Bioautography Assay Research Articles

Antibacterial, cytotoxicity and GC-MS analysis of <i>Psidium Guajava</i> extracts

Leaves of Psidium guajava was extracted with water, ethanol and chloroform using percolation technique. The extracts were subjected to thin layer chromatography, bioactivity and toxicity studies as well as GC-MS analysis. The TLC chromatogram revealed 3, 4 and 6 bands respectively for the water, ethanolic and chloroform extracts. Contact bio-autographic assay conducted on the extracts against the tested bacteria showed that, out of the thirteen (13) fractions tested only one (1) showed activity on the test bacteria (Rf3).The fraction was active against all the tested bacteria with strong zones of inhibition against S. Paratyphi B, moderate against S. Paratyphi A and weak against S. Typhi . S. typhi showed slight sensitivity, S. paratyphi moderately sensitive, while S. paratyphi B showed strong sensitivity. Result of the brine shrimp lethality assay showed that all the extracts were toxic in the order of chloroform extracts (31.112µg/mL) more toxic, and then ethanolic extracts (39.903µg/mL) and lastly water extracts (49.534µg/mL). The GC-MS analysis was conducted on the most active fraction (Rf3).In the total ion chromatograms (TIC), constituents including fatty acids, heterocyclic compounds and esters among others were found. The major constituents successfully matched and identified were Oleic acid (Peak 6, 35.56% peak area), Palmitic acid (Peak 2, 20.26% peak area)and were all reported to be active principles. Keywords: bacteria, Bioactivity, GC-MS analysis, Psidium guajava , thin layer chromatography, toxicity

Phytochemical Profiling, Antioxidant and Antibacterial Activities of Leaf Extracts From Rhus leptodictya

An integral part of nature is herbal plants which contain natural substance essential to promote health. The focal point of this research resides on the phytochemical, antioxidant and antibacterial investigations of Rhus leptoditya leaves. Hexane, dichloromethane, ethyl acetate, acetone, and methanol solvent extracts were tested for their antioxidant and antimicrobial properties. Phytochemical analysis of R. leptodictya extracts revealed the presence of phenols, tannins, saponins, and flavonoids. Acetone extract with total flavonoid contents of 18.22 ± 2.1 mg/g while methanol extract with phenol contents (0.38 ± 0.002 mg/g) exhibited the highest amount amongst the studied extracts in the preliminary phytochemicals screening. The antioxidant activity of the plant extracts was evaluated using TLC based standard qualitative 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The methanol and acetone extracts showed maximum antioxidant bands compared to other selected solvents and promising radical scavenging activity at par with the standard antioxidant ascorbic acid (IC50 = 0.125 ± 0.013 mg/ml). The bioautography assay also demonstrated the bacterial growth inhibition of active compounds present in leave extracts. Acetone and ethyl acetate fractions showed the highest number of antibacterial active compounds against S. aureus. Minimum inhibitory concentration of the extracts examined using microplates of 96 wells containing Muller-Hinton methods against some bacterial strains, revealed methanol and acetone fractions with the lowest MIC value of 0.04 mg/ml against S. pneumoniae, while methanol extract possesses 0.008 mg/ml against S. aureus and B. subtilis. Results from this study project the R. leptodictya leave extracts constituents as health supplements that could be useful in food industries and in the treatment of various infectious diseases.

Assessment of the antioxidant and antibacterial activities of different olive processing wastewaters.

