TLC Bioautographic Assay Research Articles

Phytochemical Analysis, Identification and Quantification of Antibacterial Active Compounds in Betel Leaves, Piper betle Methanolic Extract.

The problems of bacterial diseases in aquaculture are primarily controlled by antibiotics. Medicinal plants and herbs which are seemed to be candidates of replacements for conventional antibiotics have therefore gained increasing interest. Current study was performed to investigate the presence of phytochemical constituents, antibacterial activities and composition of antibacterial active compounds in methanolic extract of local herb, Piper betle . Qualitative phytochemical analysis was firstly carried out to determine the possible active compounds in P. betle leaves methanolic extract. The antibacterial activities of major compounds from this extract against nine fish pathogenic bacteria were then assessed using TLC-bioautography agar overlay assay and their quantity were determined simultaneously by HPLC method. The use of methanol has proved to be successful in extracting numerous bioactive compounds including antibacterial compounds. The TLC-bioautography assay revealed the inhibitory action of two compounds which were identified as hydroxychavicol and eugenol. The $-caryophyllene however was totally inactive against all the tested bacterial species. In this study, the concentration of hydroxychavicol in extract was found to be 374.72±2.79 mg g-1, while eugenol was 49.67±0.16 mg g-1. Based on these findings, it could be concluded that hydroxychavicol and eugenol were the responsible compounds for the promising antibacterial activity of P. betle leaves methanolic extract. This inhibitory action has significantly correlated with the amount of the compounds in extract. Due to its potential, the extract of P. betle leaves or it compounds can be alternative source of potent natural antibacterial agents for aquaculture disease management.

Antimicrobial Profiling and Molecular Identification of Alternaria arborescens CLB12, A Myco-endosymbiont Inhabiting Combretum latifolium Blume

Fungal endosymbionts are currently driving the therapeutic use of antimicrobial drugs to combat multi drug resistant infections. An endosymbiotic strain CLB12 was isolated from the root tissue of Combretum latifolium Blume (Combretaceae) collected from the Western Ghats of Southern India. Strain CLB12 was then identified as Alternaria arborescens by its characteristic cultural morphology with ITS rDNA and intervening 5.8S rRNA gene sequence. Antimicrobial profiling of ethyl acetate fraction of CLB12 was performed by disc diffusion method. Secondary metabolites produced were effectively inhibited a panel of test human pathogens including Pseudomonas aeruginosa (12.66 ± 0.33 mm), methicillin-resistant Staphylococcus aureus (12.33 ± 0.33 mm), Bacillus subtilis (16.66 ± 0.33 mm) and Candida albicans (12.00 ± 0.57 mm). TLC-bioautography assay confirmed the presence of antimicrobial compound as depicted by zone of inhibition on the intensive band. Effective inhibition of both bacterial and fungal pathogens suggests that metabolites have a broad spectrum of antimicrobial activity. The present research highlights the utility of A. arborescens CLB12 as prolific source for the biodiscovery of new antimicrobials to combat multidrug-resistant pathogens. The study also emphasizes the inter-habitat cycling of fungal endosymbiont and their possible benefits of A. arborescens to the host.

An In vitro Study on Bioactivity of Ficus racemosa

In vitro evaluation of bioactive compounds is the simplest and effective method for evaluating the antimicrobial and antioxidant activities of compound. In this study, the leaves of Ficus racemosa were extracted with chloroform. The qualitative phytochemical screening was done for the extracted sample. The extract was checked for its antioxidant and antibacterial activity using the TLC bioautography assay. The well diffusion method was carried out to study about the antibacterial activity of the plant sample against three different bacterial cultures. The chloroform extract of Ficus racemosa was showing least antioxidant and antibacterial activity.

Improved TLC Bioautographic Assay for Qualitative and Quantitative Estimation of Tyrosinase Inhibitors in Natural Products.

