Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5626_Библиотеки_им_академика_М_И_Перельмана.pdf
References 141
39 Vinatoru, M., (2001). An overview of the ultrasonically
assisted extraction of bioactive principles from herbs.
Ultrasonics Sonochemistry 8 (3): 303–13.
40 Tiwari, B.K., (2015). Ultrasound: A clean, green
extraction technology. TrAC Trends in Analytical
Chemistry 71: 100–09.
41 Chemat, F., Tomao, V., and Virot, M., (2008).
Ultrasound-assisted extraction in food analysis.
Handbook of Food Analysis Instruments 11: 85–103.
42 Baig, S., Farooq, R., and Rehman, F., (2010).
Sonochemistry and its industrial applications. World
Applied Sciences Journal 10 (8): 936–44.
43 Zeković, Z., Pintać, D., Majkić, T., et al. (2017). Utilization
of sage by-products as raw material for antioxidants
recovery—ultrasound versus microwave-assisted
extraction. Industrial Crops and Products 99: 49–59.
44 Luo, X., Cui, J., Zhang, H., et al. (2018). Ultrasound
assisted extraction of polyphenolic compounds from red
sorghum (Sorghum bicolor L.) bran and their biological
activities and polyphenolic compositions. Industrial
Crops and Products 112: 296–304.
45 Chuyen, H.V., Nguyen, M.H., Roach, P.D., et al. (2018).
Microwave‐assisted extraction and ultrasound‐assisted
extraction for recovering carotenoids from Gac peel and
their effects on antioxidant capacity of the extracts. Food
Science & Nutrition 6 (1): 189–96.
46 Mandal, V., Mohan, Y., and Hemalatha, S., (2007).
Microwave assisted extraction—an innovative and
promising extraction tool for medicinal plant research.
Pharmacognosy Reviews 1 (1): 7–18.
47 Alupului, A., Calinescu, I., and Lavric, V., (2012).
Microwave extraction of active principles from
medicinal plants. UPB Science Bulletin, Series B 74 (2):
129–42.
48 Eskilsson, C.S. and Björklund, E. (2000). Analytical-
scale microwave-assisted extraction. Journal of
Chromatography 902 (1): 227–50.
49 Henry, C. J. K., and Chapman, C. (Eds.) (2002). The
Nutrition Handbook for Food Processors. Elsevier.
50 Ellington, E., Bastida, J., Viladomat, F., et al. (2003).
Supercritical carbon dioxide extraction of colchicine and
related alkaloids from seeds of Colchicum autumnale L.
Phytochemical Analysis: An International Journal of
Plant Chemical and Biochemical Techniques 14 (3):
164–69.
51 Grigonis, D., Venskutonis, P., Sivik, B., et al. (2005).
Comparison of different extraction techniques for isolation
of antioxidants from sweet grass (Hierochloe odorata). The
Journal of Supercritical Fluids 33 (3): 223–33.
52 Wang, L. and Weller, C.L. (2006). Recent advances in
extraction of nutraceuticals from plants. Trends in Food
Science & Technology 17 (6): 300–12.
53 Pan, X., Niu, G., and Liu, H., (2002). Comparison of
microwave-assisted extraction and conventional
extraction techniques for the extraction of tanshinones
from Salvia miltiorrhiza bunge. Biochemical Engineering
Journal 12 (1): 71–7.
54 Brachet, A., Christen, P., and Veuthey, J.L., (2002).
Focused microwave‐assisted extraction of cocaine and
benzoylecgonine from coca leaves. Phytochemical
Analysis: An International Journal of Plant Chemical
and Biochemical Techniques 13 (3): 162–69.
55 Kumoro, A.C. and Hartati, I., (2015). Microwave
assisted extraction of dioscorin from gadung
(Dioscorea Hispida Dennst) tuber flour. Procedia
Chemistry 14: 47–55.
56 Farhat, A., Ginies, C., Romdhane, M., et al. (2009).
Eco-friendly and cleaner process for isolation of
essential oil using microwave energy: experimental
and theoretical study. Journal of Chromatography A
1216 (26): 5077–85.
