Boerhavia diffusa (BD) Linn. (Nyctaginaceae) is one of the most commonly used herbs in the Indian traditional system of medicine for the urinary disorders. The aim of the current investigation was to carry out initiation, development, and maintenance of BD callus cultures and quantitative estimation of punarnavine in plant and callus extracts. Leaves and stem of BD were used as explant for the tissue culture studies using Murashige and Skoog (MS) basal medium. MS Media comprising 2,4-Dichlorophenoxy acetic acid (2,4-D) (1 ppm) and 2,4-D (1 ppm) + Indole-3-acetic acid (IAA) (1.0 ppm) were found to yield friable callus from leaf explant; similarly, 2,4-D (0.3 ppm) + IAA (0.75 ppm) + Kinetin (0.3 ppm) and 2,4-D (0.5 ppm) + Naphthalene acetic acid (NAA) (1.5 ppm) + Kinetin (0.3 ppm) were found to yield friable callus from the stem explant. High-performance thin-layer chromatography method was been developed for the quantitative estimation of punarnavine (Rf = 0.73) using mobile phase containing toluene: ethyl acetate: formic acid in the ratio (7.0:2.5:0.7, v/v/v) at 262 nm. The validated method was found linear (r2 = 0.9971) in a wide range (100-1000 ng spot-1), precise, accurate, and robust. The values of limit of detection, LOD = 30.3 ng spot-1, and limit of quantification, LOQ = 100.0 ng spot-1. The robustness of the method was proved by applying the Box-Behnken design (BBD). The developed method found appropriate for the quality control of medicinal plants containing punarnavine as a constituent.
The cholesterol-lowering properties of 12 lactic acid bacteria (LAB) in the absence or presence of 0.3% bile salts were assessed and compared quantitatively and qualitatively in vitro. A new, more sensitive and cost-effective high-performance thin-layer chromatography method combined with digital image evaluation of derivatised chromatographic plates was developed and validated to quantify cholesterol in LAB culture media. The performance of the method was compared with that of the o-phthalaldehyde method. For qualitative assessment, assimilated fluorescently tagged cholesterol was visualised by confocal microscopy. All LAB strains exhibited a cholesterol-lowering effect of various degrees (19-59% in the absence and 14-69% in the presence of bile salts). Lactobacillus plantarum LAB12 and Pentosaceus pentosaceus LAB6 were the two best strains of lactobacilli and pediococci. They lowered cholesterol levels by 59% and 54%, respectively, in the absence and by 69% and 58%, respectively, in the presence of bile salts. Confocal microscopy showed that cholesterol was localised at the outermost cell membranes of LAB12 and LAB6. The present findings warrant in-depth in vivo study. Graphical abstract (A) 3D plots based on scan at 525 nm of (B) derivatized HPTLC plate of separated cholesterol and (C) confocal microscopic image showing the localisation of NBD-cholesterol assimilated by LAB.
A rapid, easy, and sensitive method is described in this paper for the assay of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase, a key enzyme in cholesterol biosynthesis. [14C]HMG CoA was used as the substrate and the product formed, i.e., [14C]mevalonate, was allowed to be converted to its lactone form (mevalonolactone) in the presence of HCl. The reaction mixture was applied to a column containing an anionic exchanger. The column was made up of QAE-Sephadex (A25, formate form) packed to a height of 4 cm in Pasteur pipets. Under these conditions, mevalonolactone was not retained by the column and was eluted with ammonium formate solution while HMG CoA, being negatively charged, was retained by the gel and eluted by HCl above 0.05 M. Determination of the amount of radioactivity in mevalonolactone was then used to quantitate the activity of HMG CoA reductase. This assay has been successfully used for determining the activity of this enzyme in a microsomal fraction prepared from the liver of the rat.
Artemisia annua L., a medicinal herb, produces secondary metabolites with antimicrobial property. In Malaysia due to the tropical hot climate, A. annua could not be planted for production of artemisinin, the main bioactive compound. In this study, the leaves of three in vitro A. annua L. clones were, extracted and two bioactive compounds, artemisinin and a precursor, were isolated by thin layer chromatography. These compounds were found to be effective in inhibiting the growth of Gram-positive and Gram-negative bacteria but not Candida albicans. Their antimicrobial activity was similar to that of antibactericidal antibiotic streptomycin. They were found to inhibit the growth of the tested microbes at the minimum inhibition concentration of 0.09 mg/mL, and toxicity test using brine shrimp showed that even the low concentration of 0.09 mg/mL was very lethal towards the brine shrimps with 100% mortality rate. This study hence indicated that in vitro cultured plantlets of A. annua can be used as the alternative method for production of artemisinin and its precursor with antimicrobial activities.