Olive processing wastewaters (OPW), namely olive mill wastewater (OMW) and table-olive wastewaters (TOW) were evaluated for their antibacterial activity against five Gram-positive and two Gram-negative bacteria using the standard disc diffusion and thin layer chromatography (TLC)-bioautography assays. Disc diffusion screening and bioautography of OMW were compared to the phenolic extracts of table-olive brines. Positive activity against S. aureus was demonstrated. The optimization of chromatographic separation revealed that hexane/acetone in the ratio of 4:6 was the most effective for phenolic compounds separation. A HPLC-MS analysis was performed showing that only two compounds, hydroxytyrosol and tyrosol, were the predominant phenolic compounds in all OPW. The phenolic extract of OMW generated by a semi-modern process showed the highest free radical-scavenging activity (DPPH assay) compared to the other phenolic extracts. It is apparent from the present study that OPW are a rich source of antioxidants suitable for use in food, cosmetic or pharmaceutical applications.

Anticholinesterase constituents from the leaves of Spondias mombin L. (Anacardiaceae).

Spondias mombin has been used in traditional medicine for the management of several diseases, including memory loss. This study aimed to evaluate the cholinesterase inhibitory activity of the methanol extract of the leaves and its derived fractions, as well as carry out detailed phytochemical investigations leading to the isolation and characterization of bioactive compounds from the plant. The acetyl cholinesterase (AChE) and butyryl cholinesterase (BUChE) inhibitory activities were evaluated by colorimetric and thin-layer chromatography bioautographic assay techniques. The ethyl acetate fraction was most active against both enzymes, with percentage inhibition of 58.10 ± 1.08% and 52.66 ± 1.34% against AChE and BUChE, respectively. Three compounds, namely, botulin, campesterol and phytol, with IC50 of 0.88 μg/mL (AChE), 4.67 μg/mL (BuChE); 1.89 μg/mL (AChE), 4.08 μg/mL (BuChE) and 12.51 μg/mL (AChE), 23.89 μg/mL (BuChE), respectively, were isolated from the supernatant of the ethyl acetate fraction. The isolated cholinesterase inhibitory compounds correlate with the known memory-enhancing property of the plant and thus support one of its uses in ethnomedicine.

Myrcia splendens (Sw.) DC. (syn. M. fallax (Rich.) DC.) (Myrtaceae) Essential Oil from Amazonian Ecuador: A Chemical Characterization and Bioactivity Profile.

In this study, we performed the chemical characterization of Myrcia splendens (Sw.) DC. (Myrtaceae) essential oil from Amazonian Ecuador and the assessment of its bioactivity in terms of cytotoxic, antibacterial, and antioxidant activity as starting point for possible applicative uses. M. splendens essential oil, obtained by hydro-distillation, was analyzed by Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID): the major components were found to be trans-nerolidol (67.81%) and α-bisabolol (17.51%). Furthermore, we assessed the cytotoxic activity against MCF-7 (breast), A549 (lung) human tumor cell lines, and HaCaT (human keratinocytes) non-tumor cell line through 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test: promising results in terms of selectivity and efficacy against the MCF-7 cell line (IC50 of 5.59 ± 0.13 μg/mL at 48 h) were obtained, mainly due to α-bisabolol. Furthermore, antibacterial activity against Gram positive and negative bacteria were performed through High Performance Thin Layer Chromatography (HPTLC) bioautographic assay and microdilution method: trans-nerolidol and β-cedren-9-one were the main molecules responsible for the low antibacterial effects against human pathogens. Nevertheless, interesting values of Minimum Inhibitory Concentration (MIC) were noticeable against phytopathogen strains. Radical scavenging activity performed by HPTLC bioautographic and spectrophotometric 1,1-diphenyl-2-picrylhydrazyl (DPPH) approaches were negligible. In conclusion, the essential oil revealed a good potential for plant defense and anti-cancer applications.