TLC bioautography for tyrosinase inhibitors has made recent progress; however, an assay with a relative low consumption of enzyme and quantitative capability would greatly advance the efficacy of related TLC bioautographic assays. An improved TLC bioautographic assay for detecting tyrosinase inhibitors was developed and validated in this study. L-DOPA (better water-solubility than L-tyrosine) was used as the substrate instead of reported L-tyrosine. The effects of enzyme and substrate concentrations, reaction temperatures and times, and pH values of the reaction system as well as different plate types on the TLC bioautographic assay were optimised. The quantitative analysis was conducted by densitometric scanning of spot areas, and expressed as the relative tyrosinase inhibitory capacity (RTIC) using a positive control (kojic acid) equivalent. The limit of detection (LOD) of this assay was 1.0ng for kojic acid. This assay has acceptable accuracy (101.73-102.90%), intra- and inter-day, and intra- and inter-plate precisions [relative standard deviation (RSD), less than 7.0%], and ruggedness (RSD, less than 3.5%). The consumption of enzyme (75U/mL) is relatively low. Two tyrosinase inhibitory compounds including naringenin and 1-O-β-D-glucopyranosyl-4-allylbenzene have been isolated from Rhodiola sacra guided by this TLC bioautographic assay. Our improved assay is a relatively low-cost, sensitive, and quantitative method compared to the reported TLC bioautographic assays. Copyright © 2016 John Wiley & Sons, Ltd.

HPTLC Bioautography Guided Isolation of α-Glucosidase Inhibiting Compounds from Justicia secunda Vahl (Acanthaceae).

α-Glucosidase inhibitors form an essential basis for the development of novel drugs in diabetes type 2 treatment. Searching for α-glucosidase inhibitors in plants, TLC bioautographic assays have been established and improved within the last years. In traditional medicine, extracts from the leaves of Justicia secunda Vahl are used to treat diabetes mellitus symptoms. To screen for α-glucosidase inhibitors in J. secunda via HPTLC bioautography. Methodology - Extracts from the leaves of J. secunda and fractions thereof were evaluated in terms of their α-glucosidase inhibiting potential by subjecting them to HPTLC bioautography. The aqueous (AQ) fraction deriving from the methanol extract was further fractionated via column chromatography on polystyrene Diaion® HP-20. Two AQ subfractions revealed active compounds, which were isolated via preparative HPTLC and semipreparative HPLC. Their identification and structure elucidation was achieved employing HPLC-ESI-MSn , HRESI-MS, and NMR analyses. α-Glucosidase inhibitors were visualised as white zones on violet background on the TLC plate. The crude water extract, the methanol extract, and the methanol extract derived AQ fraction showed α-glucosidase inhibiting effects. In the latter, two diastereomeric mixtures responsible for the α-glucosidase inhibition were enriched. They were identified as the novel 2-caffeoyloxy-4-hydroxy-glutaric acid and the diastereomers secundarellone B and C. The current study presents the α-glucosidase inhibiting potential of J. secunda supporting its traditional medicinal use in diabetes mellitus treatment. HPTLC bioautography screening for α-glucosidase inhibitors provides a simple and effective method for the investigation of complex samples, such as plant extracts. Copyright © 2016 John Wiley & Sons, Ltd.

Suppression of bacterial wilt of tomato by soil amendment with mushroom compost containing Bacillus amyloliquefaciens IUMC7

Bacillus amyloliquefaciens IUMC7 and its culture supernatant, which we previously found to suppress Fusarium wilt, were found here to have antimicrobial activity against Ralstonia solanacearum. In in vivo experiments, mushroom compost inoculated with IUMC7 significantly reduced disease severity in tomato plants over that in control soils. The R. solanacearum population decreased in soil inoculated with IUMC7. A TLC–bioautography assay showed that one of the antimicrobial substances produced by IUMC7 is likely an iturin-like lipopeptide. These results suggest that these antimicrobial compounds are responsible for disease suppression and that mushroom compost containing IUMC7 has potential as a biocontrol product.

