OBTAINING, PURIFICATION, AND IDENTIFICATION OF SECONDARY METABOLITES

Table of Contents:

Once obtained the secondary metabolites of the fractionation done in the chromatography column, they must be purified by crystallization, making sure that each compound shows a single spot in TLC. The purity of an organic compound obtained by chromatography can be proved checking the physical constants of the compound as melting or boiling point, optic activity, Ultraviolet (UV), infrared (IR), and mass spectra (MS); and nuclear magnetic resonance (NMR), and by colored tests to identify functional groups.

  • 19.3.1 DOUBLE BONDS C=C TEST OF TETRANITROMETHANE
  • 1 mg of the sample is dissolved in chloroform and add 2 mL of dissolution of tetranitromethane in chloroform, the appearance of a yellow color indicates the presence of a nonterminal double bond; cyclopropanes and aromatic compounds give this positive test.
  • 19.3.2 TEST OF BROMINE IN CARBON TETRACHLORIDE
  • 1-2 mg of the sample is dissolved in 1 mL of CC14 and is added drop by drop this solution, the test is positive, if you notice a discoloration of the solution. Test of potassium permanganate: 1-2 mg of the sample is dissolved in 1 mL of water, acetone or methanol and added dropwise KMn04 solution 2% in water. The test is positive if appear discoloration or formation of a brown precipitate (manganese dioxide) in less than 1 min.
  • 19.3.3 CARBOXYL GROUP TEST OF SODA BICARBONATE (NAHC03)

Add few drops of the solution of 10% baking soda in water, to the sample (1-2 mg) dissolved in 1 mL of water or ethanol. The test is positive if bubbles of carbon dioxide appears. Carbonyl group: 1-10 mg of the sample is dissolved in ethanol, is added to a saturated solution of 2-4-dinitrophenylhidrazine in 6N HC1; if a yellow or orange precipitate appears then a carbonyl group is present.

19.3.4 FERRIC CHLORIDE TEST (PHENOLIC HYDROXYLS)

One or two mg of the sample is dissolved in 1 mL of ethanol and then add a few drops of ferric chloride 5% in ethanol. The appearance of a red precipitate, blue-violet or green is positive for phenolic hydroxyls.

19.3.5 STEROLS AND TRITERPENES LIEBERMANNBURCHARD TEST

The reagent is prepared by adding 1 rnL of acetic anhydride to 1 rnL of chloroform and three drops of concentrated sulfuric acid. Then this reagent is added (one drop) to 1 mL of sample diluted in chloroform, sterols are present if a blue or purple color appears and triterpenes are present if appears reddish color. Test could take an hour.

19.3.6 SAPONINES

In a test tube is placed the sample (1-2 mg) dissolved in 1 mL of water, shaking and forms a lather generously, if this stays by 1 h, the test is considered positive.

  • 19.3.7 CARBOHYDRATES TEST OF THE ANTHRONE
  • 1-2 mg of sample dissolved in water are placed in a test tube, then leaves itself sliding down the walls of the tube a recent solution of Anthrone 0.2% in concentrated sulfuric acid; the test is positive if the interface shows a blue- green or purple ring.
  • 19.3.8 COUMARINS

As the coumarins are lactones, they can dissolve in alkaline solutions aqueous or alcoholic with appearance of a yellow coloration, which disappears when acid. The presence of a furan group can be determined using the test of Ehrlich. Most of the coumarins, in thin layer, chromatography present blue fluorescence when observed under UV light and when sprayed with cobalt chloride, after heating the plate, is colored green or yellow. Dissolve 1-2 mg of the sample in a solution of sodium hydroxide 10% in water; if you see a yellow coloration, which disappears to acidify, the test is positive.

19.3.8.1 SESQUITERPENLACTONES BALJET TEST

Use two solutions that are mixed in equal volumes prior to use. A solution is prepared with 1 g of picric acid dissolved in 100 mL of ethyl alcohol.

Solution В is prepared with 10 g of sodium hydroxide in 100 mL of water. Performing the test, take 3-4 mg of the compound, add 3-4 drops of reagent, is positive if it forms dark orange or red coloration. A drop of solution in ethanol or Ethereal compound placed in 4 x 50 mm tube or in a micro- Crucible, add a drop of 2 N potassium hydroxides in methanolic solution. The mixture is heated with a lighter micro for 1-2 min. Immediately cools, add hydrochloric acid 0.5 N until acid pH, and add a drop of feme chloride 1%; color purple is positive test, if the result is negative, you should dilute slightly and repeat the comment. Santonine gives a violet-pink color and alantolactone, dark violet. Coumarins, other lactones, and esters give positive this test. The simaroubolidanes and limonoids may also give positive the previous tests because they are lactones.