57 Golmakani, M-T. and Rezaei, K., (2008). Comparison of
microwave-assisted hydrodistillation withthe traditional
hydrodistillation method in the extractionof essential
oils from Thymus vulgaris L. Food Chemistry 109 (4):
925–30.
58 Jaradat, N.A., Zaid, A.N., Abuzant, A., et al. (2016).
Investigation the efficiency of various methods of
volatile oil extraction from Trichodesma africanum and
their impact on the antioxidant and antimicrobial
activities. Journal of Intercultural Ethnopharmacology
5 (3): 250–56.
59 Verma, A., Hartonen, K., and Riekkola, M.L., (2008).
Optimisation of supercritical fluid extraction of indole
alkaloids from Catharanthus roseus using experimental
design methodology—comparison with other extraction
techniques. Phytochemical Analysis: An International
Journal of Plant Chemical and Biochemical Techniques
19 (1): 52–63.
60 Verma, D.K., and Srivastav, P.P., (2018). Science and
Technology of Aroma, Flavor, and Fragrance in Rice.
CRC Press.
61 Uwineza, P.A. and Waśkiewicz, A., (2020). Recent
advances in supercritical fluid extraction of natural
bioactive compounds from natural plant materials.
Molecules 25 (17): 3847.
62 Pereira, C.G. and Meireles, M.A.A. (2010). Supercritical
fluid extraction of bioactive compounds: Fundamentals,
applications and economic perspectives. Food and
Bioprocess Technology 3: 340–72.
63 Conde-Hernández, L.A., Espinosa-Victoria, J.R., and
Guerrero-Beltrán, J.Á. (2017). Supercritical extraction of
essential oils of Piper auritum and Porophyllum
ruderale. The Journal of Supercritical Fluids 127: 97–102.
8
Qualitative and Quantitative Methods of Phytochemical Analysis
Mughisha Nagori
1
Mahakal Institute of Pharmaceutical Studies, Ujjain, Madhya Pradesh, India
2
School of Pharmacy, Devi Ahilya Vishwavidyalaya, Indore, Madhya Pradesh, India
3
Amity Institute of Pharmacy, Amity University, Noida, Madhya Pradesh, India
4
Acropolis Institute of Pharmaceutical Education and Research, Indore, Madhya Pradesh, India
&
Mughisha Nagori and Devyani Rajput have contributed equally as first authors
*Corresponding Author: rakhikhabiya@gmail.com
1,2,&
, Devyani Rajput
3,&
, Gajendra Choudhary2, Rakhi Khabiya4,*
8.1 Introduction
In the realm of herbal medicine, the importance of quality
control parameters cannot be overstated. Herbal drugs, often
derived from plant sources, offer diverse therapeutic potentials. However, their composition can vary significantly
based on factors like geographic origin, cultivation practices,
harvesting methods, and processing. This variability poses a
challenge when it comes to ensuring their safety and efficacy [1]. To address this challenge, various guidelines and
regulatory authorities have established rigorous quality control standards. These standards encompass a range of parameters, including botanical identification, quantification of
bioactive compounds, microbiological limits, heavy metal
content, pesticide residues, and more [2]. By adhering to
these guidelines, manufacturers can consistently produce
herbal drugs of high quality, potency, and safety. Moreover,
it instills confidence among healthcare professionals and
consumers regarding the reliability of these natural remedies. As a result, standardization through quality control
parameters not only safeguards public health but also promotes the integration of herbal drugs into mainstream
healthcare systems [3].
The evaluation of phytochemical properties holds a crucial role in ensuring the quality control of herbal drugs. It
involves the systematic analysis of a plant’s bioactive
compounds, offering valuable insights into its chemical
composition and therapeutic potential. One of the primary
advantages is that it helps in the authentication and botanical identification of herbal materials, ensuring that the correct plant species are used, thus mitigating the risk of
adulteration [4]. Additionally, phytochemical analysis aids
in quantifying bioactive constituents, allowing for batch-tobatch consistency and the establishment of dosage recommendations. It can also reveal the presence of potentially
harmful compounds like alkaloids or glycosides, which can
be vital for assessing safety. However, there are certain limitations to phytochemical evaluation [5]. It doesn’t provide
information on the overall pharmacological activity or the
synergistic effects of multiple compounds in the plant,
which can be a disadvantage [6]. Furthermore, the presence
of specific compounds may not always correlate with the
therapeutic efficacy of the herbal drug, and it can be challenging to standardize herbal products solely based on phytochemical data. Despite these drawbacks, phytochemical
evaluation is still a crucial instrument in the quality control
of herbal drugs and provides insightful data that helps to
guarantee the consistency, safety, and efficacy of herbal
treatments [7].
