quantitation limits, and solution stability tests. In addition, it was thought that it would be helpful in developing an analytical method by referring to the simultaneous quantitative analysis method for developing other FDC dual-layered tablets.

Phytochemical screening of Psidium guajava and Carica papaya leaves aqueous extracts cultivated in Greece and their potential as health boosters

Aim: The scope of the present study was to investigate the phytochemical profile of Psidium guajava and Carica papaya leaves aqueous extracts, from plants cultivated on Crete island in Greece. Methods: Total phenolic content (TPC) in the aqueous extracts was determined spectrometrically using the Folin-Ciocalteu (F-C) assay. The identification and quantification of different phenolic compounds in the aqueous extracts were conducted using reversed-phase high-performance liquid chromatography (RP-HPLC) analysis. Different metals were also determined (K, Fe, Zn, Ca, Mg, Pb, and Cd) to investigate the potential health claims or hazards in the water extractable infusion using inductively coupled plasma mass spectrometry (ICP-MS) method. Results: TPC in the aqueous extracts was found to be 28.0 g gallic acid equivalent (GAE)/kg dry leaves for Psidium guajava leaves aqueous extract and 15.0 g GAE/kg dry leaves for Carica papaya leaves aqueous extract. The dominant phenolic compounds in Psidium guajava leaves aqueous extract were myricetin (3,852 mg/kg dry sample) and rutin (670 mg/kg dry sample) while the dominant phenolic compounds in Carica papaya leaves aqueous extract were salicylic acid (338 mg/kg dry sample) and rutin (264 mg/kg dry sample). Different metals were also determined (K, Fe, Zn, Ca, Mg, Pb, and Cd) to investigate the potential health claims or hazards in the water extractable infusion, and it was found that no toxic metals were extracted whereas some nutritional benefits were achieved. Conclusions: Results proved that Psidium guajava and Carica papaya can be provided a strong antioxidant activity and can be used as medicinal plants.

α-AMYLASE, α-GLUCOSIDASE AND LIPASE INHIBITORY PROPERTIES AND PHYTOCHEMICAL ANALYSIS OF ENDEMIC PLANT Jurinea brevicaulis Boiss.

Obesity, defined as New World Syndrome, causes global health problems and big economic losses. Natural products have gained increasing importance because of their antiobesity potency. The genus Jurinea Cass. with approximately 200 described species worldwide has been traditionally used as a therapeutic agent for colic, fever, gout and rheumatism. The aim of this study was to analyze the volatile components, to determine phenolic compounds and to evaluate α-amylase, α-glucosidase, and lipase inhibitory activities of the endemic plant species Jurinea brevicaulis Boiss. The widely used solid-phase microextraction technique (SPME) was employed for Gas Chromatography-Mass Spectrometry (GC/MS) analysis of the volatile components. Quantitative analysis of phenolic compounds was performed using Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC). A total of 19 volatile components were specified and o-cymene (10.60 %), β-bisabolene (9.30 %), and sesquicineole (57.5 %) for different terpenes were described as major components. According to the RP-HPLC analysis, sinapic acid, p-coumaric acid and quercetin were determined for the species. IC50 values of the species were determined as 36.59 ± 2.37 μg/mL and 42.56 ± 2.83 μg/mL for α-amylase and α-glucosidase assays, respectively. IC50 value was found as 50.31 ± 3.75 μg/mL with the lipase inhibition analysis. In conclusion, it has been determined that J. brevicaulis included diverse volatile components, three phenolic compounds with antiobesity effect potential, which highlights J. brevicaulis as the up-and-coming candidate of natural product source to be used against obesity.

6-Gingerol contents of several ginger varieties of Northeast India and correlation of their antioxidant activity in respect to phenolics and flavonoids contents.