ANTIBACTERIAL CAPACITY OF Streptomyces ISOLATE FROM A MANGROVE PLANT RHIZOSPHERE <em>Avicennia marina</em>

This research was conducted to obtain Streptomyces isolates from A vicennia marina rhizosphere capable of inhibiting E. coli and S. aureus growth, to investigate the capability and the characteristics of its antibacterial compound. This study completed the isolation by applying pour plate method on SCN agar medium. Antagonistic screening and selection processes were carried out by diffusion and dilution methods. Observation on the characteristic of the antibacterial compound applied was TLC method and MIC assay. This research confirmed the antibacterial compound capability by applying bioautography assay. Parameters measured consisted of inhibition zone diameter, Rf value on a bioautography plate, and the lowest concentration capable of inhibiting bacterial growth. Out of 16 isolates of Streptomyces obtained, Streptomyces 404 showed higher antagonistic activity than others. Inhibition zone diameter reached 20–25 mm in E. coli and S. aureus growth, respectively. TLC assay showed three spots in which two of them confirmed antibacterial activity in the bioautography assay that yielded Rf values of 0.47 for E. coli and 0.72 for S. aureus , while MIC assay showed that the lowest extract concentration inhibited bacterial growth was 20%.

Chemical Composition and Acetylcholinesterase Inhibitory Activity of Essential Oils from Piper Species.

The essential oils (EOs) derived from aromatic plants such as Piper species are considered to play a role in alleviating neuronal ailments that are associated with inhibition of acetylcholinesterase (AChE). The chemical compositions of 23 EOs prepared from 16 Piper spp. were analyzed by both gas chromatography with a flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS). A total of 76 compounds were identified in the EOs from the leaves and stems of 19 samples, while 30 compounds were detected in the EOs from the fruits of four samples. Sesquiterpenes and phenylpropanoids were found to be rich in these EOs, of which asaricin, caryophyllene, caryophyllene oxide, isospathulenol, (+)-spathulenol, and β-bisabolene are the major constituents. The EOs from the leaves and stems of Piper austrosinense, P. puberulum, P. flaviflorum, P. betle, and P. hispidimervium showed strong AChE inhibitory activity with IC50 values in the range of 1.51 to 13.9 mg/mL. A thin-layer chromatography (TLC) bioautography assay was employed to identify active compound(s) in the most active EO from P. hispidimervium. The active compound was isolated and identified as asaricin, which gave an IC50 value of 0.44 ± 0.02 mg/mL against AChE, comparable to galantamine with an IC50 0.15 ± 0.01 mg/mL.

In vitro antioxidant assessment and a rapid HPTLC bioautographic method for the detection of anticholinesterase inhibitory activity of Geophila repens

ObjectiveGeophila repens (L.) I. M. Johnst. (Rubiaceae), a small, creeping, perennial herb, is claimed to have memory-enhancing property. The goal of this study was to assess its antioxidant and anticholinesterase activity and conduct a rapid bioautographic enzyme assay for screening acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition of G. repens extracts. MethodsAntioxidant activity of G. repens extracts was assessed by performing 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide (NO), superoxide (SOD), hydroxyl (OH) and total antioxidant capacity (TAC) assays. Anticholinesterase activity was investigated by quantifying the AChE and BChE inhibitory activities of chloroform (CGR), ethyl acetate (EGR) and methanol (MGR) extract fractions from G. repens leaves. A rapid high-performance thin-layer chromatography (HPTLC) bioautographic method for the detection of AChE and BChE inhibition was performed. ResultsAmong all extract fractions, EGR exhibited the highest half maximal inhibitory concentration (IC50) in DPPH, SOD, NO, OH and TAC assays, with IC50 of (38.33 ± 3.21), (45.14 ± 1.78), (59.81 ± 1.32), (39.45 ± 0.79) and (43.76 ± 0.81) µg/mL respectively. EGR displayed competitive, reversible inhibition of AChE and BChE activities with IC50 of (68.63 ± 0.45) and (59.45 ± 0.45) µg/mL, respectively. Total phenolic and flavonoids contents of EGR were found to be 360.42 mg gallic acid equivalents and 257.31 mg quercetin equivalents per gram of extract. Phytoconstituents of the EGR extract that were inhibitors of cholinesterase produced white spots on the yellow background of HPTLC plates in the bioautographic test. ConclusionThe results of this study revealed that phenols and flavonoids could be responsible for the antioxidant, anticholinesterase activities of G. repens.