Qualitative analysis of herbal drugs focuses on identifying
and characterizing the chemical constituents (Figure 8.1)
and phytochemicals present in the plant material, while
quantitative analysis involves determining the precise concentrations of specific compounds (Figure 8.2).
144 8 Qualitative and Quantitative Methods of Phytochemical Analysis
Qualitative
Phytochemical Screening via chemical test
Glycosides
Carbohydrates
Proteins
Lipids, waxes, and oils
Tannins
Renin
Alkaloids
Figure 8.1 Qualitative analysis of herbal drugs.
Quantitative
Quantification of
single/multiple
biomarkers
Total
Alkaloid
Total
protein
Total
Flavanoid
Spectroscopy
UV
IR
NMR
Mass
Total
Phenolics
Total
tannins
Through
Analysis
Total
Carbohydrates
Analysis
Analytical Screening
Chromatography
Analytical parameters
for oil/fats/waxes
TLC
HPLC
GC
HPTLC
% purity of
isolated
compounds
Chemical
Assay
Tritimetric
Analysis
Figure 8.2 Quantitative analysis of herbal drugs.
Phytochemical evaluation employs a range of techniques to analyze and identify the bioactive compounds
within plant materials. Several methods are employed for
this purpose, including chromatographic, spectroscopic,
and colorimetric techniques [8]. Chromatography is commonly employed to segregate, quantify, and identify
diverse phytochemicals by leveraging their distinct physical and chemical characteristics. Examples of this include
high-performance liquid chromatography (HPLC) and gas
chromatography (GC). To accurately detect and characterise chemicals, liquid chromatography-mass spectrometry
(LC-MS) combines chromatography with mass spectrometry (MS). Nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy are two spectroscopic methods that
provide information about the molecular structure of phytochemicals [9]. Additionally, ultraviolet-visible (UV-Vis)
spectrophotometry measures the absorbance of specific
compounds at different wavelengths, aiding in quantification [8]. Colorimetric assays utilize chemical reactions to
produce color changes, allowing for the determination of
Spectrometry
Chromatography
specific compounds. Each of these techniques plays a vital
role in phytochemical evaluation, contributing to the comprehensive understanding of plant constituents and their
potential medicinal properties. The choice of a particular
method is contingent on the nature of the compounds
being studied and the objectives of the research [10].
8.2 Phytochemical Screening Through Chemical Test
The study of plant extracts or other natural substances to
identify the existence of various types of phytochemicals,
including alkaloids, flavonoids, tannins, saponins, terpenoids, glycosides, and more, requires the important step of
phytochemical screening (Table 8.1) [11]. This screening
involves subjecting the plant material or extract to a series
of chemical tests, each specific to a particular class of phytochemical [12].
8.2 Phytochemical Screening Through Chemical Test 145
Table 8.1 Phytochemical screening for identification of various chemical constitutents in crude drugs.
Compound Chemical tests Procedure Observations
Carbohydrates Fehling’s Test Add Fehling’s A and B, heat, observe for red or
Benedict’s Test Add Benedict’s reagent, heat, look for
Iodine Test Add iodine solution, observe for color
Alkaloids Dragendorff ’s Test Add Dragendorff’s reagent, observe for
Mayer’s Test Mayer’s reagent, a potassium mercuric iodide
Wagner’s Test Iodine-potassium iodide solution is added as
Ehrlich’s Test Add a few drops of the Ehrlich’s reagent test
Glycosides Legal’s Test Add glacial acetic acid, FeCl3 solution, and
Keller-Killiani Test The test solution containing HCl is brought to
Baljet Test Before adding a drop of ferric chloride (FeCl3)
Tannins Ferric Chloride
Test
Lipids Solubility Test Add substance in various solvents,
Proteins Biuret Test Add dilute NaOH and copper sulfate, add
Flavanoids Shinoda Test Add magnesium powder and conc. HCl,
Saponins Froth Test Shake substance with water, observe for froth
Terpenoids Salkowski Test Add chloroform and conc. H
orange precipitate.
color change.
change.
orange or red-brown precipitate.
solution, needs to be combined with the test
solution.