19.3.8.2 FURANOCOUMARINES EHRLICH REAGENT

A solution 1.5% of p-dimethylaminobenzaldehyde in ethanol is used. This solution is used as developer agent on the chromatographic plates with the sample. This plate is inserted into a chamber containing atmosphere of hydrogen chloride. A violet color is indicative for furan ring. Aromaticity The reagent is prepared just in the moment to be used, adding a drop of formalin (formaldehyde 37-40%) to 1 mL of concentrated sulfuric acid, placing it in a test tube and dissolved in a nonaromatic solvent, sample is added by the walls of the tube.

The formation of a colored ring at the interface, cannine red color to black, indicates the test is positive.

19.3.9 NITRITES (NO J

A bit of ferrous sulfate is dissolved in water then add the compound, passing through the walls of the tube a few drops of concentrated sulfuric acid, the test is positive when a ring of dark brown color is formed.

19.3.10 CHLORIDES

Dissolve 1 mg of silver nitrate in double distilled water and add a drop of this solution to the sample dissolved in double distilled water, the test for chloride is positive, when a milky white precipitate is observed.

19.3.11 HALOGENS—BEILSTEIN TEST

Wet a piece of copper wire in nitric acid or sulfuric acid, passes through the flame of a lighter, then the sample is taken and we direct it to the flame; chlorine gives a green coloration and bromine color blue.

19.3.12 CYANIDES

First solution is to soak the paper filter for the determination of cyanides. Solution: 200 mg of picric acid, plus 5 mL of ethanol add 5 mL of sodium hydroxide 1 N (5 g of NaOH in 125 mL of distilled water), this solution is to impregnate filter paper diy, which will yellow coloration. In a closed and clear container offers some vegetable material to be analyzed with a small amount of water and the filter paper inside the container but without gets wet, change from yellow to red, it is indicative for cyanides.

  • 19.3.13 ALKALOIDS
  • 19.3.13.1 MAYER REAGENT

Dissolve 1.36 g of HgCl, in 60 mL of water and 5 g potassium iodide in 10 mL of water. Join the two solutions and add water until total volume 100 mL. Single reagent should be added to previously acidified with diluted HC1 or H2S04 solutions. The solution must not contain acetic acid or ethanol, because they dissolve the precipitate. Only a few drops of reagent should be added because some alkaloids are soluble in excess of reagent.

19.3.13.2 SCHEIBLER REAGENT: (PHOSPHOTUNGSTIC ACID)

Dissolve in 50 mL of water 10 g of sodium tungstate and 7 g of disodium phosphate. The solution is acidified with nitric acid. The reagent is precipitated amorphous to mingle with alkaloids in dilute H,S04 solutions. The precipitate is soluble in excess of reagent or in ethanol.

19.3.13.3 MARQUIS REAGENT

Add five drops of formalin to the sample being analyzed, add 5 mL of concentrated H,S04, heroin and morphine immediately give a reddish-purple color and then it goes to violet color and finally to blue staining.

19.3.14 FLAVONOIDS SHINODA TEST

Dissolve 1-2 mg of sample in 1 mL of ethanol by adding a few drops of concentrated hydrochloric acid and one or two magnesium filings; If the solution turns a deep red color, the test is positive, other, orange, green, or blue color may be present, flavones, flavonones, flavonols, antioxidant xanthones When there are interference pigments not flavonoids, plant material can be tested directly; for example, if the white petals of a flower change to yellow in the presence of ammonia vapors, should contain flavones or flavonols. The chalcones and the aurones going from yellow to red.

The petals that contain anthocyanins change to red in the presence of ammonia. Antimony pentachloride in carbon tetrachloride, produces characteristic colors with flavonoids (Marini-Berttolo test). The chalcones form precipitates red dark or violet and flavones, precipitates yellow or orange. Flavones and Flavonols are dissolved in concentrated sulfuric acid and originate strongly yellow solutions. The flavanones give orange colors or cherry red to bluish red.

19.3.15 QUINONES

Quinones tend to give red or purple colors with concentrated alkali and sulfuric acid, which cane used to identify them. The presence of anthraqui- nones and naphthaquinones can be identified with the reaction of Borntrager, which consists in boiling for 10 min, some material with 2-5% potassium hydroxide. The solution cooled, is acidified and extracted with benzene. The benzene layer is separated and is tossed with a little of the potassium hydroxide solution. If the phase of benzene is discolored and the alkaline gets red, is positive for Quinones.

