100 CHAPTER VII PHYTOCHEMICAL ANALYSIS Phytochemicals are chemical compounds synthesized during various metabolic processes. They are naturally synthesized in all parts of the plant body; bark, leaves, stem, root, flower, fruits, seeds, etc. These chemicals are often called secondary metabolites and serve as plant defense mechanisms against pathogenic organisms. These are classified as phenols, quinines, flavonoids, tannins alkaloids, glycosides and polysaccharides (Das et al., 2010). The quantity and quality of phytochemicals present in plant parts may differ from one part to another. In fact, there is lack of information on the distribution of the biological activity in different plant parts essentially related to the difference in distribution of active compounds which are more frequent in some plant parts than in others. Phytochemicals have been recognized as the basis for traditional herbal medicine practiced in the past and currently in vogue. In the search for phytochemicals that may be of benefit to the pharmaceutical industry, researchers sometimes follow leads provided by local healers in a region. Following such leads, plant parts are usually screened for phytochemicals that may be present. The presence of a phytochemical of interest may lead to its further isolation, purification and characterization. Then it can be used as the basis for a new pharmaceutical product. In this section, the methanol extract and ethyl acetate sub fraction of five best plants viz., Terminalia chebula, Emblica officinalis, Acaia nilotica, Rosa indica and Psidium guajava, selected out from the earlier tested activities i.e antibacterial, antioxidant, antiurease and anticollagenase, were subjected to qualitative and quantitative phytochemical analysis. 7.1 Methods 7.1.1 Qualitative phytochemical analysis The extracts were tested for the presence of bioactive compounds by using standard methods (Harborn, 1973; Trease and Evans, 1989; Sofowra, 1993). 7.1.2 Test for Flavonoids Extract was mixed with few fragments of magnesium turnings. Concentrated HCl was added drop wise. Pink scarlet colour appeared after few minutes which indicated the presence of flavonoids.
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100
CHAPTER VII
PHYTOCHEMICAL ANALYSIS
Phytochemicals are chemical compounds synthesized during various metabolic
processes. They are naturally synthesized in all parts of the plant body; bark, leaves, stem,
root, flower, fruits, seeds, etc. These chemicals are often called secondary metabolites and
serve as plant defense mechanisms against pathogenic organisms. These are classified as
phenols, quinines, flavonoids, tannins alkaloids, glycosides and polysaccharides (Das et al.,
2010). The quantity and quality of phytochemicals present in plant parts may differ from one
part to another. In fact, there is lack of information on the distribution of the biological
activity in different plant parts essentially related to the difference in distribution of active
compounds which are more frequent in some plant parts than in others.
Phytochemicals have been recognized as the basis for traditional herbal medicine
practiced in the past and currently in vogue. In the search for phytochemicals that may be of
benefit to the pharmaceutical industry, researchers sometimes follow leads provided by local
healers in a region. Following such leads, plant parts are usually screened for phytochemicals
that may be present. The presence of a phytochemical of interest may lead to its further
isolation, purification and characterization. Then it can be used as the basis for a new
pharmaceutical product.
In this section, the methanol extract and ethyl acetate sub fraction of five best plants
viz., Terminalia chebula, Emblica officinalis, Acaia nilotica, Rosa indica and Psidium
guajava, selected out from the earlier tested activities i.e antibacterial, antioxidant, antiurease
and anticollagenase, were subjected to qualitative and quantitative phytochemical analysis.
7.1 Methods
7.1.1 Qualitative phytochemical analysis
The extracts were tested for the presence of bioactive compounds by using standard
methods (Harborn, 1973; Trease and Evans, 1989; Sofowra, 1993).
7.1.2 Test for Flavonoids
Extract was mixed with few fragments of magnesium turnings. Concentrated HCl was
added drop wise. Pink scarlet colour appeared after few minutes which indicated the presence
of flavonoids.
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7.1.3 Test for Phenols and Tannins
The sample was mixed with 2ml of 2% solution of FeCl3. A blue-green or black
coloration indicated the presence of phenols and tannins.
7.1.4 Test for Saponins
5ml of distilled water was mixed with extract in a test tube and was shaken
vigorously. The formation of stable foam was taken as an indication for the presence of
saponins.
7.1.5 Test for Alkaloids
2ml of 1% HCl was mixed with crude extract and heated gently. Mayer‟s and
Wagner‟s reagent was added to the mixture. Turbidity of the resulting precipitate was taken
as evidence for the presence of alkaloids.
7.1.2 Gas Chromatography and Mass Spectroscopy (GC-MS)
GC-MS technique was used in this study to quantitatively identify the volatile
phytocomponents present in extracts. The extracts were analyzed by using Shimadzu Mass
Spectrometer-2010 series. GC-MS analysis of the best bioactive extracts was done using
Shimadzu Mass Spectrometer-2010 series. Methanol extracts of the best five plants and ethyl
acetate fraction of three plants T. chebula, E. officinalis and A. nilotica were analyzed using
this technique.
