Advantages And Disadvantages Of Phytochemicals

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CHAPTER 2 Materials and Methods
2.1. Chemicals
Table 2.0. List of chemicals
Chemicals Manufacturer/Country
ABTS(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) ) Sigma Aldrich Laboratories, Canada
Ascorbic Acid Sigma Aldrich Laboratories, Canada
Camphene Loba Chemie PVT Ltd, Mumbai
Chloroform -GPR Fisher Scientific, UK
Dichloromethane - GPR Alpha Chimie Industry (ACI), Tunisie
Dichloromethane HPLC grade Merck Millipore, France
Diphenylamine Sigma Aldrich Laboratories, Canada
DPPH(2,2-diphenyl-1-picrylhydrazyl) Sigma Aldrich Laboratories, Canada
Ethanol 98% Sigma Aldrich Laboratories, Canada
Ethyl Acetate LOBA CHEMIE PVT. Ltd, Mumbai
Formic acid Sigma Aldrich Laboratories, Canada
Glacial Acetic
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Extraction
Two methods of extractions were employed for the extraction of phytochemicals from the leaves of O.mauritiana. The percentage yield of the methanolic extract was then recorded and compared.
2.4.1. Sequential Extraction:
The powdered leaves (~10g) were subjected to the Soxhlet extractor using a series of solvents (150ml) in increasing polarity for three hours. Solvents used with increasing polarity were hexane, ethyl acetate, and methanol. The methanolic residue obtained was concentrated under vacuum at a temperature not exceeding 40oC to yield a gummy dark-brown product labeled as the methanolic extract of the leaves of O.mauritiana and kept in the refrigerator at 4oC prior to use. The experiment was repeated and a total of about 62g of powdered leaves were extracted. Figure 2.1. Extraction of phytochemicals by the Soxhlet apparatus
2.4.2. Maceration:
The powdered leaves (~450g) were first soaked in a 1:1 DCM:MeOH solvent mixture overnight, followed by 100% methanol. The combined residues were then concentrated under vacuum at a temperature not exceeding 40oC which afforded a gummy dark-greenish product, known as the crude extract of the leaves of O.mauritiana. The crude extract was kept in the refrigerator at 4oC prior to
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Hexane was added with the help of a Pasteur pipette and the sample was allowed to seep into the silica. Hexane (150ml) was then passed through the column, eluting a yellow extract which was continually allowed to be re-eluted until the yellow colouration lightened significantly before the addition of the next solvent of higher polarity. This was repeated for the remaining solvents (ethyl acetate and methanol). The methanolic extract was concentrated under vacuum using a rotary evaporator, giving a brown gummy which was stored in the refrigerator prior to use.
2.5. Qualitative Phytochemical Analysis
In order to determine the presence or absence of secondary metabolites, the methanolic extract was subjected to chemical test-tube tests as per standard methods (Evans 2002; Tasneef et al.,(2013) ).
Detection of Alkaloids
To 2ml of the extract, 3ml of dilute hydrochloric acid was added, and filtration was performed. The filtrate was then treated with Wagner’s reagent. The positive control used was quinine and a positive result is established by the formation of a reddish-brown
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