Evaluation of antibacterial and antioxidant activity of extracts of endophytic fungi isolated from Indonesian Zingiberaceous plants

Abstract. Praptiwi, Palupi KD, Fathoni A, Wulansari D, Ilyas M, Agusta A. 2016. Evaluation of antibacterial and antioxidant activity of extracts of endophytic fungi isolated from Indonesian Zingiberaceous plants. Nusantara Bioscience 8: 306-311. Thirty-two endophytic fungi isolated from rhizome of six species of Zingiberaceae from Karanganyar, Central Java, Indonesia. Based on their morphological characters, all of endophytic isolates were classified into 10 genera. All of endophytic fungi isolates were then cultivated into 200 mL of Potato Dextrose Broth and incubated at room temperature. After three weeks, each of the fungi cultures was extracted with appropriate amount of ethyl acetate, and dried under reduced pressure. The extracts then evaluated for DPPH antioxidant activity and antibacterial activities against Escherichia coli Ina-CC B5 and Staphylococcus aureus Ina-CC B4 based on non-eluted TLC bioautography assays. The results showed 19 extracts are possessing antioxidant activity, 23 extracts inhibit the growth of E. coli Ina-CC B5, and 19 extracts inhibit the growth of S. aureus Ina-CC B4. The minimum inhibitory concentration (MIC) value of active extracts against S. aureus was in the range of 80-1280 ug/mL, while MIC against E. coli was in the range of 80->1280 ug/mL. The IC50 of active extracts were in the range of 50.97-2031.26 ppm. Further analysis by the eluted TLC-bioautography assay of active extracts showed that several fungal metabolites on active extract responsible for its/their bioactivity. It can be concluded that endophytic fungi isolated from Zingiberaceae can be used as new antibacterial and antioxidant resources.

Chemical Composition and Bio-efficacy of Essential Oils from Italian Aromatic Plants: Mentha suaveolens, Coridothymus capitatus, Origanum hirtum and Rosmarinus officinalis

The chemical composition, antifungal, antioxidant and cytotoxic activities of the essential oils (EOs) of mint (Mentha suaveolens), thyme (Coridothymus capitatus), oregano (Origanum hirtum) and rosemary (Rosmarinus officinalis) were investigated. The antifingal properties of the EOs were investigated againstfour species of Candida by a microdilution method. Cytotoxicity was tested on human keratinocyte (HaCaT) and lung cancer (A549) cell lines using the MTT test. DPPH· and ABTS·+ spectrophotometric assays and DPPH-- ABTS-+(HP)TLC-bioautographic assays were used to evaluate the antioxidant activity. The main compounds of thyme and oregano EOs were carvacrol and thymol, respectively; piperitenone oxide and y-terpinene were the most abundant compounds ofmint and rosemary EOs, respectively. All EOs showed activity against all Candida species in a range between 760 ± 290 to 3120 ± 0.0 μg/mL. Among the EOs, that of M suaveolens showed a stronger cytotoxic activity on HaCaT cells. Thyme, oregano and rosemary EQs exhibited important antioxidant activities by ABTS` assay compared with trolox.

CHOLINESTERASE INHIBITORS FROM HEARTWOOD OF ARTOCARPUS FULVICORTEX F. M. JARRET (MORACEAE)

Cholinesterase inhibition activities were screened on crude extracts and isolated compounds of the heartwood of Artocarpus fulvicortex. Phytochemical studies on the heartwood of A. fulvicortex have resulted in the isolation of one flavonoid, one stilbenoid and two triterpenes. Their structures were determined by spectroscopic analysis as catechin (1), oxyresveratrol (2), lupeol-3-acetate (3) and friedelin (4). All isolates were evaluated for the first time for anti-cholinesterase activity. Inhibition of acetylcholinesterase (AChE), tested by Ellman’s method and modified TLC bioautographic assay showed catechin (1) and oxyresvetratrol (2) were active agents with detection limit of 2 mM. The microplate assay was carried out using butyrylcholinesterase (BChe) to further confirmed that catechin (1) and oxyresvetratrol (2) displayed positive cholinesterase activity with IC50 values 50.0 mM and 6.25 mM respectively in dose dependent manner.