Secondary Metabolites Production by Actinomycetes and their Antifungal Activity

<p class="Els-Abstract-text">Wilt desease of banana caused by <em>Fusarium oxysporum</em> f.sp. <em>cubense</em> (FOC) is one of the most destructive deseases of banana in the tropics. Actinomycetes are the most economically and biotechnologically valuable prokaryotes able to produce wide range of bioactive secondary metabolites. The aims of the present study are to isolate and screen the actinomycetes with high potential ability to produce secondary metabolites that have inhibitory activity against plant pathogenic fungi, <em>Fusarium oxysporum</em> f. sp. <em>cubense</em>. Two isolates from Lampung and Cianjur showed activity against fungi. The isolates designed as L.3.1 and CiIA5b. The metabolites from potent stain was produced by extraction of culture filtrate with ethyl acetate : methanol (4:1), it was tested for their antifungal activity by well diffusion method. Evidence for in vitro antibiosis of L.3.1 and CiIA5b isolates was demonstrated by the zone of fungal-growth inhibition. Production of secondary metabolites was analysis by thin layer chromatography (TLC) and bioautography assays. In this study, the metabolites from L.3.1 and CiIA5b have showed good antifungal activity.</p><div><p class="Els-keywords"><em> </em></p><p class="Els-keywords"><strong>Keywords:</strong> Actinomycetes; antifungal activity; bioautography; secondary metabolites; thin layer chromatography.</p></div>

Anticholinesterase activities of methanol extract and partitioned fractions of <i>Acanthospermum hispidum</i> DC

Age related memory loss is a common occurrence in traditional medical practice and several medicinal plants have been used over the years for managing it. Acanthospermum hispidum DC (Asteraceae) is a medicinal plant that has been included in traditional preparations used in the management of memory loss. In clinical practice however, anticholinesterases have remained relevant in managing memory and cognition dysfunction associated both with old age and certain neurodegenerative diseases. In vitro anticholinesterase activity of extracts and fractions of A. hispidum was done using Ellman’s colorimetric and TLC bioautography assay with eserine as control. The ethyl acetate fraction of A. hispidum showed highest inhibition to acetylcholinesterase and butyrylcholinesterase (91.11% and 64.31%, respectively) in a concentration-dependent manner. A. hispidum presents a good prospect in the development of cholinesterase inhibitors for the management of memory-related disorders. Keywords: Acanthospermum hispidum , Anticholinesterase, acetylcholinesterase, butyrylcholinesterase

Recent Advances in Effect-directed Enzyme Assays based on Thin-layer Chromatography.

Thin-layer chromatography (TLC) together with its more modern form high-performance thin-layer chromatography (HPTLC) is a rapid and cost effective analytical tool with a long tradition in quality control of medicinal plants, extracts and natural products. Separated compounds are fixed on the solid silica phase to form a compound library. Through direct coupling of visualisable enzyme reactions on the TLC plate, this compound library can also be used for activity screening. Such TLC-based bioautographic enzyme and enzyme inhibition assays complement first stage development activity screening assays. They provide not only phytochemical results by chromatographic separation, but also additional information about the activity of constituents or fractions in multi-compound mixtures, and thus can reveal and distinguish artefacts generated by certain compound classes. This review summarises recently introduced TLC bioautographic enzyme assays as well as advances in already existing procedures. Bioautographic enzyme and enzyme inhibitory assays offer a rapid, high-throughput method for screening of secondary metabolite profiles for potential enzyme and enzyme inhibitory activities. Copyright © 2017 John Wiley & Sons, Ltd.

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.

Reverse Phase Compatible TLC-Bioautography for Detection of Tyrosinase Inhibitors.

