Bioactive compounds from endophytic fungi exhibit diverse biological activities which include anticancer effect. Capitalising on the abundance of unexplored endophytes that reside within marine plants, this study assessed the anticancer potential of ethyl acetate endophytic fungal extracts (i.e. MBFT Tip 2.1, MBL 1.2, MBS 3.2, MKS 3 and MKS 3.1) derived from leaves, stem and fruits of marine plants that grow along Morib Beach, Malaysia. For identification of endophytic fungi, EF 4/ EF 3 and ITS 1/ ITS 4 PCR primer pairs were used to amplify the fungal 18S rDNA sequence and ITS region sequence, respectively. The resultant sequences were subjected to similarity search via the NCBI GenBank database. High-performance thin layer chromatography (HPTLC) hyphenated with bioassays was used to characterise the extracts in terms of their phytochemical profiles and bioactivity. Microchemical derivatisation was used to assess polyphenolic and phytosterol/ terpenoid content whereas biochemical derivatisation was used to establish antioxidant activities and α-amylase enzyme inhibition. The sulforhodamine B (SRB) assay was used to assess the anticancer effect of the extracts against HCT116 (a human colorectal cancer cell line). The present results indicated MBS 3.2 (Penicillium decumbens) as the most potent extract against HCT116 (IC50 = 0.16 μg/mL), approximately 3-times more potent than 5-flurouracil (IC50 = 0.46 μg/mL). Stepwise multiple regression method suggests that the anticancer effect of MBS 3.2 could be associated with high polyphenolic content and antioxidant potential. Nonlinear regression analysis confirmed that low to moderate α-amylase inhibition exhibits maximum anticancer activity. Current findings warrant further in-depth mechanistic studies.
Preparation of medicinal plants for experimental purposes is an initial step and key in achieving quality research outcome. It involves extraction and determination of quality and quantity of bioactive constituents before proceeding with the intended biological testing. The primary objective of this study was to evaluate various methods used in the preparation and screening of medicinal plants in our daily research. Although the extracts, bioactive fractions, or compounds obtained from medicinal plants are used for different purposes, the techniques involved in producing them are generally the same irrespective of the intended biological testing. The major stages included in acquiring quality bioactive molecule are the selection of an appropriate solvent, extraction methods, phytochemical screening procedures, fractionation methods, and identification techniques. The nitty-gritty of these methods and the exact road map followed solely depends on the research design. Solvents commonly used in extraction of medicinal plants are polar solvent (e.g., water, alcohols), intermediate polar (e.g., acetone, dichloromethane), and nonpolar (e.g., n-hexane, ether, chloroform). In general, extraction procedures include maceration, digestion, decoction, infusion, percolation, Soxhlet extraction, superficial extraction, ultrasound-assisted, and microwave-assisted extractions. Fractionation and purification of phytochemical substances are achieved through application of various chromatographic techniques such as paper chromatography, thin-layer chromatography, gas chromatography, and high-performance liquid chromatography. Finally, compounds obtained are characterized using diverse identification techniques such as mass spectroscopy, infrared spectroscopy, ultraviolet spectroscopy, and nuclear magnetic resonance spectroscopy. Subsequently, different methods described above can be grouped and discussed according to the intended biological testing to guide young researchers and make them more focused.
Ergosterol is the main component of the fungal membrane and is not found in plants or other microbial cells. Therefore, it can be a useful biomarker for the quantification of fungal biomass. We are now reporting the first isolation and characterisation of ergosterol from the mycelium of G. boninense. The ergosterol structure was detected by Thin Liquid Chromatography (TLC) and Ultra Performance Liquid Chromatography (UPLC) and confirmed with Gas Chromatography coupled with Mass Spectrometry (GCMS) and Nuclear Magnetic Resonance (NMR) analysis.