Cholinesterase inhibitory activity and structure elucidation of a new phytol derivative and a new cinnamic acid ester from Pycnanthus angolensis

The leaves of Pycnanthus angolensis (Welw.) Warb., Myristicaceae, are used as memory enhancer and anti-ageing in Nigerian ethnomedicine. This study aimed at evaluating the cholinesterase inhibitory property as well as isolates the bioactive compounds from the plant. The acetylcholinesterase and butyrylcholinesterase inhibitory potentials of extracts, fractions, and isolated compounds were evaluated by colorimetric and TLC bioautographic assay techniques. The extract inhibited both enzymes with activity increasing with purification, ethyl acetate fraction being most active fraction at 65.66±1.06% and 49.38±1.66% against acetylcholinesterase and butyrylcholinesterase, respectively while the supernatant had 77.44±1.18 inhibition against acetylcholinesterase. Two new bioactive compounds, (2E, 18E)-3,7,11,15,18-pentamethylhenicosa-2,18-dien-1-ol (named eluptol) and [12-(4-hydroxy-3-methyl-oxo-cyclopenta-1,3-dien-1yl)-11-methyl-dodecyl](E)-3-(3,4-dimethylphenyl)prop-2-enoate (named omifoate A) were isolated from the plant with IC50 of 22.26μg/ml (AChE), 34.61μg/ml (BuChE) and 6.51μg/ml (AChE), 9.07μg/ml (BuChE) respectively. The results showed that the plant has cholinesterase inhibitory activity which might be responsible for its memory enhancing action, thus justifying its inclusion in traditional memory enhancing preparations

Antimicrobial and antioxidant activity and chemical characterisation of Erythrina stricta Roxb. (Fabaceae)

Ethnopharmacological relevanceThe bark of Erythrina stricta Roxb. (Fabaceae) has been used in Indian indigenous systems as a remedy for rheumatism, stomach-ache, asthma, dysentery, contact dermatitis, eczema and skin infections. However, there have been limited phytochemical or biological studies on the bark of E. stricta and there are no studies that align with its traditional medicinal uses. Aim of the studyThe aim of this study was to assess the antimicrobial and antioxidant activity of the stem bark of E. stricta to support its topical use in the treatment of contact dermatitis, eczema and skin infections and to isolate and identify any bioactive compounds. Materials and methodsMTT microdilution and disc diffusion assays were used to determine the antimicrobial activities of n-hexane, dichloromethane, ethyl acetate, methanol and water extracts of the bark of E. stricta. Column and preparative thin layer chromatography were used for the purification of the dichloromethane extract. The structures of the compounds isolated were elucidated by extensive 1D and 2D NMR spectroscopic techniques and comparison with published data. The antioxidant activities of the extracts were determined by DPPH free radical scavenging and FRAP assays and the antioxidant activity of the pure compounds by dot-blot and DPPH staining methods. ResultsThe dichloromethane, ethyl acetate, and n-hexane extracts showed the most significant activity with MIC values of 7.8µg/mL, 125µg/mL, and 125µg/mL against a sensitive strain of Staphylococcus aureus. The dichloromethane and ethyl acetate extracts also showed significant activity against Candida albicans with MIC values of 125µg/mL and 1mg/mL respectively. GC-MS analysis of the n-hexane extract showed the presence of the antibacterial and antifungal compounds β-caryophyllene, caryophyllene oxide, α-selinene, β-selinene, selin-11-en-4-α-ol, α-copaene and δ-cadenine. Phytochemical studies of the dichloromethane extract led to the isolation of the novel compound erynone (1), together with six known compounds; wighteone (2), alpinum isoflavone (3), luteone (4), obovatin (5), erythrinassinate B (6) and isovanillin (7). Luteone (4) exhibited the most significant antibacterial activity with minimum inhibitory quantity (MIQ) values of 1.88µg, 1.88µg and 3.75µg, respectively, against sensitive (MSSA) and resistant strains (MRSA and MDRSA) of S. aureus using a TLC bioautography assay. Erynone (1) exhibited the greatest DPPH free radical scavenging activity. ConclusionsSeven compounds, including a new chromanone, were isolated from the antimicrobial dichloromethane extract of the stem bark of E. stricta. Six of the seven compounds showed antibacterial and/or antioxidant activities. These findings provide support for the customary (traditional and contemporary) use of E. stricta bark for the treatment of skin and wound infections.