Ginger constitutes the rhizome part of the plant Zingiber officinale from the Zingiberaceae family. A large number of ginger varieties with high sensorial and functional quality are found in Northeast India. Hence, phytopharmacological screening of different ginger varieties is essential that will serve as a guideline in applied research to develop high-end products and improve economical margins. To determine the variation in total phenolics content (TPC), total flavonoids content (TFC), and antioxidant activities and correlate that with 6-gingerol contents of different ginger varieties collected from Northeast India using Pearson's correlation analysis. The TPC and TFC values were determined using standard methods. Antioxidant activities were measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical scavenging assays, while reversed-phase high-performance liquid chromatography (RP-HPLC) analysis was utilised for quantitative determination of 6-gingerol content. The result revealed that ginger variety 6 (GV6) contains the highest 6-gingerol content and TPC value showing maximum antioxidant activity, followed by GV5, GV4, GV9, GV3, GV2, GV8, GV1, and GV7. The findings also suggested that the antioxidant activity has much better correlations with TPC as compared with TFC values. Pearson's correlation analysis showed a significant correlation between 6-gingerol contents and TPC values. This work underlines the importance of ginger varieties from Northeast India as a source of natural antioxidants with health benefits.

Phenolic Profiles, Tyrosinase Inhibitory, and Antioxidant Effects of Green Coffee, and Turkish Traditional Coffee

Coffee has been drunk for millennia due to its taste and health benefits. High levels of polyphenols, and especially flavonoids and phenolic acids, are found in coffee and contribute significantly to its flavor and health-giving properties. In this study the total phenolic contents, antioxidant, and tyrosinase inhibition of green coffee, and Turkish traditional coffee extracts were evaluated. Antioxidant activities of the coffees were examined by two different methods, radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP). Total phenolic contents were estimated by using Folin-Ciocalteu reagent as the gallic acid equivalent. The phenolic profiles were investigated by means of reverse phase-high performance liquid chromatography (RP-HPLC). At the same time, tyrosinase enzyme inhibition of extracts has also been worked. The extracts exhibited high levels of antioxidant activities associated with significant antioxidant compound contents. It was determined that the samples contain chlorogenic acid and benzoic acid in the RP-HPLC analysis. It was determined that green coffee extract exhibited tyrosinase enzyme inhibition as effective as kojic acid. The results show that green coffee especially from coffees can be regarded as a potential source of antioxidant compounds and tyrosinase inhibitors of significance in both the pharmaceutical and food industries.

Efficient Separation of C-Tetramethylcalix[4]resorcinarene Conformers by Means of Reversed-Phase Solid-Phase Extraction.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed to study the conformer formation generated during the reaction for obtaining C-tetramethylcalix[4]resorcinarene. The chromatographic method was used to design a strategy for purifying the reaction products, using solid-phase extraction columns (RP-SPE) and gradient elution. The chromatographic profiles of the cyclocondensation reaction between resorcinol and acetaldehyde show the presence of three products under the different reaction and precipitation conditions studied. Using RP-SPE, it was possible to enrich the products, which were later characterized by means of RP-HPLC and 1H nuclear magnetic resonance (NMR). This investigation explored and established a new method for RP-HPLC analysis and RP-SPE separation of conformational isomers obtained in the formation reaction of C-tetramethylcalix[4]resorcinarene.

Effects of in vitro digestion on protein degradation, phenolic compound release, and bioactivity of black bean tempeh.

The nutritional value and bioactivity of black beans are enhanced when fermented as tempeh, but their bioaccessibility and bioactivity after ingestion remain unclear. In this study, black bean tempeh and unfermented black beans were digested in vitro and changes in protein degradation, phenolic compound release, angiotensin I-converting enzyme (ACE)-inhibitory activity, and antioxidant activity between the two groups were compared. We observed that the soluble protein content of digested black bean tempeh was generally significantly higher than that of digested unfermented black beans at the same digestion stage (P < 0.05). The degree of protein hydrolysis and the content of <10 kDa peptides were also significantly higher in the digested black bean tempeh than in digested unfermented black beans (P < 0.05). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and reversed-phase high-performance liquid chromatography (RP-HPLC) analysis showed that most macromolecular proteins in tempeh had been degraded during fermentation and more of the small peptides were released from black bean tempeh during digestion, respectively. Compared to that of the unfermented black beans, the level of ACE inhibition of black bean tempeh was lower, but this significantly increased to 82.51% following digestion, closing the gap with unfermented black beans. In addition, the total respective levels of phenolics, flavonoids, and proanthocyanidins released from black bean tempeh were 1.21, 1.40, and 1.55 times those of unfermented black beans following in vitro digestion, respectively. Antioxidant activity was also significantly higher in digested black bean tempeh than in digested unfermented black beans and showed a positive correlation with phenolic compound contents (P < 0.05). The results of this study proved that, compared to unfermented black beans, black bean tempeh retained protein and phenolic compound bioaccessibility and antioxidant activity and showed an improved ACE-inhibitory activity even after consumption.

