Further, it highlights the isolation of active molecules by chromatographic techniques like TLC, column chromatography. The most important step toward analysis of bioactive compounds present in the plant extracts is characterization, which includes phytochemical screening assays that give ideas about the presence of secondary metabolites used to cure the health problems. These techniques are the heart and key challenges in research of natural drug discovery giving rise to natural products in drug discovery.
The combination of various types of bioactive compound or phytochemicals is usually present in different plant extracts. The different bioactive compounds have different polarities.
Separation, identification, and characterization of bioactive compounds are a big challenging job in the herbal drug development process. Phytochemical screening assay is a simple, quick, and inexpensive procedure that tells about various types of phytochemicals in a mixture and an important tool in bioactive compound analyses. Phytochemical examinations are carried out for all extracts as per the standard methods [ 15 ].
Preparation of test solution: the test extract was prepared by dissolving with water. Observe for yellow and then brick red precipitate.
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The solution may appear green, yellow, or red depending on the amount of reducing sugar present in test solution. Observe for red precipitate.https://poeworllesle.cf
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Precipitate if warm, turns brick red or precipitate dissolves giving red color. Observe for purple or bluish color. Heat the solution and observe for dark red color. Black ppt. Observe for first red, then blue, and finally green color. Heat and cool. Add few drops of concentrated H 2 SO 4 and observe for blue color. Preparation of test solution: the test solution was prepared by dissolving extract in the alcohol or hydro-alcoholic solution. Tests for cardiac glycosides:. Observe for pink to red color.
Observe for reddish brown color at the junction of the two liquid and upper layers bluish green. Foam test: the extract was mixed with water and shaken vigorously. Persistent foam was observed. Hemolytic test: add test solution to one drop of blood placed on the glass slide. Hemolytic zone appears. Boil and filter. To cold filtrate, add equal volume benzene or chloroform. Shake well. Separate the organic solvent. Add ammonia. Ammoniacal layer turns pink or red. Cool and add benzene, shake well, and separate organic layer.
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Add equal volume dil. Ammoniacal layer shows pinkish red color.
Natural Products Phytochemistry Botany And Metabolism Of Alkaloids Phe ...
Flavonoids are present in hydrolyzed plant extracts. Its presence is maximum in parts of the leaves and they are highly soluble in methanol. The flavonoids are all derived structurally from the important substance called flavone. The flavonoids occur in the free form as well as bound to sugars as glycosides. Flavonoids are found maximum in herbal plants and have good phytopharmacological activities. Pink color was observed. To small quantity of residue, acetate solution was added and observed for yellow colored precipitate. Addition of sodium hydroxide to the residue showed yellow coloration, which was decolorized after addition of dilute hydrochloric acid.
Chromatography is a technique where the molecules are separated based on their shape, size, and charge. In any extract, there are hundreds of unknown components and many of them are in very low amount.
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During chromatography, analyte in solvent and move through solid phase that acts as a sieving material. As molecule proceeds further through molecular sieve, it gets separated. Moreover, there usually exists variability within the same herbal materials. Hence, it is very important to obtain reliable chromatographic fingerprints that represent pharmacologically active and chemically characteristic components of the herbal medicine. Thin layer chromatography is a chromatographic technique that readily provides qualitative information and through which it becomes possible to obtain quantitative data [ 17 ].
Stahl gave the first practical application of thin layer chromatography. TLC is a most versatile technique and it shows its separation with good speed. Advantage of TLC is its sensitivity. TLC works on the principle of an adsorption chromatography in which samples were separated. Separation is based on the interaction between a thin layer of adsorbent attached on the plate and solvent system. The technique is mostly used for the separation of low molecular weight compounds. Many different adsorbents are used in TLC like silica gel, aluminum, cellulose powder, starch, etc.
It is being implemented extensively due to the following reasons: It carries out good speedy separation and rapid analysis of herbal extracts. It has the ability for calculating qualitative and semiquantitative information of the separated compounds with Rf values. It enables the quantification of chemical constituents Table 1. TLC mobile phase for important classes of phytoconstituents [ 15 ].
كتاب: Natural Products: Phytochemistry, Botany and Metabolism of Alkaloids, Phenolics and Terpenes
HPTLC is a more powerful separation tool for quantitative analysis and it uses the technique in a more optimized way. High performance thin layer chromatography HPTLC is based on the principle of planar chromatography where separation of sample components is achieved on high performance layers with detection and data evaluation. These high performance layers on TLC plates are precoated with an adsorbent of 6 micron particle size and a microns layer thickness.
The lesser the thickness of layer and particle size results in increased plate efficiency as well as nature of separation. HPTLC has an ability to show its performance on graphical representation in the form of chromatogram. Separation can be easily visualized by pictorial representation, which is possible only in case of HPTLC. In TLC fingerprinting analysis, the information can be stored and recorded using specific highly sophisticated instruments like high performance TLC scanner. After derivatization and using different visualization reagents, snaps of TLC plates can be obtained and saved for further process.
Thus, this represents TLC fingerprint profile of the provided sample. The information so generated has a potential application in the identification of an authentic drug when compared with bioactive marker and it helps in maintaining the quality and consistency of the isolates or herbal drugs in natural drug discovery [ 18 ].
Column chromatography works on the principle of ion exchange, molecular sieves, and adsorption phenomenon. CC is a most useful technique for separation of active constituents with larger concentration. Sometimes fractions require another step for concentration.
Displacement chromatography is a newer method that contains elution of bioactive compounds that have great affinity for the adsorbent. Fractions of elute materials can be more concentrated than the original solution placed in column. The column was prepared using silica for column chromatography. The fraction was dissolved in smallest possible volume of solvent and it was mixed with 2 gms of silica for column chromatography. Wet column or dry column packing can be done.
Packing of column plays an important role in good quality separation. The mixture was dried to obtain free flowing powder and it was added to column. Then, the column was eluted with solvent of various proportions. Every eluent was collected in properly cleaned test tube separately for further studies to be carried out. High performance liquid chromatography HPLC plays a mandatory role in isolation of natural products.
It is a versatile and widely used technique for the isolation and identification. In the modern era, HPLC technique is becoming popular for studying separation, identification, and fingerprinting study for the quality control of herbal plants. Currently, this technique works as the main choice for research scientists. The multicomponent samples on both an analytical and preparative scale can be separated and studied more easily by HPLC. Thus resolving power of HPLC is ideally used for the rapid processing of herbal extracts.
HPLC instruments are designed in modular ways and they contain delivery pump for solvents and manual injection valve along with an auto-sampler. As sample is introduced in autosample, it carries toward the important part or heart of HPLC that is an analytical column, a guard column. Further, a detector, recorder, and printer are used to show a graphical representation on the software based or installed computer device. In every chemical separation, the working and result production differ due to the fact that certain compounds have different migration rates, which can be fulfilled using HPLC by utilizing a particular column and mobile phase as per the requirement for separations.
Trial and error concept is applied for developing new mobile phase along with prior knowledge of separation, its structure, and required solvent polarity or nonpolarity. Thus, the extent or degree of separation is based upon the choice of stationary phase or mobile phase.