Thin Layer Chromatography Analysis Research Articles

Evaluation of in vitro Cholesterol Esterase Inhibitory Activity of Few Selected Vegetables – An Indication of Cardioprotective Property

Pharmacognosy Research,2022,14,2,188-194.DOI:10.5530/pres.14.2.27Published:April 2022Type:Original ArticleAuthors:Thiyagarajan Sathishkumar, Muruvanda Bhuvan Uthaiah, Ravichandran Vinu Varshith, Kuppamuthu Kumaresan, and Vinohar Stephen Rapheal Author(s) affiliations:Thiyagarajan Sathishkumar, Muruvanda Bhuvan Uthaiah*, Ravichandran Vinu Varshith, Kuppamuthu Kumaresan, Vinohar Stephen Rapheal Department of Biotechnology, Kumaraguru College of Technology, Coimbatore, Tamil Nadu, INDIA. Abstract:Background: Cardiovascular diseases are responsible for severe mortality rate amongst the population. Several drugs available to control and/ or cure the same, cause severe adverse effects amidst human population. Contextually, the present study was devised to screen the in vitro cholesterol esterase (CEase) activity of few selected vegetables as an indicator of cardioprotective property. Objectives: To screen and purify the polyphenolic fractionate from the vegetable(s) extract that record an optimal CEase activity. Materials and Methods: CEase inhibitory activity was investigated using standard spectrophotometric assay. Microwave assisted central composite design based response surface methodology (CCD based RSM) was executed to quantify an improvement in the CEase inhibitory activity of Solanum melongena extract. One dimensional thin layer chromatography (1D TLC) and two-dimensional preparative thin layer chromatography (2D PTLC) analyses were carried out for Brassica oleracea var. botrytis hot water extract to identify and eluate the polyphenolic fractionate, respectively. Results: Significant CEase inhibitory activity was noticed in the hot water extract of Brassica oleracea var. botrytis (84.65 ± 3.29%) than the other vegetable extracts. Even though the optimal process model was validated (at 5% level), no significant improvement was recorded in the CEase inhibition. The presence of polyphenolic content in the hot water extract of Brassica oleracea var. botrytis (Rf = 0.69) was confirmed by comparing with standard quercetin (Rf = 0.60). Strong blue and moderate brownish yellow color spots revealed the presence of phenolic acid and flavonoid eluates respectively, were purified using 2D PTLC technique. The eluate has recorded a near moderate (28.57 ± 2.12%) CEase inhibitory activity. Conclusion: Brassica oleracea var. botrytis possess significant CEase inhibitory activity and proved to be a good diet to protect from various cardiovascular diseases. Key words: Brassica oleracea var. botrytis, Cholesterol esterase, Solanum melongena, Microwave Assisted Response Surface Methodology, Flavonoid. View:PDF (245.05 KB)

Analysis of Phytochemical compounds present in some Traditional Anti-dermatophytic plants by Thin Layer, Column Chromatographic Methods

Traditionally, various medicinal plants were known to possess anti-dermatophytic potential and have been used for many years to treat skin diseases. We are aimed to examine anti-dermatophytic plants like Calotropis gigantea (L.) R. Br., Cassia alata (L) Roxb., Kaempferia galanga L., Melia azedarach L. and Sesbania grandiflora (L) Pers. for the quantification and separation of phyto-chemical compounds through thin layer and column chromatography. Five plant samples comprising of leaves, stem fiber and rhizome were separately extracted using five solvents such as, ethanol, methanol, acetone, chloroform and petroleum ether. The solvent extracts were subsequently investigated for qualitative, quantitative phytochemical, thin layer and column chromatographic analysis using standard assays. The experimental findings revealed the varied presence of compounds such as sugar, amino acid, protein, phenol, alkaloid and flavonoid in the solvent extracts used. In TLC profiling, a number of phyto chemical compounds, at different ranges of visible spots, with different Rf values at varying solvent system, were identified. Also, column fractions at different solvent run ratio (hexane: acetone = 8:2, 6:4, 4:6, 2:8) were identified with a wide range of color variations at different retention time and were collected sequentially. The present analysis exhibit a significant role to identify and differentiate therapeutic phyto-compounds which helps for further study on isolation and characterization with anti-dermatophytic potential.

