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: 

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. 

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.

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.

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.

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:

  1. Harborne J. B. 1973. Phytochemical Methods A Guide to Modern Techniques of Plant Analysis.   Chapman & Hall, London, UK.
  2. Trease G E. and  Evans I C.  1983. Pharmacognosy. Bailliere Tindall,  London, UK.

Comments

Post a Comment

Popular posts from this blog

How we performed the estimation of total flavonoid content ?

-
Image
This time I am sharing a method for the estimation of total flavonoid content in our experiments. We Flowchart. the process of estimating total flavonoids content. followed a previously done standard protocol  (Quettier  et al.  2000 and Stankovic 2011)  with slight modification.  Firstly, we  mixed  e ach plant extract (2 ml; 0.5 mg/ml) separately  with aluminium chloride solution (1 ml; 2 %). Then, we allowed the reaction mixture to stand for an hour at room temperature. Again, we measured the absorbance of the mixture at 415 nm using the UV-spectrophotometer. We  obtained  calibration curve (see Fig 1.) using quercetin solution series (25, 50 75, and 100 µg/ml) as a standard. Here, we used the reagent mixture with the same volume of ethanol instead of plant extract solution as a blank. Thus, the obtained curve was calibrated to estimate the concentration of total flavonoid in each plant extract. The total flavonoids content was expressed in terms of the milligram of qu
Read more

The Rapid Method of Estimating Antioxidant Activity and Total Polyphenols Content of Sample Plant Extracts

-
Image
Estimation of total polyphenols content Figure1. The appearance of bluish-black colour after the addition of reagents on sample plant extracts during the estimation of total polyphenols content.  We estimated  total polyphenol content  by a standard protocol (Singleton et al . 1999 and Stankovic 2011) with slight modification. Briefly, we  separately mixed  each plant extract (0.5 ml; 1 mg/ml)  with the Folin-Ciocalteu phenol reagent (2.5 ml; 10%) and aqueous sodium bicarbonate solution (2.5 ml; 7.5%). Then, the reaction mixture was allowed to stand for about 45 minutes and the absorbance was measured at 765 nm using the UV- spectrophotometer (Thermo Fisher Scientific, Genesystem -10.5).The calibration curve was plotted using gallic acid as standard in ethanol (absolute) using the concentration series of 25, 50, 75, and 100 µg/ml. Here, the reagent mixture with the  same volume of ethanol instead of plant extract solution was used as a blank. On the basis of this calibration
Read more

How to Align Fastq Files of Sample Genomic DNA Sequence to Reference Genome

-
Image
Since the aim of this blog is to share the information on various aspects of plants and plant sciences, this time, I am blogging about the bioinformatics tools that help study about the genome of organisms including plants.    The application of  BWA  software or any other software that allows alignment of fastq (.fq) files of the sample genomic DNA sequence to  reference genome  is an essential step before carrying out further investigations such as the genome analysis. In this post, I am going to present the instruction for the alignment of quality trimmed fastq (.fq) files of a sample genome to the reference genome using BWA (Burrows-Wheeler Aligner) software. This instruction covers installing of BWA software, indexing the reference genome, quality trimming the raw fastq files, and aligning the quality trimmed  fastq  files to reference genome to get  SAM  (Sequence Alignment Map) file of a sample genome.  Image 1. The vines of Vitis vinifera cv. 'Pinot Noir' with be
Read more