Development and Validation of Analytical RP HPLC Method for Benidipine Hydrochloride

The aim of the present study is development and validation of analytical reverse phase (RP) high performance liquid chromatography (HPLC) from Benidipine Hydrochloride. HPLC method is used for quantification, identification and purification of particular analyte or compound. The developed HPLC chromatographic technique was validated based on international conference on harmonization (ICH) Q2 guidelines for accuracy, precision, linearity, range and robustness. High performance liquid chromatography technique is rapid and precise technique. Advance for the validated of Benidipine Hydrochloride in pure and tablet dosage form. Diethyl ether was extracted from plasma in the presence of 5M NaOH (Sodium Hydroxide) Diethyl ether is used as internal standard like Benidipine and Benidipine d5. Benidipine is dihydropyridine calcium channel blocker, which is used in the treatment of hypertension and angina pectoris and is used to examine the efficacy and safety of therapy with Benidipine in elderly hypertensive patients.

Microbial transglutaminase induced modification of wheat gliadin based nanoparticles and its impact on their air-water interfacial properties

There is a growing interest in plant protein based nanoparticles (NPs) such as wheat gliadin NPs (GNPs) for stabilizing foams in food systems. To increase the range of their applications, it is necessary to understand the role of different gliadin types and to tailor their behavior at air-water (A-W) interfaces. The latter is here done by modifying GNPs with microbial transglutaminase (TG). The most pronounced solubility loss of ω-gliadins (by 36.1%) after TG treatment at pH 4.5, measured by reversed phase high performance liquid chromatography (RP-HPLC), indicated that this protein type was more susceptible to TG action than α-gliadins and γ-gliadins, the solubility losses of which were 11.2 and 11.4% respectively. The lack of intramolecular disulfide bonds in ω-gliadins but not in the other gliadin types likely resulted in a greater accessibility of the lysine residues and thus more pronounced susceptibility of ω-gliadins to the enzyme. Interestingly, TG treatment of GNPs notably improved their foaming properties at pH 4.5 (and thus below the gliadin pI). The high surface dilatational modulus value of TG treated GNPs, when compared to control GNPs (53.9 and 32.7 mN/m, respectively), indicated formation of a strong viscoelastic A-W interfacial protein film, presumably due to the presence of TG catalyzed intermolecular isopeptide bonds. Fractionation of foams stabilized by (un-)treated GNPs and subsequent RP-HPLC analysis of the foam and liquid fractions showed that γ-gliadins were more important for A-W interface stability than ω-gliadins even if the latter were most affected by the TG treatment. Elimination of positively charged (at pH 4.5) lysine residues and conformational changes (both as a result of TG treatment) may have altered the ability of ω-gliadins to adsorb at the interface and to interact with other proteins thereat. Overall, TG treatment of GNPs resulted in formation of very strong protein films at the A-W interface stabilized by both covalent bonds and non-covalent interactions which in turn led to highly stable foams.

Identification of Bioactive Phytochemicals from Six Plants: Mechanistic Insights into the Inhibition of Rumen Protozoa, Ammoniagenesis, and α-Glucosidase.