Radical scavenging activity of extruded corn gruels with addition of linden inflorescence

Abstract Antioxidant activity is one of the most desirable properties of natural compounds. Among these substances are phenolic compounds which exhibit excellent antiradical activity. The main aim of the present study was determination of the free radical scavenging activity of gruels with 5, 10 and 20% addition of linden inflorescence. The studies were based on two methods: TLC-bioautographic assay and spectrophotometric analysis using DPPH (2,2-diphenyl-1-picrylhydrazyl radical). The obtained results indicate that the radical scavenging properties of the extracts are positively correlated with the content of phenolic compounds in gruels and that a high-temperature extrusion process does not deactivate antioxidant polyphenolic compounds.

A new TLC bioautographic assay for qualitative and quantitative estimation of lipase inhibitors.

Lipase inhibitory assays based on TLC bioautography have made recent progress; however, an assay with greater substrate specificity and quantitative capabilities would advance the efficacy of this particular bioassay. To address these limitations, a new TLC bioautographic assay for detecting lipase inhibitors was developed and validated in this study. The new TLC bioautographic assay was based on reaction of lipase with β-naphthyl myristate and the subsequent formation of the purple dye between β-naphthol and Fast Blue B salt (FBB). The relative lipase inhibitory capacity (RLIC) was determined by a TLC densitometry with fluorescence detection, expressed as orlistat equivalents in millimoles on a per sample weight basis. Six pure compounds and three natural extracts were evaluated for their potential lipase inhibitory activities by this TLC bioautographic assay. The β-naphthyl myristate as the substrate improved the detection sensitivity and specificity significantly. The limit of detection (LOD) of this assay was 0.01 ng for orlistat, the current treatment for obesity. This assay has acceptable accuracy (92.07-105.39%), intra-day and inter-day precisions [relative standard deviation (RSD), 2.64-4.40%], as well as intra-plate and inter-plate precisions (RSD, 1.8-4.9%). The developed method is rapid, simple, stable, and specific for screening and estimation of the potential lipase inhibitors.

Evaluation of Synergistic Antibacterial and Antioxidant Efficacy of Essential Oils of Spices and Herbs in Combination.

The present study was carried out to evaluate the possible synergistic interactions on antibacterial and antioxidant efficacy of essential oils of some selected spices and herbs [bay leaf, black pepper, coriander (seed and leaf), cumin, garlic, ginger, mustard, onion and turmeric] in combination. Antibacterial combination effect was evaluated against six important food-borne bacteria (Bacillus cereus, Listeria monocytogenes, Micrococcus luteus, Staphylococcus aureus, Escherichia coli and Salmonella typhimurium) using microbroth dilution, checkerboard titration and time-kill methods. Antioxidant combination effect was assessed by DPPH free radical scavenging method. Total phenolic content was measured by Folin-Ciocalteu method. Bioactivity –guided fractionation of active essential oils for isolation of bioactive compounds was done using TLC-bioautography assay and chemical characterization (qualitative and quantitative) of bioactive compounds was performed using DART-MS and HPLC analyses. Cytotoxic potential was evaluated by brine shrimp lethality assay as well as MTT assay using human normal colon cell line. Results showed that among the possible combinations tested only coriander/cumin seed oil combination showed synergistic interactions both in antibacterial (FICI : 0.25-0.50) and antioxidant (CI : 0.79) activities. A high positive correlation between total phenolic content and antibacterial activity against most of the studied bacteria (R2 = 0.688 – 0.917) as well as antioxidant capacity (R2 = 0.828) was also observed. TLC-bioautography-guided screening and subsequent combination studies revealed that two compounds corresponding to Rf values 0.35 from coriander seed oil and 0.53 from cumin seed oil exhibited both synergistic antibacterial and antioxidant activities. The bioactive compound corresponding to Rf 0.35 from coriander seed oil was identified as linalool (68.69%) and the bioactive compound corresponding to Rf 0.53 from cumin seed oil was identified as p-coumaric acid (7.14%) by DART-MS and HPLC analyses. The coriander/cumin seed oil combination did not show any cytotoxic effect both in brine shrimp lethality as well as human normal colon cell line assays. The LC50 in brine shrimp lethality assay was found to be 4945.30 μg/ml and IC50 in human normal colon cell line was > 1000 μg/ml. The results provide evidence that coriander/cumin seed oil combination might indeed be used as a potential source of safe and effective natural antimicrobial and antioxidant agents in pharmaceutical and food industries.