Investigation of Flavonoids in Stereospermum suaveolens DC Leaves (Patala) Using HPTLC Analysis for Inflammatory Pain, Swelling, and Edema Treatment

Background: As per extensive research study, it was observed that Stereospermum suaveolens DC (Patala) is rich sources of flavonoids. These phytoconstituents have played an important role in prevention and maintenance of acute and chronic diseases of pain and inflammation. There is need to explore method for identification and quantification of the presence of flavonoids in S. suaveolens DC leaves (SSL) by qualitatively and quantitatively and to establish its mechanism in pain, swelling and edema Aim: The aim of the study was to identify and assess role of flavonoids in SSL by high-performance thin-layer chromatography (HPTLC) analysis and animal screening models of inflammatory pain, swelling, and edema. Materials and Methods: HPTLC to quantify flavonoid in the ethyl acetate fraction of plant component in comparison with Quarcetin. The inflammatory pain, swelling, and edema method were investigated by acetic acid induced writhing method in mice, histamine-induced edema in Wistar rats, and croton oil-induced ear edema in mice. The test doses 125 mg/kg, 250 mg/kg, and 500 mg/kg oral administration (p.o.) of ethyl acetate fraction of SSL were selected by oral acute toxicity OECD 423. Results: HPTLC analytical method estimated presence of flavonoids and estimated amount was 22.64%. However, inhibition in histamine-induced paw edema and reduction inhibition in ear edema significantly (P < 0.05) observed by dose-dependent effect and antinociceptive activity detected on acetic acid induced writhing response method in mild to moderately using one-way analysis of variance method. Conclusion: It was clear that flavonoids were responsible for direct and indirect release of intermediate inflammatory mediators and promote its role in the treatment of pain and swelling.

Study on the Biochemical Characterization and Selectivity of Three β-Glucosidases From Bifidobacterium adolescentis ATCC15703.

Three β-glucosidases from Bifidobacterium adolescentis ATCC15703, namely, BaBgl1A, BaBgl3A, and BaBgl3B, were overexpressed in Escherichia coli. The recombinant β-glucosidases were sufficiently purified using Ni2+ affinity chromatography, and BaBgl1A exhibited the best purification efficiency with a purification factor of 2.3-fold and specific activity of 71.2 U/mg. Three recombinant β-glucosidases acted on p-nitrophenyl-β-glucopyranoside (pNPβGlc) at around pH 7.0 and 30–50°C. The results of the substrate specificity assay suggested that BaBgl1A acted exclusively as β-1,2-glucosidase, while BaBgl3A and BaBgl3B acted mostly as β-1,3-glucosidase and β-1,4-glucosidase, respectively. The substrate specificity of the three recombinant enzymes was further studied using the ginsenosides Rb1 and Rd as substrates. The results of thin-layer chromatography and high-performance liquid chromatography analyses showed that BaBgl1A exhibited the highest bioconversion ability on Rb1 and Rd, where it hydrolyzed the outer C-3 glucose moieties of Rb1 and Rd into the rare ginsenosides Gypenoside XVII and F2; BaBgl3A exhibited medium bioconversion ability on Rb1, where it hydrolyzed both the outer C-3 and C-20 glucose moieties of Rb1 into Gyp XVII and Rd; and BaBgl3B was not active on Rb1 and Rd. These β-glucosidases will act as new biocatalytic tools for transforming ginsenosides and preparing active glycosides and aglycone.

Phytochemical Profile and Evaluation of Acute Toxicity of the Crude Extract from Acanthospermum hispidum Roots

Acanthospermum hispidum DC (Asteraceae), popularly known as "espinho-de-cigano", is a plant species used in folk medicine to treat gastrointestinal and respiratory disorders. These activities can be related to the presence of secondary metabolites, which can also play toxicity. Considering that, the objective of this work was to evaluate the toxicity of the crude extract from roots of A. hispidum. The herbal material was collected, ground, and the crude extract was obtained with acetone: water (7:3, v/v) at 10% (w/v) by turbo extraction. Phytochemical analysis by thin-layer chromatography (TLC) was performed, and the results were used to obtain the HPLC fingerprint. A single oral dose (2,000 mg/kg) was administered daily over 14 consecutive days to mice. Bodyweight, food and water intake, blood biochemical, and hematological parameters were measured. The results obtained in the TLC analysis suggested the presence of cinnamic derivatives, flavonoids, and terpenes and were confirmed by HPLC analysis. Additionally, the total polyphenol content was 10.1% ± 0.0093%, expressed as pyrogallol. According to the acute toxicity study, the data demonstrated the low toxicity of the crude extract of A. hispidum roots, considered very low acute toxicity or non-toxic, proved safe when administered orally.

