Bioassay-guided purification

More recently, an abalone sperm attractant has been identified. Sperm of the red abalone Haliotis rufescens responds to soluble factors released into the seawater by conspecific eggs. Bioassay-guided purification revealed... 

A typical extraction scheme for marine invertebrates (frozen, dried, or lyophilized) Invertebrates are cut into small pieces and macerated in a high-speed blender with solvent (see Note 4). The resulting extracts are filtered either through Whatman no. 1 filter paper (gravity or vacuum), or a bed of Celite 521 packed in a Bilchner funnel (see Note 5). AAct filtration, the tissue residue (marc) is returned to the blender and extracted with a second portion of solvent. This process is continued until no further color is extracted. In cases where the extracts are colorless, the successive extracts can be concentrated separately and the mass of residue after concentration determined. Extraction can be considered complete when little or no additional residue is obtained after concentration. When following bioassay-guided purification, successive extracts can be concentrated and assayed separately. Extraction is considered complete when no ftirther activity is detectaf in the extracts. In our lab, sanqiles are extracted three to six times to ensure complete extraction. 

During bioassay-guided purifications, never discard a fraction until an analytical method is available that distinguishes the active compound(s) from the other components of the mixture. The compounds may be present but their effects masked by other components of the mixture. This is especially important in instances where the supply of organism is limited and recollection is difficult if not impossible. It is important not to waste any of the specimen. 

In the course of screening extracts from Bolivian plants against Trypanosoma cruzi, Leishmania spp., Bacillus subtilis and Staphylococcus aureus, Dunalia brachyacantha (Griseb.) Sleumer was found to be active. The bioassay-guided purification of the leaf extract led to the isolation of two known acetoxywithanolides (136 and 137), which displayed antiparasitic and antimicrobial activity (Table 4) [25]. This constitutes the first report of antileishmanial and antitrypanosomial (Chagas disease) activities for steroidal lactones. 

This material was further resolved by LH-20 (chloroform-methanol, 1 1). Bioassay guided purification of these fractions will continue imtil the chemical promoter(s) responsible for titer augmentation are isolated and characterized. This has proven more difficult than expected because the broth extracts are a complex mixture. Each column fraction must be tested in triplicate at several different concentrations, because a taxoid promoter may actually depress production if added at too high of a concentration. If the concentration is too low, then the actuating threshold may not be reached. Finding the proper test concentration for each extract is a slow process. With time, the process may yield an effective promoter of fungal taxol biosynthesis. 

Aloe exhibited antitumor, anticarcinogenic, and cancer chemopreventive properties in animal models. According to a Russian report, treatment of experimental tumors in mice and rats with aloe juice contributed to reduction of tumor mass, metastatic foci, and metastasis frequency. Bioassay-guided purification of an aloe species afforded a hydroxyanthraquinone compound called aloe-emodine, which significantly inhibited P-388 lymphocytic leukemia and human neuroectodermal tumors implanted in mice 157,158 inhibited human hepatoma cell lines, Hep G2 and Hep 3B, human... 

The diabrotidns were isolated from fermentation broths of B. subtilis (isolate ALB 102) obtained from a soil sample collected in an oat field in South Dakota. Bioassay guided purification versus D. undecimpunctata afforded 1, 13 mg/L, and 2, 3 mg/L, as colorless oils. The 0.45pm filtered broth was purified by open column, gradient cation... 

Si W et al (2006) Bioassay-guided purification and identification of antimicrobial components in Chinese green tea extract. J Chromatogr A 1125 204-210... 

Ultimately, the definitive structure elucidation of unknown molecules is most often accomplished via NMR. NMR has traditionally been performed on the purified natural product isolated using bioassay-guided fractionation. With the advent of hyphenated techniques such as LC/NMR, these data can now be obtained prior to purification [130,131]. LC/NMR can prove useful even in the dereplication phase, particularly when LC/UV/MS data are insufficient for unambiguous peak identification. LC/NMR has played an important role in natural products structure elucidation, where several related compounds (factors) are often encountered in a single sample. For example, isobaric or isomeric mixtures that may prove difficult or impossible to differentiate by MS, can often be readily distinguished by NMR. Several thorough reviews of LC/NMR in natural products discovery and phytochemical analysis have recently appeared [132,133]. 

Direct Purification, Orthogonal Purification, ot Bioassay-Guided... 

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... 

The extraction and purification, leading to the crystallization of abscisin II, were guided by an abscission bioassay (1). Seedlings of... 

Spore germination and protonemal growth and morphogenesis are therefore, useful systems to test in vitro, with good reproducibility, potential allelochemicals both through direct co-existence test in vitro or using the described bioassays to monitor/guide isolation, purification, characterization of chemical structure of bioactive compounds. 

Development of bioassays for the isolation of bioactive compounds from natural sources has played an important role in recent natural products chemistry. For the isolation and purification of BRs from plant sources, highly sensitive and specific bioassays are indispensable, because of the very low concentration of BRs in plants. The following three bioassays have been employed for the BRs purification procedure to guide the fractionation. 

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