Acetylcholinesterase inhibitors and compounds promoting SIRT1 expression from Curcuma xanthorrhiza

Curcuma xanthorrhiza is a well-known traditional medicine with anti-inflammatory and anticancer activities, as well as protective effects against neurodegenerative disorders. A previous study revealed the acetylcholinesterase (AChE) inhibitory activity of some sesquiterpenoids from C. xanthorrhiza. In this study, further bioassay-guided isolation led to the identification of nine compounds for the first time from C. xanthorrhiza, including a new Guaiane-type sesquiterpene, zedoaraldehyde (1). Their structures were elucidated using NMR and MS techniques. The AChE inhibitory activities of compounds 1, 3, 4 and 7 were detected as minimum inhibitory quantities of 3, 4, 6 and 1μg, respectively, using a TLC bioautography assay. Meanwhile, compounds 1, 3, 4 and 8 could promote SIRT1 expression by 1.37-, 1.71-, 1.73- and 1.27-fold, respectively, in HEK293 cell lines exposed to compounds at a concentration of 20μM for 24h. SIRT1 is becoming an important drug target for new therapies in the treatment of neurodegenerative diseases. This study indicates the potential of sesquiterpenoids from C. xanthorrhiza for use against Alzheimer's disease.

Sesquiterpenoids from the Fruits of Alpinia oxyphylla and Their Anti‐Acetylcholinesterase Activity

AbstractFourteen sesquiterpenoids were isolated from the fruits of Alpinia oxyphylla Miq. Their structures were elucidated based on NMR analyses (1H, 13C, DEPT, 1H,1H‐COSY, HMQC, HMBC, and NOESY) and identified as 12‐nornootkaton‐6‐en‐11‐one (3), (+)‐(3S,4aS,5R)‐2,3,4,4a,5,6‐hexahydro‐3‐isopropenyl‐4a,5‐dimethyl‐1,7‐naphthoquinone (5), nootkatene (6), 9β‐hydroxynootkatone (7), 2β‐hydroxy‐δ‐cadinol (8), 4‐isopropyl‐6‐methyl‐1‐tetralone (11), oxyphyllone E (12), oxyphyllone D (13), oxyphyllanene B (15), oxyphyllone A (16), oxyphyllol E (17), (9E)‐humulene‐2,3;6,7‐diepoxide (18), mustakone (20), and pubescone (21). Among them, 3 was a new norsesquiterpenoid, 8 was a new natural product, and 5, 6, 11, 20, 21 were isolated from A. oxyphylla for the first time. Twenty sesquiterpenoids, 1–5 and 7–21, were investigated for their in vitro acetylcholinesterase (AChE) inhibitory activities, including previously isolated seven sesquiterpenoids from A. oxyphylla, (11S)‐12‐chloronootkaton‐11‐ol (1), (11R)‐12‐chloronootkaton‐11‐ol (2), nootkatone (4), oxyphyllenodiol A (9), oxyphyllenodiol B (10), 7‐epiteucrenone B (14), and alpinenone (19). TLC‐Bioautographic assay indicated that 1–4, 7, 14, 16, 18, 19, and 21 displayed anti‐AChE activities at 10 nmol. Microplate assay confirmed that 19, 18, 16, and 21 displayed moderate‐to‐weak anti‐AChE activities at the concentration of 100 μM, and 19 was the most potent inhibitor with an IC50 value of 81.6±3.5 μM. The presence of anti‐AChE sesquiterpenoids in A. oxyphylla may partially support the traditional use of this fruit for the treatment of dementia.