19.3.16 CARDIOTONIC GLYCOSIDES

Glucosides or glycosides are metabolites formed of two parts: one is a carbohydrate and the other no-sugar or aglycone. The link between the two is hydrolysable and must break which is activated compound; this rupture is catalyzed by enzymes containing the same plant. Glycosides are classified according to the structural characteristics of the aglycone:

19.3.16.1 RAYMOND REAGENT

Dissolve 1-2 mg of extract in 5-8 drops of ethanol at 50% then add 1-2 drops of 1% of m-dinitrobenzene in ethanol and then add 2-3 drops of an aqueous solution NaOH 20%. A violet color is formed. Legal test: Treat the extract with pyridine and add alkaline sodium nitroprusside solution, blood red color appears. Tollens test: In a small, clean test tube 5-10 mg of extract dissolve in 5-8 drops of pyridine and add 4-6 drops of very recent Tollens reagent. (Mix 0.5 ml of 10% AgN03 and

0.5 mL of NaOH 10%; after ammonium hydroxide is added dropwise to complete dissolve of the precipitate.) This reagent must decompose before one hour, since explosive nitrides are formed. Positive if is formed a mirror on the tube.

CONCLUSIONS

The general techniques have been described in this chapter applied to phytochemical analysis of a plant whose therapeutic or toxic properties have been described by the ethnobotanic and through an analytical procedure and applying the scientific method is it aims to validate the hypothesis of herbalists. The main objective is the pursuit of secondaiy metabolites that possess biological activity and the description of the main extraction methods and techniques of separation and structure elucidation of these compounds.

The phytochemical screening is a tool that guides us for the separation and purification of compounds and through a bioassay determine the biological activity and identify by spectroscopic methods the bioactive compounds, validating scientifically its use in traditional medicine; This information would provide knowledge for the pathways of biosynthesis of the active principles isolated, and their further use as a drug of natural origin.

KEYWORDS

  • medicinal plants
  • phytochemistry
  • extraction techniques
  • biological activity
  • antioxidant
  • chemotaxonomy
  • identification techniques

REFERENCES

Bonatti, A. Formulation of Plant Extracts into Dosage Form. In The Medicinal Plant Industry-, Wijesekera, R. О. B., Ed.; CRC Press: London, 1991; pp 106-107.

Butler, M. S. The Role of Natural Product Chemistry in Drug Discovery. J. Nat. Prod. 2004, 67, 2141-2153. http://doi.org/10.1021 np040106y.

Colegate, S. M.; Molyneux, R. J. Bioactive Natural Products: Detection, Isolation, and Structural Determination, 2nd Ed.; CRC Press, 2007.

Der Marderosian, A.: Beutler. J. A. The Review of Natural Products: The Most Complete Source of Natural Product Information. Facts & Comparisons. Saint Louis, USA, 2005, 1343.

Dewick. P. M. Medicinal Natural Products: A Biosynthetic Approach', John Wiley & Sons: New Jersey, USA 2002.

Dominguez, X. A. Metodos de investigacion fitoquimica; Limusa, 1988.

Garcia Gonzalez, S. Estudio Quimico de Cnidoscolus wens; Instituto Tecnologico у de Estudios Superiores de Monterrey, 1992.

Harborne, A. J. Phytochemical Methods A Guide to Modern Techniques of Plant Analysis; Springer Science & Business Media. 1998.

Lambert. J.; Srivastava, J.; Vietmeyer, N. Medicinal Plants: Rescuing a Globed Heritage; World Bank. 1997.

Lewis, H. W.; Christopher, J. Moody. Experimental Organic Chemistiy: Principles and Practice (Illustrateded.); WileyBlackwell, 1989; pp 159-173. ISBN 978-0-632-02017-1. Luque de Castro, M. D.; Priego-Capote, F. Soxhlet extraction: Past and Present Panacea. J.

Chromatogr. A 2010,1217(16), 2383-2389. http://doi.Org/10.1016/j.chroma.2009.ll.027. Maplestone. R. A.; Stone, M. J.; Williams. D. H. The Evolutionary Role of Secondary Metabolites—AReview. Gene 1992, 775 (1-2), 151-157.

Mislira, В. B.; Tiwari, V. K. Natural Products: An Evolving Role in Future Drug Discovery.

Eur. J.Med. Chem. 2011,46(10),4769-4807. http://doi.Org/10.1016/j.ejmech.2011.07.057. Paech, K.; Tracey, M. V. Modern Methods of Plant Analysis/Moderne Methoden der Pflanzenanalyse; Springer: Berlin Heidelberg. 2012.

Sarker, S. D.; Latif, Z.; Gray, A. I. Natural Products Isolation; Humana Press, 2005.

Verde-Star. Maria Julia: Rivas-Morales, Catalina; Garcia, Gonzalez Sergio. Metodologia cientifica para el estudio deplantas medicinales. In Investigation en plantas de importancia medica, la Ed.; OmniaScience. 2016; p37. ISBN: 978-84-944673-7-0.

Vogel, A. I.; Tatchell, A. R.; Furnis, B. S.; Hannaford, A. J.; Smith, P. W. G. Vogel's Textbook of Practical Organic Chemistry, 5th ed. ISBN 0-582-46236-3.

Walton, N. J.; Brown, D. E. Chemicals from Plants: Perspectives on Plant Secondaty Products-, Imperial College Press, 1999.

 
Source
< Prev   CONTENTS   Source   Next >