How did we perform preliminary screening for the phytochemical classes
While performing the laboratory tasks for my dissertation work, I searched many articles and books to have a reliable protocol which would be easy to follow. However, I found either very standard books which compiled the test methods in lengthy literature or research articles mentioning test procedures only briefly; easy to understand but not easy to follow. At last, I consulted protocols by three authors (which are named below) and did according to the state of our laboratory. In this post, I am going to share the information about how we (me and my junior fellow) detected the presence of phytochemical classes in orchid sample extracts. The aim of this post is to provide reliable and easy to follow protocol for undergraduate and graduate students who wish to detect phytochemical classes in sample plant extracts.
Basically, we followed protocols established by Habourne (1998), Trease and Evans (1983) with minor modifications according to the condition of our laboratory and availability of resources to reveal the presence of five phytochemical classes – tannins, saponins, steroids and terpenoids, flavonoids, and alkaloids. We tested these orchid extracts by following ways:
A. Test for tannins
We dissolved the plant extract (100 mg) in distilled water (10 ml) in a clean test tube. Then, we taken the resulted aqueous plant extract solution (2 ml) a separate test tube. Again, we added ferric chloride solution (FeCl3; 0.1%; 2 ml). Finally, we observed for the appearance of blue-black or greenish brown colouration.
B. Test for saponins
We added the aqueous solution of plant extract (0.5 ml) which was prepared by plant extract (100 mg) and distilled water (10 ml) to with distilled water (5 ml) in a clean test tube. Then, we shook it vigorously for five minutes. Finally, we observed for the persistent froth for 5 minutes.
C. Test for steroids and terpenoids
We dissolved the plant extract (100 mg) in methanol (4 ml) in a clean test tube. We, then, completely dried up resulted methanol solution by the help of bioling hot water bath.Then, we poured chloroform (2 ml) into methanolic solution dried test tube. Again, we added concentrated sulfuric acid (2 ml) to it from side wall of a test tube in order to make two layers. Finally, we made an observation for the presence of yellowish ring between two layers and converting yellow colour to red or dark red.
D. Test for flavonoids
We dissolved the plant extract (100 mg) into methanol (10 ml). We took resulted methanolic solution of plant extract (2 ml) in a separate test tube and added concentrated hydrochloric acid (HCl; 2 – 3 drops) to it. Then, we plunged magnesium ribbon pieces (3 mm×5 mm) into the resulted solution. Then, we allowed the resulted mixture to stand for 10 minutes. We did observation for the appearance of pinkish colouration on a mixture. We carried a portion of methanolic solution (2 ml) in another test tube as positive control to distinguish pinkish colouration appearance on a test solution.
E. Test for alkaloids
We dissolved the plant extract (100 mg) in methanol (10 ml) in a test tube. Then, a mixture was filtered through Whatman Number 1 filter paper. Thus, obtained filtrate was mixed with HCl (1 %, 2 ml) in a test tube. Then, the mixture was heated in a steam bath. After that, we treated the mixture with Mayer’s reagent (6 drops in each test ). Finally, we observed for the appearance of turbidity on a mixture.
References:
- Harborne J. B. 1973. Phytochemical Methods A Guide to Modern Techniques of Plant Analysis. Chapman & Hall, London, UK.
- Trease G E. and Evans I C. 1983. Pharmacognosy. Bailliere Tindall, London, UK.
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