Hyperlipidemia is defined as the presence of either hypertriglyceridemia or hypercholesterolemia, which could cause atherosclerosis. Although hyperlipidemia can be treated by hypolipidemic drugs, they are limited due to lack of effectiveness and safety. Previous studies demonstrated that xanthorrhizol (XNT) isolated from Curcuma xanthorrhizza Roxb. reduced the levels of free fatty acid and triglyceride in vivo. However, its ability to inhibit cholesterol uptake in HT29 colon cells and adipogenesis in 3T3-L1 cells are yet to be reported. In this study, XNT purified from centrifugal TLC demonstrated 98.3% purity, indicating it could be an alternative purification method. The IC50 values of XNT were 30.81 ± 0.78 μg/mL in HT29 cells and 35.07 ± 0.24 μg/mL in 3T3-L1 adipocytes, respectively. Cholesterol uptake inhibition study using HT29 colon cells showed that XNT (15 μg/mL) significantly inhibited the fluorescent cholesterol analogue NBD uptake by up to 27 ± 3.1% relative to control. On the other hand, higher concentration of XNT (50 μg/mL) significantly suppressed the growth of 3T3-L1 adipocytes (5.9 ± 0.58%) compared to 3T3-L1 preadipocytes (81.31 ± 0.55%). XNT was found to impede adipogenesis of 3T3-L1 adipocytes in a dose-dependent manner from 3.125 to 12.5 μg/mL, where 12.5 μg/mL significantly suppressed 36.13 ± 2.1% of lipid accumulation. We postulate that inhibition of cholesterol uptake, adipogenesis, preadipocyte and adipocyte number may be utilized as treatment modalities to reduce the prevalence of lipidemia. To conclude, XNT could be a potential hypolipidemic agent to improve cardiovascular health in the future.
Multivariate analysis of thin-layer chromatography (TLC) images was modeled to predict antioxidant activity of Pereskia bleo leaves and to identify the contributing compounds of the activity. TLC was developed in optimized mobile phase using the 'PRISMA' optimization method and the image was then converted to wavelet signals and imported for multivariate analysis. An orthogonal partial least square (OPLS) model was developed consisting of a wavelet-converted TLC image and 2,2-diphynyl-picrylhydrazyl free radical scavenging activity of 24 different preparations of P. bleo as the x- and y-variables, respectively. The quality of the constructed OPLS model (1 + 1 + 0) with one predictive and one orthogonal component was evaluated by internal and external validity tests. The validated model was then used to identify the contributing spot from the TLC plate that was then analyzed by GC-MS after trimethylsilyl derivatization. Glycerol and amine compounds were mainly found to contribute to the antioxidant activity of the sample. An alternative method to predict the antioxidant activity of a new sample of P. bleo leaves has been developed.
The role of aldose reductase (ALR2) in diabetes mellitus is well-established. Our interest in finding ALR2 inhibitors led us to explore the inhibitory potential of new thiosemicarbazones. In this study, we have synthesized adamantyl-thiosemicarbazones and screened them as aldehyde reductase (ALR1) and aldose reductase (ALR2) inhibitors. The compounds bearing phenyl 3a, 2-methylphenyl 3g and 2,6-dimethylphenyl 3m have been identified as most potent ALR2 inhibitors with IC50 values of 3.99 ± 0.38, 3.55 ± 0.26 and 1.37 ± 0.92 µM, respectively, compared with sorbinil (IC50 = 3.14 ± 0.02 μM). The compounds 3a, 3g, and 3m also inhibit ALR1 with IC50 value of 7.75 ± 0.28, 7.26 ± 0.39 and 7.04 ± 2.23 µM, respectively. Molecular docking was also performed for putative binding of potent inhibitors with target enzyme ALR2. The most potent 2,6-dimethylphenyl bearing thiosemicarbazone 3m (IC50 = 1.37 ± 0.92 µM for ALR2) and other two compound 3a and 3g could potentially lead for the development of new therapeutic agents.