Simple SummaryRumen protozoa have some contribution to feed digestibility in the rumen, but Entodinium, the most predominant genus, is the main culprit of inefficient nitrogen utilization in ruminants. Using chemical drugs, many studies have attempted to inhibit the rumen protozoa, but few of the approaches are either effective or practical. In this study, we investigated the nutritional and functional properties of Adansonia digitata (baobab), Flemingia macrophylla (waras tree), Kalimeris indica (Indian aster), Brassica rapa subsp. chinensis (bok choy), Portulaca oleracea (common purslane), and Calotropis gigantea (giant milkweed) for their potential as feed additives in animal husbandry. The plants were also analyzed for their major phytochemicals using reversed phase-high performance liquid chromatography (HPLC) and then evaluated for their ability to inhibit rumen protozoa, ammoniagenesis, and microbial α-glucosidase activity in vitro. C. gigantea inhibited the rumen protozoa and reduced the wasteful ammoniagenesis, thereby indicating improved nitrogen utilization. A. digitata also reduced the microbial α-glucosidase activity that can potentially contribute to rumen acidosis. The tested plants, especially C. gigantea and A. digitata, could be used as potential alternatives to chemicals or antibiotics to ensure sustainable and green animal husbandry.Rumen protozoa prey on feed-degrading bacteria synthesizing microbial protein, lowering nitrogen utilization efficiency in ruminants. In this in vitro study, we evaluated six plants (Adansonia digitata, Flemingia macrophylla, Kalimeris indica, Brassica rapa subsp. chinensis, Portulaca oleracea, and Calotropis gigantea) for their potential to inhibit rumen protozoa and identified the phytochemicals potentially responsible for protozoa inhibition. Rumen protozoa were anaerobically cultured in vitro in the presence of each plant at four doses. All of the tested plants reduced total rumen protozoa (p ≤ 0.05), but C. gigantea and B. rapa were the most inhibitory, inhibiting rumen protozoa by 45.6 and 65.7%, respectively, at the dose of 1.1 mg/mL. Scanning electron microscopy revealed a disruption of the extracellular structure of protozoa cells. Only C. gigantea also decreased the wasteful ammoniagenesis (p ≤ 0.05). Moreover, the A. digitata extract inhibited α-glucosidase activity by about 70% at 100 µg/mL. Reversed-phase high-performance liquid chromatography analysis detected quercetin, anthraquinone, 3-hydroxybenzoic acid, astragaloside, and myricetin in the tested plant leaves. These plants may hold potential as feed additives to reduce rumen protozoa and α- glucosidase activity. Future research is needed to identify the specific anti-protozoal compound(s), the effects on the rumen microbiome, and its fermentation characteristics.

Fungal Solubilisation and Subsequent Microbial Methanation of Coal Processing Wastes.

Large quantities of rejects from coal processing plants are currently disposed of as waste piles or in ponds and rivers, resulting in environmental concerns including pollution of rivers, and ground and surface water contamination. This work investigates for the first time, a two-stage microbial process for converting coal processing wastes (coal rejects) to methane, involving (1) fungal solubilisation of coal rejects and (2) microbial methanation of the solubilised products. Phanerochaete chrysosporium, Trichoderma viride and Neurospora discreta were screened for their ability to solubilise coal rejects. N. discreta was found to be the most suitable candidate based on the extent of bio-solubilisation, laccase activity and reversed-phase high-performance liquid chromatography (RP-HPLC) analysis. Bio-methanation of fungal-solubilised coal rejects was carried out in mesophilic anaerobic reactors with no additional carbon source, using inoculum from an anaerobic food digester. Coal rejects solubilised by N. discreta produced 3- to 6-fold higher methane compared to rejects solubilised by the other two fungi. No methane was produced from untreated coal rejects, demonstrating the importance of the fungal solubilisation stage. A total of 3.7mmol of methane was generated per gram of carbon in 15days from N. discreta-solubilised coal rejects. This process offers a timely, environment-friendly, and sustainable solution for the treatment of coal rejects and the generation of value-added products such as methane and volatile fatty acids.

Effect of the Histone Methyltransferase Specific Probe BRD4770 on Metabolic Profiling of the Endophytic Fungus Diaporthe longicolla.