Saponins from Albizia procera extract: Surfactant activity and preliminary analysis

We report our results on surface activity and preliminary analytical data of natural surfactant (saponin) from Albizia procera extract. The plant-based extract exhibited moderate reduction of surface tension, low alkalinity, moderate foaming and emulsifying characteristics. The critical micelle concentration is 7.00 × 10−3 g/cc, where the emulsifying power reached a maximum. An abrupt increase in viscosity and conductivity with surfactant concentration was seen in this region. Dirt dispersion results displayed a sharp maximum at the Critical Micelle Concentration. The extracts were analyzed to understand the constituents. The presence of polar and non-polar compounds was seen in Thin Layer Chromatography analysis. Using methanol water solvent system, a good separation of compounds was observed in High Performance Liquid Chromatography. The presence of saponin along with some traces of flavonoids was observed by UV-Visible spectroscopy. Occurrence of triterpenoid saponins were indicated by Fourier Transform Infrared spectroscopy analysis. The results obtained from surface tension, foaming and emulsification highlights the efficiency of Albizia procera extract to be employed as a possible environment friendly surface-active agent. This natural surfactant could find various industrial and domestic applications.

A biosurfactant-producing yeast Rhodotorula sp.CC01 utilizing landfill leachate as nitrogen source and its broad degradation spectra of petroleum hydrocarbons.

Biosurfactants (BSs) are known for their remarkable properties, however, their commercial applications are hampered partly by the high production cost. To overcome this issue, a biosurfactant producing strain, Rhodotorula sp.CC01 was isolated using landfill leachate as nitrogen source, while olive oil was determined as the best sole carbon source. The BS produced by Rhodotorula sp.CC01 had oil displacement diameter of 19.90 ± 0.10cm and could reduce the surface tension of water to 34.77 ± 0.63 mN/m. It was characterized as glycolipids by thin layer chromatography, FTIR spectra, and GC-MS analysis, with the critical micelle concentration of 70mg/L. Meanwhile, the BS showed stability over a wide range of pH (2-12), salinity (0-100g/L), and temperature (20-100°C). During the cultivation process, BS was produced with a maximum rate of 163.33mgL-1h-1 and a maximum yield of 1360mg/L at 50h. In addition, the removal efficiency of NH4+-N reached 84.2% after 75h cultivation with a maximum NH4+-N removal rate of 3.92mgL-1h-1. Moreover, Rhodotorula sp.CC01 has proven to be of great potential in remediating petroleum hydrocarbons, as revealed by chromogenic assays. Furthermore, genes related to nitrogen metabolism and glycolipid metabolism were found in this strain CC01 after annotating the genome data with KEGG database, such as narB, glycoprotein glucosyltransferase, acetyl-CoA C-acetyltransferase, LRA1, LRA3, and LRA4. The findings of this study prove a cost-effective strategy for the production of BS by yeast through the utilization of landfill leachate.

Potentials of N-(2, 4-dinitro-1-naphthyl)-p-toluenesulphonamide and 2, 4-dinitro-1-naphthol as novel charge transfer acceptors in pharmaceutical analysis of some quinolones and cephalosporins

BackgroundQuinolones and cephalosporins are antibiotic agents with activity against Gram-positive and Gram-negative bacteria. They contain chromophores and amine groups which are electron-rich centres capable of donating electrons to electron-deficient compounds. A survey of the literature revealed that 2, 4-dinitro-1-naphthol, a nitroaromatic useful in chemical synthesis, can accept electrons in charge transfer reactions. This work investigates N-(2, 4-dinitro-1-naphthyl)-p-toluenesulphonamide and 2, 4-dinitro-1-naphthol in the formation of charge transfer complexes by accepting electrons from selected quinolones and cephalosporins. Five other nitroaromatics (i.e. 4-nitro-1-naphthylamine, 2-nitro-1-naphthol, 2,4-dinitro-1-naphthylamine, 1-nitronaphthalene and 1,4-dinitronaphthalene) were screened in addition to the aforementioned and compared for charge transfer complexes formation. Spot test was used to establish charge transfer complex formation at room and elevated temperatures with determination done by visual inspection and thin layer chromatographic analysis of the reaction mixture. Ultraviolet visible absorption spectroscopy was used to estimate the extent of complexes.ResultsOnly solutions of adducts of N-(2, 4-dinitro-1-naphthyl)-p-toluenesulphonamide and 2, 4-dinitro-1-naphthol gave instant and distinct colour with each drug used at room and elevated temperature. While the former gave deep golden yellow, the latter gave golden yellow against their blank reagent solutions which were, lemon and greenish yellow respectively. Visual inspections of 2-nitro-1-naphthol adduct solutions showed no colour change from the yellow colour of the blank reagent solution, even though the Ultraviolet visible absorption spectra revealed the formation of charge transfer complexes. The adducts solutions of 4-nitro-1-naphthylamine, 2,4-dinitro-1-naphthylamine, 1-nitronaphthalene and 1,4-dinitronaphthalene showed no colour change from their blank reagent solutions and their Ultraviolet visible absorption spectra revealed no formation of charge transfer complexes.ConclusionUltraviolet visible absorption spectral analysis shows superiority of N-(2, 4-dinitro-1-naphthyl)-p-toluenesulphonamide and 2, 4-dinitro-1-naphthol in charge-transfer complex formation over other nitroaromatics screened. N-(2, 4-dinitro-1-naphthyl)-p-toluenesulphonamide and 2, 4-dinitro-1-naphthol are good acceptors of electrons from these drugs, hence could be useful as charge transfer reagents in ultraviolet visible spectrophotometric analysis of these drugs.