Reverse phase chromatography and bioautographic assays are key tools for natural product bioguided isolation; however, their direct coupling has not been fully achieved. To develop a bioautographic assay to detect tyrosinase inhibitors present in complex matrices sorbed on reverse phase (RP) TLC-plates that can be used for bioguided isolation of bioactive compounds. Enzyme gel entrapment with an amphiphilic copolymer was used for assay development. The gel turns into a brown "skin like" colour due to tyrosinase catalysed oxidation of l-tyrosine. The inhibitors are visualised as clear spots against a brown coloured background. The assay was able to localise cinnamaldehyde in Cinnamomum cassia essential oil, as its main constituent with known tyrosinase inhibition properties. The assay allowed the detection of 0.03% (w/w) of kojic acid co-spotted with a methanolic extract of Sphaeralcea bonariensis and chromatographed on RP-TLC. The developed assay is able to detect, with high sensitivity, tyrosinase inhibitors present in complex matrices that were chromatographed in RP-TLC. Results can be easily read by colour change, inhibitors appear as clear spots in a darker background. 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, antioxidant and antimicrobial activities and characterization of bioactive compounds from essential oil of Cinnamomum tamala grown in north-western Himalaya

The essential oil (EO) obtained from the fresh and dried leaves of Cinnamomum tamala was analysed by gas chromatography/mass spectrometry. EO from fresh leaves showed the presence of 21 compounds, whereas, EO from the dried leaves of C. tamala showed the presence of 20 compounds. In vitro assays namely scavenging ability against 1,1-diphenyl-2-picrylhydrazyl, reducing power and chelating ability on Fe2+ ions were used to determine the antioxidant potential of EO of C. tamala. With regard to antifungal activity, EO from dried leaves was more effective against Alternaria alternata and Curvularia lunata than the EO from fresh leaves. Similarly, EO from C. tamala leaves also showed potent antibacterial activity against two Gram negative and two Gram positive bacteria namely, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus and Pseudomonas aeruginosa. Furthermore, bioactive molecule from C. tamala EO having antifungal and antioxidant activity was isolated and characterized using bioautography, preparative thin layer chromatography and GC/MS analysis and was determined as eugenol. Its minimum inhibitory amount against A. alternata and C. lunata was determined using bioautography assay and was found to be 9.5 and 8.2 µg respectively.

Bioautography assay of Caesalpinia coriaria (Jacq) wild, as antifungal agent.

Bioautography assay of Caesalpinia coriaria (Jacq) wild, as antifungal agent.

Targeted Isolation of Indolopyridoquinazoline Alkaloids from Conchocarpus fontanesianus Based on Molecular Networks.

A dichloromethane-soluble fraction of the stem bark of Conchocarpus fontanesianus showed antifungal activity against Candida albicans in a bioautography assay. Off-line high-pressure liquid chromatography activity-based profiling of this extract enabled a precise localization of the compounds responsible for the antifungal activity that were isolated and identified as the known compounds flindersine (17) and 8-methoxyflindersine (18). As well as the identification of the bioactive principles, the ultra-high-pressure liquid chromatography-high-resolution mass spectrometry metabolite profiling of the dichloromethane stem bark fraction allowed the detection of more than 1000 components. Some of these could be assigned putatively to secondary metabolites previously isolated from the family Rutaceae. Generation of a molecular network based on MS(2) spectra indicated the presence of indolopyridoquinazoline alkaloids and related scaffolds. Efficient targeted isolation of these compounds was performed by geometric transfer of the analytical high-pressure liquid chromatography profiling conditions to preparative medium-pressure liquid chromatography. This yielded six new indolopyridoquinazoline alkaloids (5, 16, 19-22) that were assigned structurally. The medium-pressure liquid chromatography separations afforded additionally 16 other compounds. This work has demonstrated the usefulness of molecular networks to target the isolation of new natural products and the value of this approach for dereplication. A detailed analysis of the constituents of the stem bark of C. fontanesianus was conducted.