Bioassay Guided Isolation

As screening capacity in the U.S. National Cancer Institute s P388 lymphocytic leukemia (PS) test increased in the 1965-1968 period it began to have a very beneficial impact on the success of our research directed at the discovery of marine animal antineoplastic constituents. The early (6) and more recent 13) evolutionary direction of the NCI antineoplastic and cytostatic evaluation systems has been reviewed. By 1968 extracts of B. neritina reached the confirmed active level and consistently allowed over a 100% increase in life span. Bioassay guided isolation was begun with the PS in vivo system and supplemented from 1975 on with the PS in vitro cell line. The in vivo and in vitro activity 

The first two years spent on exploring the active fractions from A. convoluta were devoted to evaluating separation methods that ranged 

1) HPLC, Partisil-10 ODS-2 (C-18 reverse phase), 4 1 CH3OH-H2O to CH3OH 

2) HPLC, Partisil-10 silical gel n-heptane-ethyl acetate-CH3OH-H2O (600 400 10 1.5) 

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Other studies show the presence of a diversity of compounds other than limonoids as defenses in the tissues of Meliaceae. Woody tissues of Trichilia trifolia afforded three novel dolabellanes with flexible Curing structures. These substances were very active antifeedants in the Sitophilus bioassay (Ramirez et al., 2000). T. martiana seeds yielded large amounts of 2-((Z,Z)-6,9-heptadecadienyl)furan. T. hirta and T. americana bark have yielded novel steroids by insect bioassay-guided isolation and application of a nanoprobe nuclear magnetic resonance (NMR) technique for structure elucidation (Chaurest etal.,1996). Compounds isolated included hydroxyandrosta-l,4-diene-3,16-dione (Fig. 1.4) and derivatives. However, studies by Wheeler et al. (2001) suggest that other unidentified compounds may also be... 

Rimando, A.M., Olofsdotter, M., Dayan, F.E., Duke, S.O. Searching for rice allelochemicals An example of bioassay-guided isolation. Agron J 2001 93 16-20. 

Compounds chemically related with the triterpenoid group such as triterpene saponins were also isolated as antimicrobial constituents in the Anthemideae tribe. Seven saponins were bioassay-guided isolated from the flowers of Beilis perennis L. [215]. Compounds had an antimicrobial... 

Enniatins J2 and J3 were isolated by Pohanka et al. in 2004 from Fusarium sp. strain F31 by bioassay-guided isolation directed against Botrytis cinerea . ... 

The majority of the active constituents from the family Amaryllidaceae are alkaloids. These alkaloids have been isolated first and thereafter evaluated for their biological activities. A bioassay guided isolation of active extracts may lead to the isolation of interesting non-nitrogenons compounds that could be used as leads for the synthesis of nseful therapentic dmgs. 

Bioassay-guided Isolation and Anti-inflammatory Studies... 

Several examples of bioassay-guided isolation are given in the literature. Recio et al [47] used a topical irritant agent to track the activity during the isolation of the anti-inflammatory triterpenes from Diospyros leucomelas, whereas Cuellar et al. [48] employed a combined system of in vivo and in vitro experiments, consisting of the inhibition of an enzyme activity Fig. (1) . Other authors prefer in vitro models. Ammon and Safayhi [49] used the inhibition of leukotriene formation in rat peritoneal neutrophils to isolate the boswellic acids of gum resin exudate of Boswellia serrata, whereas Takaishi et al. [42] studied the inhibition of interleukin-1 secretion, and Jain et al. [50] studied the in vitro inhibition of phospholipase A2 activity. The main results of these studies are summarised in the pharmacological activity section of this chapter. 

Using an affinity probe technique it has become possible to identify natural products that bond to DNA (79). Extracts derived from Albizia amara (Leguminosae) were found to demonstrate activity in a recently developed HPLC system designed to detect compounds capable of interacting with DNA. Further investigation led to the procurement of four sets of alkaloid isolates X1-X4 that were found to be macrocyclic pithecolobine alkaloids. Isolate Xi found to inhibit the catalytic activity of DNA polymerase, RNA polymerase, and HIV-1 reverse transcriptase. It has been identified as a mixture of budmunchiamines A (89), B (90), and C (91), in the ratio 4 1 1 80), whose structures were determined by spectroscopic methods 81). In further detailed bioassay-guided isolation, six more spermine macrocyclic alkaloids, budmunchiamines D-I (92-97) were obtained 82). The structures of these substances were confirmed by spectral analysis and comparison with the related alkaloids budmunchiamines A (89)-C (91). (See Fig. 10.)... 