A study on antibacterial efficacy of different extracts of Artocarpus chama fruits and identification of bioactive compounds in the most potent extract

This study is designed to evaluate the antibacterial potential of various extracts of mature green fruits of Artocarpus chama with the identification of bioactive compounds. The antibacterial efficacy was tested against eight pathogenic bacteria using standard antibiotics as the positive control. Antibacterial bioassay was performed by measuring zone of inhibition, Minimum Inhibitory Concentrations (MIC), and Minimum Bactericidal Concentrations (MBC). The most effective extract was further subjected to preliminary phytochemical tests, Thin Layer Chromatography (TLC), and Fourier Transform Infrared analysis. Ethyl acetate extract (EAAC) was found to be the most effective extract producing an inhibition zone of 45.50 ± 0.50 mm against Pseudomonas aeruginosa MTCC 2453. Effective spots (ES) (Rf=0.37) of EAAC separated by TLC analysis were subjected to indirect contact bio-autography for evaluating their efficacies against the tested bacterial strains. Values of MBC/MIC for the extract ranged from 1.20 to 1.80 (<2) indicating their bactericidal property. Gas Chromatography-Mass Spectroscopy analysis of ES was performed for the identification of bioactive antibacterial compounds. Alkaloids, saponins, steroids, terpenoids, and an array of functional groups in EAAC were detected. MICs of ES ranged from 6 - 10 µg/ml. Four bioactive compounds in ES were detected in GC-MS analysis. Thus, this study revealed that ethyl acetate extract of A. chama fruits contained several bioactive compounds with promising bactericidal properties against tested strains of both Gram-positive and Gram-negative pathogenic bacteria.

Micellar Thin Layer Chromatography and Computer-Aided Analysis of Empagliflozin, Linagliptin and Metformin HCl Ternary Mixture.

The present work was performed in order to study the mechanism of micellar thin layer chromatography (MTLC) and to develop a new simple and sensitive simultaneous MTLC method for separation of empagliflozin, Linagliptin and metformin hydrochloride ternary mixture. The study was done using three different surfactants; sodium dodecyl sulphate (SDS), benzalkonium chloride (BAC) and polysorbate 80 (tween 80). Chromatographic procedure was performed using micellar mobile phase that composed of aqueous solution of each surfactant and methanol (6: 4v/v) and micellar TLC determination at λmax 237nm. Separation using SDS (anionic surfactant) and BAC (cationic surfactant) depends on ionization potential (AMI-IP), partition coefficient (logP (o/w)) and hydrogen bond donor atoms (a-don), whereas separation using tween 80 depends mainly on the lipophilicity (RM0), solvation energy (E-sol) and Van der Waals energy (E-vdw). Quantitative structure-retention relationships study was carried out, modeled, evaluated and validated using molecular operating environment software.