The endophytic fungus Diaporthe longicolla was isolated from the stem of Saraca asoca (Roxb.) Willd., commonly known as Ashok plant in India and Sri Lanka. Since no reports are available regarding epigenetic modulations by BRD4770 in microbial entities, D. longicolla was treated with different concentrations of BRD4770 for this purpose and evaluated for its antioxidant and antibacterial potential against five human pathogenic bacteria, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Shigella boydii, Klebsiella pneumoniae, and Escherichia coli. The crude extract obtained from cultures treated with 100 nM concentration of BRD4770 showed increased antioxidant activity and inhibition zone against S. aureus and MRSA, compared to the non-treated control. The composition of the non-treated and treated crude extract was analyzed, and induced compounds were identified with the help of Gas chromatography–mass spectrometry (GC-MS) and LC-ESI-MS/MS. LC-ESI-MS/MS analysis showed that berberine (antibacterial)-, caffeine-, and theobromine (antioxidant)-like compounds were induced in the BRD4770-treated crude extract. The presence of particular absorbance at a wavelength of 346.5 nm for berberine, 259.4 nm for caffeine, and 278.4 nm for theobromine in the reverse-phase high-performance liquid chromatography (HPLC) analysis of both BRD4770-treated crude metabolites and standard solution of the above compounds strongly supported the increased antibacterial and antioxidant activities that may be due to inducing the alterations in bioactivities of the BRD4770-treated culture.

Sedative-hypnotic, anxiolytic and possible side effects of Salvia limbata C. A. Mey. Extracts and the effects of phenological stage and altitude on the rosmarinic acid content

Ethnopharmacological relevanceSalvia limbata C. A. Mey. (Persian name: Maryam Goli-e-labeh dar) has been used for treating central nervous disorders such as insomnia, anxiety and depression in Persian traditional medicine. S. limbata is known for its pharmacological activities which could be at least in a part, upon the presence of rosmarinic acid (RA). However, the sedative-hypnotic effect, anxiolytic activity, possible side effects, and the mechanism of action of S. limbata extract has not yet been examined. Aim of the studyIn the current study the sedative-hypnotic effect, anxiolytic activity, possible side effects, and the mechanism of action of S. limbata extracts were evaluated. Besides, the effects of altitude and phenological stage on the RA content of S. limbata were investigated. Materials and methodsSedative-hypnotic and anxiolytic effects were evaluated through the pentobarbital induced loss of righting reflex test and open field test, respectively. Flumazenil was used to reveal the mechanism of action. Possible side effects were investigated in the passive avoidance and grip strength tests. Besides, the effects of altitude and phenological stage (vegetative, flowering, and seed setting) on the RA content of S. limbata were evaluated using reversed-phase high-performance liquid chromatography (RP-HPLC). ResultsFollowing behavioral tests, sedative-hypnotic and anxiolytic effects were observed. Since the observed effects were reversed by flumazenil and no side effect on the memory and muscle strength was reported, modulation of the α1-containing GABA-A receptors could be proposed as one of the involved mechanisms. According to the RP-HPLC analysis, harvesting S. limbata in the vegetative stage at the altitude of 2500 m led to the highest content of RA (8.67 ± 0.13 mg/g dry matter). Among different extract of the plant samples collected in the vegetative stage at the altitude of 2500 m, the hydroalcoholic extract showed the highest rosmarinic acid content. ConclusionThe obtained results help to find the optimum situation to gain the highest content of RA as well as the pharmacological activity that could be economically important for the pharmaceutical industries.

Isolation and Characterization of a Plant Growth-Promoting Bacterium Acinetobacter sp. SuKIC24 From in vitro-Grown Basilicum polystachyon (L.) Moench.

In the present study, a plant growth-promoting bacterium isolated from contaminated in vitro-grown Basilicum polystachyon (L.) Moench, an herbaceous plant, was studied and identified as Acinetobacter strain SuKIC24 based on the 16S rRNA gene sequence analysis. The results revealed that the identified strain exhibited a strong ability to solubilize the phosphate with phosphate solubilization index of 4.0 and produced 387µg/ml of soluble phosphate in Pikovskaya media after 7days of incubation, whereas zinc solubilization efficacy was determined to be 175%. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis revealed that the bacterium strain produced 77.36µg/ml of indole-3-acetic acid. Plant inoculated with the bacterium strain was found to demonstrate a significant increase in height of B. polystachyon as compared to the control plant. The present findings indicated that the identified bacterium strain might act as plant growth-promoting bacterium (PGPB).