The new fungus Phoma etheridgei isolated from black galls of aspen was known to be strongly antagonistic to the aspen decay fungus Phellinus tremulae. The bioassay guided isolation of metabolites from... 

Ceratinamides A (68) and B (69), antifouling bromotyrosine derivatives, were isolated from marine sponge Pseudoceratina purpurea through a bioassay -guided isolation (Figure 4) [38]. Ceratinamide A (68) is structurally similar to psammaplysin A (27) and has one formyl group at the A-terminus. 

Liu X, Cui Y, Yu Q, Yu B (2005) Triterpenoids from Sanguisorba officinalis. Phytochemistry 66(14) 1671-1679 Logendra S, Ribnicky DM, Yang H, Poulev A, Ma J, Kennelly EJ, Raskin I (2006) Bioassay-guided isolation of aldose reductase inhibitors from Artemisia dracunculus. Phytochemistry 67(14) 1539-1546... 

Microsphaerins A-D (1074-1077, Fig. 13.31) were found to be produced by the anamorphic soil fungus Microsphaeropsis sp. via a bioassay-guided isolation process (685). These species were identified as being inhibitory for methicillin-resistant Staphylococcus aureus (MRSA), which has been demonstrated to have an almost 20% lethality rate among infected patients in one study of U.S. hospitals (686). 

Yang CY, Yang Y, Aisa HA, Xin XL, Ma HR, Yili A, Zhao YX (2012) Bioassay-guided isolation of antioxidants from Astragalus altaicus by combination of chromatographic techniques. J Sep Sci 35 977-983... 

Geng Y, Lu Z-M, Huang W, Xu H-Y, Shi J-S, Xu Z-H (2013) Bioassay-guided isolation of DPP-4 inhibitory fractions from extracts of submerged cultured of Inonotus obliquus. Molecules 18 1150-1161... 

Bioassay-guided fractionation Bioassay-guided isolation involves the use of some well-defined protocol for the isolation of pure compounds with defined biological activity from a crude extract of a natural product which could be plant or animal parts or a cocktail of mixture. It involves step-by-step separation of fractions/constituents contained within the crude extracts or mixture based on their physicochemical activities and through assaying for biological activity. Several rounds of separation of fractions/constituents and bioassay may be required to finally identify pure molecular hit compound(s). 

The cytoprotective effects of various solvent extracts of Artocarpus altilis (Parkinson) Fosberg were evaluated. These effects were determined in human U937 cells incubated with oxidized LDL (OxLDL) using the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-l, 3-benzene disulfonate (WST-1) assay. Results demonstrated that the EtOAc extract showed cytoprotective activities. To identify the main cytoprotective components, a bioassay guided isolation of the ethyl acetate extract afforded P-sitosterol and six flavonoids. Their chemical structures were established on the basis of spectroscopic evidence and comparison with literature data. One of these compounds was obtained from A. altilis for the first time. The cytoprotective effect offers good prospects for the medicinal applications of. Altilis [34]. 

Wang, Y Deng, T Lin, L Pan, Y Zheng, X. Bioassay-guided isolation of antiatherosclerotic phytochemicals from Artocarpus altilis . Phytotherapy Research, 2006, 20,(12), 1052-1055. 

Bioassay-guided isolation of African ethnobotanical anthelmintics... 

Traditional Chinese medicines (TCMs) serve as a major source of a variety of drug lead compounds. In the process of natural products development, bioassay-guided isolation is a rapid and validated method for isolation of compounds with bioactivities. This chapter describes bioassay-guided separation and purification of compounds from the crude extracts of TCMs. Two approaches including size-exclusion chromatography (SEC) and high performance liquid chromatography (HPLC) are described in detail. 

HPLC is a technique in analytic chemistry used to separate the components in a mixture, to identify each component, and to quantify each component. HPLC has been used for medical, legal, research, and manufacmring purposes. HPLC has been widely used for both separation and quantification of natural products (NPs) from the active fractions ofTCMs [18-22]. The efficiency of HPLC analysis and preparation might be affected by many factors, including different support materials in the column, mobile phase, and others [23]. This chapter describes purification of bioactive molecules from TCMs based on bioassay-guided isolation integrated with SEC and HPLC preparation approaches. 

Ejele AE et al. (2014) Bioassay-guided isolation, purification and charaterization of antimicrobial compoimd from acidic metabolite of piper umbellatum seed extract. Int J Chem 6 61—70... 

Fitch RW et al (2003) Bioassay-guided isolation of epiquinamide, a novel quinolizidine alkaloid and nicotinic agonist from an Ecuadoran poison frog, epipedobates tricolor. J Nat Prod 66 1345-1350... 

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