Comparative Physicochemical and Antioxidant Activities of Eight Commercially Available Antidiabetic Polyherbal Products

Background: High cost and side effects of current drugs has led to increased use of herbal preparations for Diabetes Mellitus, which is a chronic multifactorial disease associated with several complications and more than 422 million people affected worldwide. Objectives: To comparatively evaluate physicochemical and antioxidant properties of eight commercially available antidiabetic polyherbal products. Material and methods: Samples were subjected to physicochemical analysis; organoleptic tests, uniformity of weight, thin layer chromatography (TLC), phytochemical screening, heavy metals, extractive values, moisture content, ash values, antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) inhibition and total phenolic content (TPC). Statistical analysis was done at p < 0.05.Results: The samples which ranged from tablets, capsules to tea sachets were of varying colour, odour and taste, with obtained average weights significantly different from the labelled weight for most products The TLC analysis gave varied number of spots with the identified phytochemicals (tannins, flavonoids, phenols and proteins) confirmed by phytochemical screening. Moisture contents ranged from 5.99 to 8.84 %w/w, with water and alcohol-soluble extractives 13.0-27.7%w/w and 15.4-47.5 %w/w respectively. Lead, arsenic and cadmium contents were within WHO specification. Total ash, water-soluble ash and acid-insoluble ash ranged from 1.27 ±0.02 to 42.40 ±0.04; 0.36 ±0.01 to 37.65 ±0.05 and 0.29 ±0.02 to 7.10 ±0.03 %w/w respectively. The TPC ranged from 0.015 ±0.006 to 0.277 ±0.006 mgGAE/mL with DPPH scavenging activity (IC50) ranging from 8.87 ±0.113 to 825.24 ±2.03 µg/mL. Conclusion: Only two brands complied with all the WHO herbal products specifications, while varied antioxidant activities were observed across all the polyherbal products.

Xylan Deconstruction by Thermophilic Thermoanaerobacterium bryantii Hemicellulases Is Stimulated by Two Oxidoreductases

Thermoanaerobacterium bryantii strain mel9T is a thermophilic bacterium isolated from a waste pile of a corn-canning factory. The genome of T. bryantii mel9T was sequenced and a hemicellulase gene cluster was identified. The cluster encodes seven putative enzymes, which are likely an endoxylanase, an α-glucuronidase, two oxidoreductases, two β-xylosidases, and one acetyl xylan esterase. These genes were designated tbxyn10A, tbagu67A, tbheoA, tbheoB, tbxyl52A, tbxyl39A, and tbaxe1A, respectively. Only TbXyn10A released reducing sugars from birchwood xylan, as shown by thin-layer chromatography analysis. The five components of the hemicellulase cluster (TbXyn10A, TbXyl39A, TbXyl52A, TbAgu67A, and TbAxe1A) functioned in synergy to hydrolyze birchwood xylan. Surprisingly, the two putative oxidoreductases increased the enzymatic activities of the gene products from the xylanolytic gene cluster in the presence of NADH and manganese ions. The two oxidoreductases were therefore named Hemicellulase-Enhancing Oxidoreductases (HEOs). All seven enzymes were thermophilic and acted in synergy to degrade xylans at 60 °C. Except for TbXyn10A, the other enzymes encoded by the gene cluster were conserved with high amino acid identities (85–100%) in three other Thermoanaerobacterium species. The conservation of the gene cluster is, therefore, suggestive of an important role of these enzymes in xylan degradation by these bacteria. The mechanism for enhancement of hemicellulose degradation by the HEOs is under investigation. It is anticipated, however, that the discovery of these new actors in hemicellulose deconstruction will have a significant impact on plant cell wall deconstruction in the biofuel industry.

Isolation, Screening, Characterization And Application Of Biosurfactant By Achromobacter Xylos Strain GSR21 Producing Bacteria From Hydrocarbons Contaminated Soil

: Biosurfactants plays a key role in tertiary recovery (EOR), and production, excessive minerals discharge from water during petroleum refinery process, environmental utilization and eco-friendly. Biosurfactant producing bacteria found to be sufficient in hydrocarbon-polluted soil samples; it is expected to more amounts of agrichemicals contemporary in the clay. These bacteria establish itself soil and region specific. So, in this study we made an attempt to identify and characterize biosurfactant produced by achromobacter xylos strain GSR21 from hydrocarbon polluted soil in Andhrapradesh, India. A battery of biosurfactant screening methods engaged were haemolytic activity, oil spreading technique, lipase activity, emulsification index (E24), emulsification assay, tilting glass slide, blood haemolysis test, drop-collapsed assay, and foaming activity. The organism isolate was studied based on molecular, phenotypic, and biochemical methods. Thin-layer chromatography I(TLC), Fourier transforms infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC–MS) investigates were used to identify and characterize the biosurfactant produced. The isolated biosurfactant was applied on chosen hydrocarbons to measure its emulsifying capacity.The phylogeny study of the 16S rRNA classified the isolate as A chromobacter xylos strain GSR21. The sequence secured from the isolate has been accumulated in GenBank covered by the accession number JQ746488. The result obtained from the study acknowledge high biosurfactant action with a maximum emulsification index (E24) of 62 % compared to emulsification index (E24) of 72% by sodium lauryl sulfate (SLS). Moreover, the biosurfactant appear emulsifying activity against the following hydrocarbons: diesel, methylbenzene, kerosene, dimethylbenzene, and petrol. The optimum cultural conditions (incubation time, carbon, pH, hydrocarbon, inoculum concentration, nitrogen, and temperature) for growth and biosurfactant produced by A chromobacter xylos GSR21 were analysed. The biosurfactant was characterized as a glycolipid using thin layer chromatography (TLC), while the gas chromatography-mass spectrometry (GC–MS) technique analyzed the glycolipid as dodecanoic acid-undecyl ester.Present study has exhibited the magnitude of Achromobacter xylos strain GSR21 isolated from hydrocarbon-polluted soil to produce biosurfactant and the effectiveness of the produced biosurfactant in emulsifying different hydrocarbons. Moreover, the biosurfactant produced was established to be held by the class, glycolipid based on the thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC–MS) analyses.