1 µl of sample was injected in GC-MS equipped with a split injector and a PE Auto
system XL gas chromatograph interfaced with a Turbo-mass spectrometric mass selective
detector system. The MS was operated in the EI mode (70 eV). Helium was employed as the
carrier gas and its flow rate was adjusted to 1.2 ml/min. The analytical column connected to
the system was an Rtx-5 capillary column (length-60m × 0.25mm i.d., 0.25 µm film
thickness). The column head pressure was adjusted to 196.6 kPa. Column temperature
programmed from 100˚C (2 min) to 200˚C at 10˚C/min and from 200˚ to 300˚C at 15˚C/min
withhold time 5 and 22 min respectively. A solvent delay of 6 min was selected. The injector
temperature was set at 270°C. The GC-MS interface was maintained at 280°C. The MS was
operated in the ACQ mode scanning from m/z 40 to 600.0. In the full scan mode, electron
ionization (EI) mass spectra in the range of 40–600 (m/z) were recorded at electron energy of
70 eV. Compounds were identified by comparing mass spectra with library of the National
Institute of Standard and Technology (NIST), USA/Wiley.
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7.2 Results and Discussion
7.2.1 Qualitative Phytochemical Analysis
The phytochemical characteristics of medicinal plants tested are summarized in Table
7.1. The results revealed the presence of medically active compounds in the plants screened.
From the table, it could be observed that, tannins, phenols and flavonoids were present in all
the extracts of tested plants. Saponins were absent only from the leaves of A. nilotica.
Alkaloids were present only in the leaves of P. guajava and fruit of E. officinalis.
Present results are in accordance with Dhiman et al., 2011, Manjari et al., 2011, Chang et al.,
2012 and Dahiya et al., 2012.
Phytochemical analysis conducted on the plant extracts revealed the presence of
constituents which are known to exhibit medicinal as well as physiological activities.
Analysis of the plant extracts revealed the presence of phytochemicals such as phenols,
tannins, flavonoids, saponins, and alkaloids. It is evident from the results that phenols,
tannins and flavonoids present in the plant samples tested could be responsible for the
activities of these plants. Since phenolic compounds are one of the largest and most
ubiquitous groups of plant metabolites (Singh et al., 2007). They possess biological
properties such as antiapoptosis, antiaging, anticarcinogen, antiinflammation,
antiatherosclerosis, cardiovascular protection and improvement of endothelial function, as
well as inhibition of angiogenesis and cell proliferation activities (Han et al., 2007). Several
studies have described the antioxidant properties of medicinal plants which are rich in
phenolic compounds. The site and the number of hydroxyl groups on the phenol group are
thought to be related to their relative toxicity to microorganisms, with evidence that increased
hydroxylation results in increased toxicity (Geissman, 1963). The mechanisms thought to be
responsible for phenolic toxicity to microorganisms include enzyme inhibition by the
oxidized compounds, possibly through reaction with sulfhydryl groups or through more
nonspecific interaction with proteins (Mason and Wasserman, 1987).
Tannin is a general descriptive name for a group of polymeric phenolic substances.
Their mode of antimicrobial action may be related to their ability to inactivate microbial
adhesions, enzymes, cell envelope, transport-proteins etc. A number of studies indicated that
tannins can be toxic to filamentous fungi, yeasts and bacteria (Scalbert, 1991).
Flavonoids are hydroxylated phenolic substances and occur as a C6- C3 unit linked to
an aromatic ring. Their activity is probably due to their ability to complex with extracellular
soluble proteins and with bacterial cell walls. Lipophilic flavonoids may also disrupt
microbial membranes (Tsuchiya et al., 1996).
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Table 7.1 Phytochemical Analysis of Plant Extracts
Tested Plants
Extracts
T.chebula E. officinalis P.guajava R.indica A. nilotica
Met Et Met Et Met Et Met Et Met Et
Alkaloids
- - + + + + - - - -
Flavanoids
+ + + + + + + + + +
Saponins + + + + + + + + - -
Tannins
+ + + + + + + + + +
Phenols
+ + + + + + + + + +
Met: Methanol extract; Et: Ethyl acetate fraction
7.2.2 Gas Chromatography and Mass Spectroscopy (GC-MS)
There is growing awareness in correlating the phytochemical constituents of a
medicinal plant with its pharmacological activity. The demand for medicinal plant products
has increased considerably because phytocompounds target the biochemical pathway which
makes them safer than synthetic medicines. Lead compounds of the modern medicines are
directly or indirectly obtained from the medicinal plants. So the active principles in
medicinal plants need to be identified.
Gas chromatography and mass spectrometry (GC/MS) are an effective combination
for the analysis of volatile chemicals. Gas chromatography uses a carrier gas to move
analytes through a coated, fused silica capillary. Separation occurs based on differential
partition between the gas phase and the coating inside the capillary. GC/MS requires the
analyte to be vaporized in order for migration through the capillary to occur. Analytes,
therefore, must be volatile or amenable to chemical derivatization to render them volatile.
Certain types of samples are particularly well suited to GC/MS analyses. These include plant