ISOLATION

In natural product drug discovery, isolation of the active pure compound is an essential step in the identification of a new chemical entity. The isolation of secondary metabolites from a crude extract is known to be one of the bottlenecks in natural product drug discoveiy. A purified compound allows for chemical characterization, confirmation, and further evaluation of its bioactivity. Isolation can be rather easy and rapid when the desired compound is present as the major metabolite in the extract. However, it is typically not the case, as the target compounds often exist in trace quantities in a matrix of dozens of other constituents.

If the chemical property of the active compound is not known, then the process of isolation becomes complicated making it impossible to design the isolation procedure for a compound in many cases. Besides, the reactive functional groups of the active principles can readily undergo reaction. The combination of classical techniques (such as thin-layer chromatography, HPLC, Column chromatography, etc.) will help in the partial purification and identification of the active components. Analysis of samples by U'-visible spectroscopy, IR spectroscopy, mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR) often enables the unambiguous structural elucidation of pure compounds.

THIN LAYER CHROMATOGRAPHY(TLC)

Analytical TLC is used in natural product isolation to identify the degree of polarity of various chemical components. TLC is also widely used for the detection of unknown compounds; and assists in measuring the degree of purity of isolated compounds. Spraying reagents will specifically react with certain classes of compounds on TLC plates to impart color changes, which can be detected easily. Combining TLC with bioassay (bio-autography) is an effective method to get information on the active component within the extract, especially in the case of antimicrobial agents [109]. Following are most commonly used spraying reagents for marine natural products [40]:

  • 1. Alkaloids:
    • • The developed TLC plates were sprayed with 37% formaldehyde in concentrated H,S04 (1:10) immediately after removing from the chamber. No heating was required. The occurrence of various colored spots indicates the presence of alkaloids.
    • • Dragendorff’s reagent: 40% aqueous solution of potassium iodide (10 ml) was added to 10 ml of the solution containing 0.85 g of basic bismuth sub-nitrate in acetic acid (10 ml) and 50 ml of distilled water. The resulting solution was diluted with acetic acid and water in the ratio 1:2:10. The plates were heated if the reaction is not spontaneous, a dark orange to red coloration indicates the presence of alkaloids.
  • 2. Phenolics:
    • • The developed TLC plate was sprayed with 5% ferric chloride in 0.5 N HC1. No heat was required or gently heat.
  • 3. Flavonoids:
    • • Spray the developed TLC plate with 10% solution of antimony (III) chloride in chloroform. Fluorescing spots at 360 nm.
  • • Spray TLC plate with 1% aluminum chloride. Yellow fluorescence spots at 360 run.
  • 4. Steroids and Terpenes:
    • Vanillin / sulfuric acid: The spray reagent was made by dissolving 4 g of vanillin in concentrated H1SOJ (100 ml). The plates on subsequent development were sprayed in fume cupboard and heated at 100°C until the color appears. It is a universal spray reagent for terpenes; and many of them give red and blue colors.
    • Perchloric acid: A 20% (w/v) aqueous perchloric acid solution was prepared and sprayed it on the developed TLC plate using a spraying apparatus. The plates were heated at 100°C until the coloration appears. Violet and pink coloration indicate the presence of steroids and terpenes.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

Several chromatographic techniques can be utilized for the separation and purification of biologically active molecules from complex mixtures. When the natural product is a known compound and if the standards are available, then HPLC may be used as a method of purification to get a high degree of the outcome. HPLC is the most versatile and robust technique for secondary metabolite isolation and offers high resolving power and can be scaled up as well as automated.

Among the available chromatographic techniques, preparative HPLC has emerged as the method of choice for secondary metabolite isolation. The term “preparative” refers to a chromatographic analysis, where the objective is to collect a valuable product after it is separated from the other sample constituents. Preparative HPLC is the most convenient tool for separating mixtures of the crude extract [122]. Other chromatography detectors include: flame ionization detector (FID), flame photometric detector (FPD) and nitrogen phosphorus detector (NPD), etc.

7.5.6.2.1 UV Diode Array Detector (DAD)

HPLC and UY diode arrays have become indispensable components of the natural product separation process. Today most natural product experts analyze hundreds of purified natural products using HPLC-UV. With UY photodiode array detectors, we can collect UV-absorbance data simultaneously at different wavelengths and thus facilitate purity assessment peaks. Most modem DADs contain UY spectral libraries of previously reported molecules. The operating software assists in the generation of spectral library allowing rapid identification of known biocompounds [280].

7.5.6.2.2 Evaporative Light-Scattering Detector (ELSD)

ELSD has the advantage of detecting compounds with weak UY absorption. Here, the HPLC effluent is allowed to nebulize and then vaporize in a heated dr ift tube. It results in a cloud of analyte particles which then pass through a beam of light. The analyte particles disperse the light eliciting a signal.

A wide range of preparative HPLC columns are available. The surface modification of the column packing material determines the kind of interactions between the sample analytes and the stationary phase. For the isolation of secondary metabolites, RP columns are most frequently utilized, because most drug-like compounds can be purified using RP-HPLC [36]. Among the available RP column packing material surface modifications, octadecyl (Cl8), bonded silica is most widely used. In addition, a wide range of other RP column packing material surface modifications exists like phenyl-hexyl, fluoropherryl, and dihydroxypropane [35]. The isolation process is often initiated by trial and error, where various HPLC columns and elution gradients are tested for their ability to separate the desir ed compound from the rest of the sample matrix.

If two compounds show different retention time values (tR (HPLC)) on similar chromatographic systems, they are never the same and can be regarded as two different compounds. If more than one peak is detected, then the compounds are entirely different. However, in rare cases different compounds show the same retention time; in such cases, additional identification methods should be adopted to verify the result.

 
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