The use of biomaterials or microorganisms in PAHs degradation had presented an eye-catching performance. Pleurotus eryngii is a white rot fungus, which is easily isolated from the decayed woods in the tropical rain forest, used to determine the capability to utilize naphthalene, a two-ring polycyclic aromatic hydrocarbon as source of carbon and energy. In the meantime, biotransformation of naphthalene to intermediates and other by-products during degradation was investigated in this study. Pleurotus eryngii had been incubated in liquid medium formulated with naphthalene for 14 days. The presence of metabolites of naphthalene suggests that Pleurotus eryngii begin the ring cleavage by dioxygenation on C1 and C4 position to give 1,4-naphthaquinone. 1,4-Naphthaquinone was further degraded to benzoic acid, where the proposed terepthalic acid is absent in the cultured extract. Further degradation of benzoic acid by Pleurotus eryngii shows the existence of catechol as a result of the combination of decarboxylation and hydroxylation process. Unfortunately, phthalic acid was not detected in this study. Several enzymes, including manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase and 2,3-dioxygenase are enzymes responsible for naphthalene degradation. Reduction of naphthalene and the presence of metabolites in liquid medium showed the ability of Pleurotus eryngii to utilize naphthalene as carbon source instead of a limited glucose amount.
Armillaria sp. F022, a white-rot fungus isolated from a tropical rain forest in Samarinda, Indonesia, was used to biodegrade benzo[a]pyrene (BaP). Transformation of BaP, a 5-ring polycyclic aromatic hydrocarbon (PAH), by Armillaria sp. F022, which uses BaP as a source of carbon and energy, was investigated. However, biodegradation of BaP has been limited because of its bioavailability and toxicity. Five cosubstrates were selected as cometabolic carbon and energy sources. The results showed that Armillaria sp. F022 used BaP with and without cosubstrates. A 2.5-fold increase in degradation efficiency was achieved after addition of glucose. Meanwhile, the use of glucose as a cosubstrate could significantly stimulate laccase production compared with other cosubstrates and not using any cosubstrate. The metabolic pathway was elucidated by identifying metabolites, conducting biotransformation studies, and monitoring enzyme activities in cell-free extracts. The degradation mechanism was determined through the identification of several metabolites: benzo[a]pyrene-1,6-quinone, 1-hydroxy-2-benzoic acid, and benzoic acid.
The authors selected 38 thin-layer-chromatography (TLC) systems described in the available literature published over the last 10 years and evaluated those systems with respect to their suitability for detection and identification of opiates in urine, opium and heroin, as well as adulterants in heroin. A total of 14 substances: 8 opiates (morphine, 6-monoacetylmorphine, diacetylmorphine, codeine, acetylcodeine, noscapine, papaverine and thebaine) and 6 adulterants (ephedrine, quinine, methadone, caffeine, cocaine and strychnine) were used as test samples for this research. Using laboratory-coated plates and pre-coated plates, 15 and 13 TLC systems, respectively, were found to be able to detect and identify morphine and codeine in urine without interference from the remaining 12 substances. For the detection of opiates in opium samples as well as opiates and adulterants in illicit heroin samples the TLC system: chloroform-n-hexane-triethylamine (9:9:4) which was developed by the National Drug Research Centre, Penang, Malaysia, was found to be most suitable on both laboratory-coated and pre-coated plates. In addition, the following two systems, one on laboratory-coated plates--hexane-chloroform-diethylamine (50:30:7)--and the other on pre-coated plates--benzene-dioxane-ethanol-ammonia (50:40:5:5; T-7)--were also found to be among most suitable TLC systems for the analysis of opiates in opium samples. The article also presents the relative cost of each of the 38 evaluated TLC systems.
Trichoderma spp. are regularly found as a constituent of the mycoflora of many soils and are noted for their antagonistic activity against bacteria and other fungi. This latter property is the basis for the widespread interest in their use in the biological control of soil-borne fungal plant pathogens. This antagonism is partly based on their ability to produce an impressive inventory of secondary metabolites. An important group of bioactive metabolites produced by Trichoderma spp. are the non-ribosomal peptides (NRPs), especially the peptaibols. A virulent antagonistic strain, T. asperellum, which had been used in biological control strategies in Malaysia and previously examined for mycolytic enzyme production, has been studied for its potential for peptaibol production. The present research demonstrated the ability of T. asperellum to produce at least two metabolites which were identified as acid trichotoxin 1704E (Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Ala-Ala-Aib-Pro-Leu-Aib-Iva-Glu-Vol) and neutral trichotoxin 1717A (Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Ala-Aib-Aib-Pro-Leu-Aib-Iva-Gln-Vol). Addition of free Aib to the culture medium enhanced the production of trichotoxins. Biological activity of these substances was investigated against Bacillus stearothermophilus. The general characteristics of peptaibols, also found in the trichotoxins, include the presence of high proportions of the uncommon amino acid Aib, the protection of the N- and C-termini by an acetyl group and reduction of the C-terminus to 2-amino alcohols, respectively, amphipathy and microheterogeneity.