Chemical Composition of the Bark of Tetrapterys mucronata and Identification of Acetylcholinesterase Inhibitory Constituents

The secondary metabolite content of Tetrapterys mucronata, a poorly studied plant that is used occasionally in Brazil for the preparation of a psychotropic plant decoction called "Ayahuasca", was determined to establish its chemical composition and to search for acetylcholinesterase (AChE) inhibitors. The ethanolic extract of the bark of T. mucronata exhibited in vitro AChE inhibition in a TLC bioautography assay. To localize the active compounds, biological profiling for AChE inhibition was performed using at-line HPLC-microfractionation in 96-well plates and subsequent AChE inhibition bioautography. The analytical HPLC-PDA conditions were transferred geometrically to a preparative medium-pressure liquid chromatography column using chromatographic calculations for the efficient isolation of the active compounds at the milligram scale. Twenty-two compounds were isolated, of which six are new natural products. The structures of the new compounds (9, 10, 16-18, and 20) were elucidated by spectroscopic data interpretation. Compounds 1, 5, 6, 9, and 10 inhibited AChE with IC50 values below 15 μM.

Development of a simple TLC-bioautographic assay based on the inhibition of hemozoin synthesis for screening of natural sources antimalarial compounds

Development of a simple TLC-bioautographic assay based on the inhibition of hemozoin synthesis for screening of natural sources antimalarial compounds

An approach based on antioxidant fingerprint–efficacy relationship and TLC bioautography assay to quality evaluation of Rubia cordifolia from various sources

In this work, an approach based on antioxidant fingerprint-efficacy relationships and TLC bioautography assay was developed for quality evaluation of traditional Chinese medicine (TCM). First, chemical fingerprints of 20 batches of different sources of Rubia cordifolia were established by HPLC. The antioxidant activities of these samples were evaluated by 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay. Then, five active components from fingerprint peaks were determined by both multiple correlation analysis and TLC bioautography assay. Similarity analysis and hierarchical clustering analysis (HCA) based on these active peaks were chosen to evaluate the quality of R. cordifolia from different sources. The samples with similarities below 0.9 had poorer antioxidant activities and those having stronger activities fell into the same cluster in HCA. Finally, five batches of commercial samples were processed in the same way to verify the feasibility of this method. The results suggested that chemical fingerprinting combined with similarity analysis based on fingerprint-efficacy relationship and TLC bioautography could evaluate the antioxidant activity of R. cordifolia from various sources. The established technique would also provide an easy method of evaluating the quality of other TCMs.

Antimicrobial, antioxidant activity and phytochemical analysis of an endophytic species of Nigrospora isolated from living fossil Ginkgo biloba

Nigropsora sp. was isolated as an endophyte from a medicinal plant, Ginkgo biloba. Ethyl acetate extract of the fungus was evaluated for antimicrobial and free radical scavenging (DPPH) activity. Ethyl acetate extract was effective against Escherichia coli (R-2046), Klebsiella sp. (R2434), Staphylococcus aureus (ATCC 6538), Candida albicans (NCIM 3471) and Geotrichum sp. (NFCCI- 2521) while it did not show activity against Saccharomyces cerevisiae (NFCCI-1248), Nodulisporium sp. (NFCCI- 2264) and Syncephalastrum sp. (NFCCI-2269). The minimum inhibitory concentration (MIC) of ethyl acetate extract evaluated by tube broth dilution method was recoerded as 2.5mg/ml against all test organisms while by agar dilution method it was recorded as 1.25mg/ml against E. coli, 0.15625mg/ml against S. aureus and 0.3125mg/ml against Klebsiella sp. and C. albicans. The TLC-bioautography assay, used to screen the plant extracts for antimicrobial activity and localization of the active compounds indicated the presence of a number of inhibitory compounds with activity against tested microorganisms. Phytochemical analysis revealed the presence phenolic compounds and anthraquinones. Nigrospora sp. was also examined for in vitro antioxidant activity by DPPH radical scavenging assay. The ethyl acetate extract of the fungus showed potent antioxidant activity with IC50 value of 9.28µg/ml compared to the IC50 value of standard ascorbic acid, 1.74μg/ml.