Pharmacognostic evaluation and coagulant analysis of the ethanol extracts of the leaves of Ocimum gratissimum L. (Lamiaceae)

Ocimum gratissimum belongs to the group of plants known as spices. The plant is an erect small plumb with many branches usually not more than 1m high. It is of the familylabiatae, genus Ocimum and species gratissimum. It is commonly known as scent leaf. This project is focused on the pharmacognostic and coagulant activities of the ethanol extract of the leaves of Ocimum gratissimum. The ethanol leaf extract of the plant was obtained using cold maceration with ethanol and then filtered and concentrated using rotary evaporator. The extract was used for coagulant evaluations, pharmacognostic evaluation and chromatographic (thin layer chromatography) analysis. The LD50 was determined in rats using Lorkes method. Microscopic result shows the presence of a covering or uniseratetrichomes, sclerenchyma none pitted with lumen and diacytic stomata. The numerical data obtained were 8.75%, 12% and 8.45% for moisture content, alcohol extractive value and total cash value. The LD50 shows that there was no death recoded because of the holistic use of the plants. The result of the coagulation research are as follow: Group 1 clotting time 1: 69 min and bleeding time 2: 62min, group 2 clotting time 1: 39 min and bleeding time 4: 79min, group 3 clotting time 1: 37 min and bleeding time 1: 99min, group 4 clotting time 1: 81mins and bleeding time is 2: 28mins, group 5 clotting time is 1: 47mins and bleeding time is 2: 49mins) This shows that the ethanol extract of Ocimum gratissimumon rats exhibited coagulant property. The phytochemical analysis of this plant reveals that it contains alkaloids, flavonoids, saponins, terpenoids, tannins, glycosides and resins. It can therefore be concluded that the ethanol extract of Ocimum gratissimum possesses coagulant activity. This study can be of great importance to the development of new coagulant drugs. Citation: Osualaet al.(2021) Pharmacognostic Evaluation and Coagulant screening of Ethanol Extract of the Leaves ofOcimumgratissimumL. (Lamiaceae) Journal

Mycoflora and Aflatoxin Producing Fungi from Some Storage Cereal Grains Sold in Darki Market, Wudil Local Government Area, Kano State, Nigeria

Aim: The study was aimed at determining the mycoflora and aflatoxin producing fungi in some storage cereals sold at Darki market, Wudil Local Government Area, Kano State. Nigeria.
 Study Design: A total of thirty samples (ten samples containing 250g each of maize, millet and wheat) were obtained from storage facilities of traders in different location of Darki, market.
 Place and Duration of Study: Storage facilities of Darki market, Wudil, Kano State, between May 2021 and July 2021.
 Methodology: Samples were subjected to serial dilution, enumeration of fungal load using pour plate method, inoculation and isolation of fungi using direct plating method, extraction and detection of aflatoxin using thin layer chromatography and molecular analysis using PCR.
 Result: Total fungal (mold) count on Potato Dextrose Agar recorded the highest count of 6.5103 in wheat sample 4 (WH4) while the least count of 2.2102 was recorded in wheat sample 6 (WH6). The fungal isolates obtained from this study were Aspergillus niger, Aspergillus flavus, Fusarium spp, Mucor spp, and Rhizopus spp. The percentage of occurrence of fungi isolated from the cereal samples was highest in Aspergillus niger (34.43%) and the least (11.48%) was recorded in Mucor spp. Extraction and detection methods revealed a blue fluorescence on the TLC plates indicating the presence of aflatoxin B in the samples. The standard calculated range of Retention factor value was between 0.58 and 0.75. After DNA extraction, the fragment size obtained by PCR reactions with primer pairs ITS1/ITS4 showed 600bp for most of the isolates. All the Aspergillus spp, Fusarium spp. and Rhizopus spp showed positivity for the molecular markers ITS1/ITS4 by having positive bands. Only Mucor spp showed negative results for ITS1/ITS4 primers and showed no band.
 Conclusion: This study shows that most of the cereals were contaminated with either one or more molds but within the satisfactory and marginal limits. However, aflatoxin contamination is a threatening issue Hence aflatoxins contamination of crops at harvest conditions should be controlled to some extent by the implementation of good agricultural practices and good storage practices.