The emergence of novel diseases caused by microbial pathogens and the undesirable side effects of certain antibiotics has been a recent dilemma in the medical arena. Consequently, it has stirred the discovery of many naturally occurring agents which could possibly provide important ramifications against various pharmacological targets and to combat various ailments. The main aim of the present study was to determine the antimicrobial activity of the crude methanolic extract of Piper (P.) sarmentosum against methicillin resistant Staphylococcus aureus (MRSA), Escherichia coli, Vibrio cholera and Streptococcus pneumoniae.
Background. Quercus infectoria (QI) is a plant used in traditional medicines in Asia. The plant was reported to contain various active phytochemical compounds that have potential to stimulate bone formation. However, the precise mechanism of the stimulation effect of QI on osteoblast has not been elucidated. The present study was carried out to isolate QI semipurified fractions from aqueous QI extract and to delineate the molecular mechanism of QI semipurified fraction that enhanced bone formation by using hFOB1.19 human fetal osteoblast cell model. Methods. Isolation of QI semipurified fractions was established by means of column chromatography and thin layer chromatography. Established QI semipurified fractions were identified using Liquid Chromatography-Mass Spectrometry (LC-MS). Cells were treated with derived QI semipurified fractions and investigated for mineralization deposition and protein expression level of BMP-2, Runx2, and OPN by ELISA followed gene expression analysis of BMP-2 and Runx2 by RT-PCR. Results. Column chromatography isolation and purification yield Fractions A, B, and C. LC-MS analysis reveals the presence of polyphenols in each fraction. Results show that QI semipurified fractions increased the activity and upregulated the gene expression of BMP-2 and Runx2 at day 1, day 3, and day 7. OPN activity increased in cells treated with QI semipurified fractions at day 1 and day 3. Meanwhile, at day 7, expression of OPN decreased in activity. Furthermore, the study showed that combination of Fractions A, B, and C with osteoporotic drug (pamidronate) further increased the activity and upregulated the gene expression of BMP-2 and Runx2. Conclusions. These findings demonstrated that polyphenols from semipurified fractions of QI enhanced bone formation through expression of the investigated bone-related marker that is its potential role when combined with readily available osteoporotic drug.
The ethyl acetate and methanol bark extracts of Melicope glabra were evaluated for their antioxidant capacities by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity and β-carotene bleaching/linoleic acid system. Both extracts exhibited strong inhibition against the DPPH radical (IC50 values of 24.81 and 13.01 μg ml(-1), respectively) and strong antioxidant activity in β-carotene bleaching assay. Both samples were found to have high phenolic content with values of 39 and 44 mg GAE/g as indicated by Follin-Ciocalteau's reagent. Antioxidant TLC assay-guided isolation on the methanol extract led to the isolation of a new pyranocoumarin, glabranin (1), umbelliferone (2), scopoletin (3) and sesamin (4), and their structures were determined by spectroscopy. Compounds (1-3) showed significant activities on DPPH free radical with the IC50 of 240.20, 810.02 and 413.19 μg ml(-1), respectively. However, in β-carotene bleaching assay, sesamin (4) showed higher inhibitory activity (1 mg ml(-1), 95%) than glabranin (1) (1 mg ml(-1), 74%), whilst umbelliferone (2) and scopoletin (3) were slightly pro-oxidant.
The application of preparative thin layer chromatography-2,2-diphenyl-1-picrylhydrazyl (PTLC-DPPH) bioautography technique successfully isolated a lignan sesamin (1), two prenylated coumarins (2 and 3) and a marmesin glycosides (4) from Micromelum minutum methanol bark extract. Compounds 2 and 3 were identified as new compounds whereas 1 and 4 were first isolated from Micromelum genus. Structural identification of all compounds were done by detailed spectroscopic analyses and comparison with literature data. Antioxidant capacities of extract, active fraction and compounds were measured based on DPPH free radical savenging activity, oxygen radical absorbance capacity (ORAC) and β-carotene bleaching. The DPPH activity of methanol extract and its fraction present the IC50 values of 54.3 and 168.9 µg/mL meanwhile the β-carotene bleaching results were 55.19% and 5.75% respectively. The ORAC measurements of M. minutum extract, compounds 2 and 4 showed potent antioxidant activity with the values of 5123, 5539 and 4031 µmol TE/g respectively.
Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe necrotizing skin disease endemic in tropical countries. Clinical evidence suggests that M. ulcerans isolates from Asia, Mexico, and Australia may be less virulent than isolates from Africa. In vivo studies suggest that mycolactone, a polyketide-derived macrolide toxin, plays a major role in the tissue destruction and immune suppression which occur in cases of Buruli ulcer. Mycolactones were extracted from 34 isolates of M. ulcerans representing strains from Africa, Malaysia, Asia, Australia, and Mexico. Thin-layer chromatography, mass spectroscopic analysis, and cytopathic assays of partially purified mycolactones from these isolates revealed that M. ulcerans produces a heterogeneous mixture of mycolactone variants. Mycolactone A/B, the most biologically active mycolactone species, was identified by mass spectroscopy as [M(+)Na](+) at m/z 765.5 in all cytotoxic isolates except for those from Mexico. Mycolactone C [M+Na](+) at m/z 726.3 was the dominant mycolactone species in eight Australian isolates, and mycolactone D [M+Na](+) m/z 781.2 was characteristic of two Asian strains. Mycolactone species are conserved within specific geographic areas, suggesting that there may be a correlation between mycolactone profile and virulence. In addition, the core lactone, [M+Na](+) m/z 447.4, was identified as a minor species, supporting the hypothesis that mycolactones are synthesized by two polyketide synthases. A cytopathic assay of the core lactone showed that this molecule is sufficient for cytotoxicity, although it is much less potent than the complete mycolactone.
This study aimed at investigating the presence of alkaloids and other chemical constituents in Datura stramonium (Saikaran, Jimson weed). All parts of the plant were dried, crushed and then underwent extraction by soxhlet and maceration methods. The solvents used in these methods were normal hexane (nonpolar) and ethanol (polar). Thin Layer Chromatography (TLC) and FTIR techniques were used to analyse the chemical components of jimson weed. The results showed the presence of hyoscine in all plant parts while atropine in the seeds only. The best separation was found to be when the solvent system was acetone: water: ammonia (90:07:03). Maceration method is the best and cost effective procedure for extraction.
The study aimed to isolate and elucidate the chemical compounds that are found in banana
(Musa balbisiana cv. Saba) inflorescences. Banana inflorescence buds were extracted using
methanol and the resulted methanolic extract was partitioned using chloroform, ethyl acetate
and butanol against deionized water. The chloroform partition was further separated into
fractions using column chromatography assisted by thin layer chromatography. The structure
elucidation was performed using nuclear magnetic resonance spectrometry (NMR). Three
triterpenes were isolated namely 31-norcyclolaudenone (1), cycloartenol (2) and (24R)-4a,24-
trimethyl-5a-cholesta-8,25(27)-dien-3b-ol (3). This is the first report on the isolation of these
triterpenes from Musa balbisiana inflorescence. The discovery of new triterpenes from banana
inflorescence should be further explored to open a new perspective that banana by-products
might serve as new source of natural products for food and pharmaceutical applications.
Rigidoporus microporus, Ganoderma philippii, and Phellinus noxius are root rot rubber diseases and these fungi should be kept under control with environmentally safe compounds from the plant sources. Thus, an antifungal compound isolated from Catharanthus roseus was screened for its effectiveness in controlling the growth of these fungi. The antifungal compound isolated from C. roseus extract was determined through thin layer chromatography (TLC) and nuclear magnetic resonance (NMR) analysis. Each C. roseus of the DCM extracts was marked as CRD1, CRD2, CRD3, CRD4, CRD5, CRD6, and CRD7, respectively. TLC results showed that all of the C. roseus extracts peaked with red colour at Rf = 0.61 at 366 nm wavelength, except for CRD7. The CRD4 extract was found to be the most effective against R. microporus and G. philippii with inhibition zones of 3.5 and 1.9 mm, respectively, compared to that of other extracts. These extracts, however, were not effective against P. noxius. The CRD4 extract contained ursolic acid that was detected by NMR analysis and the compound could be developed as a biocontrol agent for controlling R. microporus and G. philippii. Moreover, little or no research has been done to study the effectiveness of C. roseus in controlling these fungi.