Basal stem rot (BSR) is a devastating disease to Malaysian oil palm. Current techniques employed for BSR disease detection on oil palm are laborious, time consuming, costly, and subjected to accuracy limitations. An ergosterol detection method was developed, whereby it correlated well with the degree of infection in oil palm. This current study was designed to study the relationship between Ganoderma biomass, ergosterol concentration, BSR disease progress and to validate the efficiency of microwave assisted extraction (MAE) method for extraction of ergosterol compound. In addition, testing on the sensitivity of thin layer chromatography (TLC) analysis for detection of ergosterol was also the aim of this study. The optimised procedure involved extracting a small amount of Ganoderma-infected oil palm root tissues suspended in low volumes of solvent followed by irradiation in a conventional microwave oven at 70°C and medium high power for 30 s, resulting in simultaneous extraction and saponification. Based on the results obtained, MAE method may be effective in extracting low to high yields of ergosterol from infected oil palm roots demonstrating disease scale 2, 3 and 4. Positive relationship was observed between ergosterol content and inoculation period starting day 3 in the inoculated oil palm seedlings and hour 6 in germinated seeds. TLC analysis demonstrated a good correlation with high performance liquid chromatography (HPLC) quantification. Therefore, a semi-quantitative TLC analysis may be applied for handling a large amount of samples during onset field survey.
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.
In this study, the unprecedented extraction of the Vitex pouch was performed. The compounds from
methanolic and chloroform extracts were isolated by using thin layer chromatography (TLC). The
compound of interest was investigated by using 1H-Nuclear Magnetic Resonance (NMR, 500 MHz)
spectroscopy. From the NMR spectral examination, the compound from the methanolic extract was
suggested as glucononitol. Indeed, there are some parameters that could enhance the attainment of this
research, which include high performance liquid chromatographic supplies. Nevertheless, more
information and understanding on the pharmaceutical and chemical analysis of the Vitex species were
obtained. To sum up, it is anticipated that incoming research with advanced technology for this
natural product could be explored in the future.
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.
This article discusses on the natural compounds from the ant plant (Myrmecodia species, family: Rubiaceae). The ethyl
acetate (EtOAc) extract from the tuber of M. platytyrea was fractionated by using medium pressure liquid chromatography,
giving eight fractions (F1-F8). Those fractions were evaluated using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH)
assay. Fraction F5 was recorded as potent (EC50 = 21.57 ± 1.40 µg/mL). Then, it was purified by using column
chromatography (CC) (mobile phase = chloroform: EtOAc). From the CC, ten fractions (F5F1-F5F10) were obtained
and compound (1) was isolated from F5F3 via preparative thin layer chromatography (TLC). After spraying with
anisaldehyde-sulphuric reagent, compound (1) gave a green TLC spot (Rf
= 0.65, 100% CHCl3
, multiple development).
The 1
H-Nuclear Magnetic Resonance (NMR) spectroscopy (500 MHz, CDCl3
) was performed to determine the chemical
framework of (1). This compound was identified as morindolide, having an iridoid structure. Meanwhile, the mass
spectra for compounds (2) and (3) were analysed. The data presented the molecular ion at m/z 375 [M-H]- and 255,
suggesting the formulation of 2-(2-methylbutyryl)phloroglucinol glucoside and a flavanone, respectively. From the
literature, compound (1) was firstly isolated from a Chinese natural medicine, the dried root of Morinda officinalis
(family: Rubiaceae). The flavonoids are also included as the biologically active compounds from Myrmecodia. In
short, this is the first occurrence of morindolide from the ant plant.
Detection of basal stem rot (BSR) by Ganoderma of oil palms was based on foliar symptoms and production of basidiomata. Enzyme-Linked Immunosorbent Assays-Polyclonal Antibody (ELISA-PAB) and PCR have been proposed as early detection methods for the disease. These techniques are complex, time consuming and have accuracy limitations. An ergosterol method was developed which correlated well with the degree of infection in oil palms, including samples growing in plantations. However, the method was capable of being optimised. This current study was designed to develop a simpler, more rapid and efficient ergosterol method with utility in the field that involved the use of microwave extraction. The optimised procedure involved extracting a small amount of Ganoderma, or Ganoderma-infected oil palm suspended in low volumes of solvent followed by irradiation in a conventional microwave oven at 70°C and medium high power for 30s, resulting in simultaneous extraction and saponification. Ergosterol was detected by thin layer chromatography (TLC) and quantified using high performance liquid chromatography with diode array detection. The TLC method was novel and provided a simple, inexpensive method with utility in the field. The new method was particularly effective at extracting high yields of ergosterol from infected oil palm and enables rapid analysis of field samples on site, allowing infected oil palms to be treated or culled very rapidly. Some limitations of the method are discussed herein. The procedures lend themselves to controlling the disease more effectively and allowing more effective use of land currently employed to grow oil palms, thereby reducing pressure to develop new plantations.