Extracts of Musa basjoo induce growth inhibition and changes in the protein expression of cell cycle control molecules in human colorectal cancer cell lines.

Musa basjoo (MB) is a species of the banana plant belonging to the genus Musa that has been used as a folk medicine. However, evidence-based biological activities and the molecular mechanism of action of MB are unknown. Thus, the aim of the present study was to examine whether the crude dried leaf extracts of MB inhibit the growth of colorectal (HT29 and HCT116) and other types (HepG2, MCF-7 and PC-3) of human cancer cell lines. Crude extracts of MB inhibited the growth of cells with IC50 values of 136 µg/ml (acetone extract, HT29), 51 µg/ml (acetone extract, HCT116), 45 µg/ml (acetone extract, HepG2), 40 µg/ml (acetone extract, MCF-7), 29 µg/ml (acetone extract, PC-3), 175 µg/ml (methanol extract, HT29), 137 µg/ml (methanol extract, HCT116), 102 µg/ml (methanol extract, HepG2), 85 µg/ml (methanol extract, MCF-7), and 85 µg/ml (methanol extract, PC-3) in colony formation assays, and 126 µg/ml (acetone extract, HT29), 68 µg/ml (acetone extract, HCT116), 260 µg/ml (methanol extract, HT29), and 216 µg/ml (methanol extract, HCT116) in MTT assays. Thin layer chromatography analysis revealed the potential existence of aromatic compounds in the acetone extract of MB. Flow cytometric analysis indicated that the percentage of cells in G1 increased, and this was associated with a concomitant decrease of cells in the S and/or G2-M phases of the cell cycle. When colorectal cancer cells were treated with acetone extract of MB, there was a marked decrease in the levels of expression of the cyclin D1, cyclin E, cdk2 and cdk4 proteins and a marked increase in the levels of the expression of the p21CIP1, p27KIP1, and p53 proteins, but those of apoptosis-associated protein PARP did not change. There was a tendency for acetone extract of MB to inhibit xenograft tumor growth in mice. Collectively, the crude extracts of MB contain active components that exert growth inhibition of human cancer cells. This is the first systematic study of the anticancer activity of MB and may broaden insights into the possible clinical approach of specific herbal medicines.

Antioxidant, Cytotoxic Activities and Characterization of Secondary Metabolites of Endophytic Fungus Schizophyllum commune isolated from Coleus amboinicus (Lour.) Leaves

Schizophyllum commune (fam Schizophyllaceae) is an endophytic fungus isolated from a medicinal plant Coleus amboinicus (Lour.) Spreng. This study was aimed to identify metabolite profiles, characterize bioactivities (antioxidant and cytotoxic), and analyze metabolite production of the fungus due to light exposure and initial fungal culture age. Fungal cultures aged 7, 14, and 21 days old on PDA were incubated in Potato Dextrose Broth (PDB) fermentation medium for 2 weeks either in full light or dark condition. Metabolite profiles were analyzed using thin-layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were used to determine antioxidant and cytotoxic activity. The results showed that light exposure and different age of fungal cultures affected metabolite production based on TLC analysis. Phytochemical screening of ethyl acetate extract of fermentation indicated the presence of flavonoids, terpenoids, steroids, phenols, and alkaloids. There are five major compounds detected by GC-MS. Bioactive screening showed that this extract had DPPH scavenging activity (IC50 = 109.19 ± 0.77 µg/mL) and cytotoxic activities, the best cytotoxic activity was on HeLa cell (IC50 = 492.23 ± 24,43 µg/mL) with selectivity index (SI) value is 2.33.

Antioxidative-, Antimicrobial-, Enzyme Inhibition-, and Cytotoxicity-Based Fractionation and Isolation of Active Components from Monotheca buxifolia (Falc.) A. DC. Stem Extracts.