The Malaysian level of health care has greatly improved so that many of the infectious diseases are now under control. However, perinatal death or death due to unknown childhood diseases remains high (10.3%) being second on the list of causes of death amongst Malaysians. Could inborn metabolic diseases be the main cause of death among these children? Recently, with our success in the development of confirmatory techniques for amino acid disorders using high performance liquid chromatography (HPLC), we have examined 404 samples received from all over the country in 1993. Each specimen with abnormal findings from screening tests by one-dimensional thin layer chromatography was confirmed using HPLC. 41% had generalized aminoacidurias and 4.2% had maple syrup urine disease (MSUD). Patients were aged between 11 days to 6 years. Most of them were Malay males and presented with a history suggestive of MSUD. With this preliminary finding, further studies will be carried out in order to have an investigation and management protocol for the diseases and more importantly to formulate a strategy of screening for the country.
A quantitative assay using high-performance thin-layer chromatography (HPTLC) was developed to investigate bile salt hydrolase (BSH) activity in Pediococcus pentosaceus LAB6 and Lactobacillus plantarum LAB12 probiotic bacteria isolated from Malaysian fermented food. Lactic acid bacteria (LAB) were cultured in de Man Rogosa and Sharpe (MRS) broth containing 1 mmol/L of sodium-based glyco- and tauro-conjugated bile salts for 24 h. The cultures were centrifuged and the resultant cell free supernatant was subjected to chromatographic separation on a HPTLC plate. Conjugated bile salts were quantified by densitometric scans at 550 nm and results were compared to digital image analysis of chromatographic plates after derivatisation with anisaldehyde/sulfuric acid. Standard curves for bile salts determination with both methods show good linearity with high coefficient of determination (R2) between 0.97 and 0.99. Method validation indicates good sensitivity with low relative standard deviation (RSD) (<10%), low limits of detection (LOD) of 0.4 versus 0.2 μg and limit of quantification (LOQ) of 1.4 versus 0.7 μg, for densitometric vs digital image analysis method, respectively. The bile salt hydrolase activity was found to be higher against glyco- than tauro-conjugated bile salts (LAB6; 100% vs >38%: LAB12; 100% vs >75%). The present findings strongly show that quantitative analysis via digitally-enhanced HPTLC offers a rapid quantitative analysis for deconjugation of bile salts by probiotics.
Abelmoschus esculentus L. (ladies finger, okra) is a well-known tropical vegetable, widely planted from Africa to Asia and from South Europe to America. In the present study, we investigated the in vitro antioxidant capacity and in vivo protective effect of the aqueous and methanolic seed extracts of Abelmoschus esculentus against scopolamine-induced cognitive impairment using passive avoidance task and acute restraining stress-induced behavioural and biochemical changes using elevated plus maze (EPM) and forced swimming test (FST) in mice. Our results demonstrated that the pretreatment of mice with aqueous and methanolic seed extracts of Abelmoschus esculentus (200 mg/kg, p.o.) for seven days significantly (P < 0.01) attenuated scopolamine-induced cognitive impairment in the passive avoidance test. In addition, these extracts significantly reduced the blood glucose, corticosterone, cholesterol, and triglyceride levels elevated by acute restraint stress and also significantly increased the time spent in open arm in EPM and decreased the immobility time in FST. It has also been revealed that these extracts showed a significant antioxidant activity and no signs of toxicity or death up to a dose of 2000 mg/kg, p.o. These results suggest that the seed extracts of Abelmoschus esculentus L. possess antioxidant, antistress, and nootropic activities which promisingly support the medicinal values of ladies finger as a vegetable.
High performance liquid chromatography (HPLC) with phenylisothiocyanate (PITC) is recently used for confirming the diagnosis of inborn errors of metabolism (IEM) especially amino acid disorders in Malaysian children. The method of HPLC used is a precolumn derivatization of amino acids with phenylisothiocyanate and is separated by reversed phase chromatography using 3.9 x 300 mm free amino acid columns and is detected by a UV/Vis detector. The samples are obtained from cases suspected of inborn errors of metabolism, especially of amino acid disorders, which are detected clinically by pediatricians. Initially, samples from patients suspected of inborn errors of metabolism, either urine or serum, are run on one-dimensional thin layer chromatography and supplementary chemical tests to detect the abnormal bands and associated abnormalities respectively. Positive samples are further run on HPLC to determine the specific amino acids abnormality. An examples of a case of maple syrup urine disease is discussed, based on the thin layer chromatography findings and HPLC findings.
The steady increase of diabetes is becoming a major burden on health care systems. As diabetic complications arise from oxidative stress, an antioxidant therapy along with anti-diabetic drugs is recommended. Myrmecodia or ant plant is highly valued as a traditional medicine in West Papua. It is used as an alternative treatment for diabetes, as the substances produced by ants can reduce blood sugar levels. The aim of this study was to develop and establish high-performance thin-layer chromatographic (HPTLC)-bioautographic methods to measure the antioxidant and hypoglycemic effects in different extracts from Myrmecodia platytyrea and to compare them with sterol content. Antioxidant activity in methanol, ethanol, dichloromethane (DCM) and ethyl acetate (EA) extracts were measured with a direct HPTLC-2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) assay, while hypoglycemic effects were assessed using a newly developed α-amylase inhibitory activity assay. Stigmasterol is observed, after derivatization with anisaldehyde, as purple colored zones under visible light at hRF values of 0.66. The highest antioxidant activity was observed in the ethanol extract which is rich in polyphenols and flavonoids, while the DCM extract did not show antioxidant activity, but had significant α-amylase inhibitory activity. The highest α-amylase inhibitory activity was observed in the EA and DCM extracts and was related to their stigmasterol content.
The present study describes a simple high performance thin layer chromatographic (HPTLC) method for the simultaneous quantification of apigenin, chamazulene, bisabolol and the use of DPPH free radical as a post-chromatographic derivatization agent to compare the free radical scavenging activities of these components in leaf and flower head extracts from feverfew, German chamomile and marigold from the Asteraceae family. Feverfew (Tanacetum parthenium) leaves have been traditionally used in the treatment of migraine with parthenolide being the main bioactive compound. However, due to similar flowers, feverfew is sometimes mistaken for the German chamomile (Matricaria recutita). Bisabolol and chamazulene are the main components in chamomile essential oil. Marigold (Calendula officinalis) was included in the study for comparison, as it belongs to the same family. Parthenolide was found to be present in all leaf extracts but was not detected in calendula flower extract. Chamazulene and bisabolol were found to be present in higher concentrations in chamomile and Calendula flowers. Apigenin was detected and quantified only in chamomile extracts (highest concentration in flower head extracts). Antioxidant activity in sample extracts was compared by superimposing the chromatograms obtained after post-chromatographic derivatization with DPPH and post-chromatographic derivatization with anisaldehyde. It was found that extracts from chamomile flower heads and leaves have the most prominent antioxidant activity, with bisabolol and chamazulene being the most effective antioxidants.
Quorum sensing is a well-studied cell-to-cell communication method that involves a cell-density dependent regulation of genes expression mediated by signalling molecules. In this study, a bacterium isolated from a plant material compost pile was found to possess quorum sensing activity based on bioassay screening. Isolate YL12 was identified using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and molecular typing using rpoD gene which identified the isolate as Aeromonas caviae. High resolution tandem mass spectrometry was subsequently employed to identify the N-acyl homoserine lactone profile of Aeromonas caviae YL12 and confirmed that this isolate produced two short chain N-acyl homoserine lactones, namely C4-HSL and C6, and the production was observed to be cell density-dependent. Using the thin layer chromatography (TLC) bioassay, both AHLs were found to activate C. violaceum CV026, whereas only C6-HSL was revealed to induce bioluminescence expression of E. coli [pSB401]. The data presented in this study will be the leading steps in understanding the role of quorum sensing in Aeromonas caviae strain YL12.
Quorum sensing (QS) is a mechanism adopted by bacteria to regulate expression of genes according to population density. N-acylhomoserine lactones (AHLs) are a type of QS signalling molecules commonly found in Gram-negative bacteria which have been reported to play a role in microbial spoilage of foods and pathogenesis. In this study, we isolated an AHL-producing Hafnia alvei strain (FB1) from spherical fish pastes. Analysis via high resolution triple quadrupole liquid chromatography/mass spectrometry (LC/MS) on extracts from the spent supernatant of H. alvei FB1 revealed the existence of two short chain AHLs: N-(3-oxohexanoyl) homoserine lactone (3-oxo-C6-HSL) and N-(3-oxo- octanoyl) homoserine lactone (3-oxo-C8-HSL). To our knowledge, this is the first report of the production of AHLs, especially 3-oxo-C8-HSL, by H. alvei.