The current study elaborates the pharmacological potential of the methanolic extract and its fractions of the stems of Monotheca buxifolia based on thin-layer chromatography and column chromatography analyses, exploiting biological and phytochemical assays. The results suggest that bioassay-guided isolation and fractionation led to the accumulation of biologically active components in the most active fractions that resulted in the isolation of different compounds. Structural elucidation of the purified compounds was accomplished using spectroscopic one-dimensional (1H, 13C) and two-dimensional NMR (heteronuclear multiple quantum coherence, heteronuclear multiple bond coherence, and correlation spectroscopy) and spectrometric (electron ionization mass spectrometry and high-resolution electron ionization mass spectrometry) techniques. The n-hexane, CHCl3, and EtAOc fractions led to the isolation of lupeol from different fractions. 1-Triacontanol was also isolated from the n-hexane fraction, while benzoic acid, methyl benzoate, ursolic acid, and 3-hydroxybenzoic acid were obtained from the EtOAc fraction. The compounds depicted good-to-moderate total antioxidative potential and total reducing power activity and significant free-radical scavenging activity. All the compounds showed significant urease and lipase inhibitory activity with poor-to-moderate amylase inhibition. Significant zone of inhibition was observed against different bacterial strains by the isolated compounds. This work therefore states that bioassay-guided isolation plays a vital role in the isolation of biologically active constituents that can be exploited for drug development.

Elucidation of Chemical Interactions between Crude Drugs Using Quantitative Thin-Layer Chromatography Analysis.

To elucidate the interactions between crude drugs in Kampo medicines (traditional Japanese medicines), it is important to determine the content of the constituents in a cost-effective and simple manner. In this study, we quantified the constituents in crude drug extracts using thin-layer chromatography (TLC), an inexpensive and simple analytical method, to elucidate the chemical interactions between crude drugs. We focused on five crude drugs, for which quantitative high-performance liquid chromatography (HPLC) methods are stipulated in the Japanese Pharmacopoeia XVIII (JP XVIII) and compared the analytical data of HPLC and TLC, confirming that the TLC results corresponded with the HPLC data and satisfied the criteria of JP XVIII. (Z)-ligustilide, a major constituent in Japanese Angelica Root, for which a method of quantification has not been stipulated in JP XVIII, was also quantitatively analyzed using HPLC and TLC. Furthermore, Japanese Angelica Root was combined with 26 crude drugs to observe the variation in the (Z)-ligustilide content from each combination by TLC. The results revealed that combinations with Phellodendron Bark, Citrus Unshiu Peel, Scutellaria Root, Coptis Rhizome, Gardenia Fruit, and Peony Root increased the (Z)-ligustilide content. Quantifying the constituents in crude drug extracts using the inexpensive and simple TLC method can contribute to elucidating interactions between crude drugs in Kampo medicines, as proposed by the herbal-pair theory.

Antioxidant Activity and Bioactive Compounds of Ethyl Acetate Fractions from Syzygium cumini Wood Stem

Syzygium cumini is one of the endemic plants in Indonesia that has the potential to be developed. The fruit was known to have potential as a diabetes drug. However, very few studies to determine the medicinal potential and identification of compounds of Syzygium cumini wood stem has never been studied before. Therefore, this study aimed to determine the antioxidant activity and content of compounds in Syzygium cumini wood stem ethyl acetate fraction. The crude ethanol extract was prepared from the wood stem powder of Syzygium cumini in ethanol using maceration. Furthermore, it was carried out by the liquid-liquid fractionation process using solvents of different polarity. Antioxidant activity of the crude extract, hexane fraction, and ethyl acetate fraction was determined by spectrophotometric methods using 1,1-diphenyl-2-picrylhydrazyl (DPPH). Analysis of bioactive compounds by using Thin Layer Chromatography (TLC). Identification of compounds functional groups was carried out to support the results of TLC analysis by using Fourier-transform infrared (FTIR). Ethyl acetate fraction showed the highest antioxidant activity (IC50 13,62 µg/mL), followed by ethanol extract (IC50 19,64 µg/mL) and hexane fraction (IC50 61,25 µg/mL). The antioxidant activity of the acetate fraction was lower than that of ascorbic acid with an IC50 value of 2,85 µg/mL. However, this fraction has very strong antioxidant activity. The TLC analysis results showed that the sample was thought to contain an alkaloid, phenolic, flavonoid, and terpenoid. The assumption on agreement with FTIR analysis which showed functional groups were C-H, O-H, N-H, C=O, and C=C aromatics as constituents of these compounds.