Wagner’s reagent to the test solution.
solution, which is alcohol-based
para-dimethylaminobenzaldehyde.
conc. H
a boil before being cooled. Add a small amount
of the ferric chloride (FeCl3) solution.
solution, glacial acetic acid should be added to
the test solution.
Add ferric chloride solution, observe for color
change.
observe for solubility.
substance, observe color change.
observe for color change.
formation.
for color change.
. Observe color change.
2SO4
2SO4
, observe
The formation of a red precipitate
indicates the presence of reducing sugars.
Reducing sugars are indicated by colour
shifts from blue to green, yellow, orange,
or red.
Starch is present and is indicated by the
blue-black colour.
Formation of an orange or reddish brown\
sprecipitate implies alkaloids.
Alkaloids are confirmed to exist when a
creamy or yellowish precipitate forms.
The presence of alkaloids is indicated
when a reddish-brown precipitate forms.
Alkaloids are present when a violet or
purple colouring develops.
There are glycosides present when a blue
or green colour develops.
The presence of cardiac glycosides is
indicated by a red, violet, or purple tint
(e.g. digitalis glycosides).
Cardenolides are indicated by a green
colouring or a blue-green fluorescence
under UV light.
When a bluish-black or greenish-black
precipitate forms, tannins are present.
Solubility, transparency, or emulsion
formation indicates lipid presence.
Proteins are present when violet or pink
colour development occurs.
Flavanoids are present as shown by the
colour red.
Formation of stable froth indicates
saponins.
The presence of terpenoids is indicated by
a red color in the chloroform layer.
8.2.1 Alkaloids
Phytochemical tests for alkaloids are chemical assays
employed to figure out if something is therealkaloids in
plant extracts or natural compounds. Alkaloids represent a
diverse category of naturally occurring organic compounds, often characterized by their significant physiological effects [11]. These assays are useful instruments for
phytochemical analysis and are essential for determining
the chemical make-up of various plant components. They
provide preliminary indications of the presence of alka-
loids and guide further, more specific analyses. Several
common phytochemical tests are available to detect the
existence of alkaloids, and these tests are often employed
in combination for a more comprehensive evaluation [13].
Dragendorff’s Test: Dragendorff’s reagent is a widely
employed test for alkaloid detection in plant materials.
This technique, which uses thiourea to create a yellow
bismuth complex in a nitric acid medium, is praised for
its efficiency and speed. It has successfully detected alkaloids in a range of plants, including species like Buddleia
146 8 Qualitative and Quantitative Methods of Phytochemical Analysis
and Piper methysticum. However, while alkaloids have
been detected, the specific types have not always been
isolated and fully characterized.
For example, in field daisy flowers, Dragendorff’s reagent was used to isolate pyrrolizidine alkaloids and choline. The simultaneous detection of numerous hazardous
plant alkaloids, including aconitum alkaloids, solanaceous tropane alkaloids, sophora alkaloids, strychnos
alkaloids, and colchicine, in herbal and urine samples
has also been accomplished using a liquid chromatography-tandem MS method [11].
Dragendorff’s reagent and related methods remain
invaluable for the detection and analysis of alkaloids in
diverse plant materials. These assays are essential for
determining whether alkaloids are present and serve as
a starting point for further isolating and characterizing
these substances [14].
Procedure: Add a few milliliters of Dragendorff’s reagent
(potassium bismuth iodide solution) to the test solution.
Observation: Formation of an orange-red or brown pre-
cipitate indicates the presence of alkaloids.
Mayer’s Test: A sensitive diagnostic test called the Mayers
test can be performed to find opium alkaloids in bodily
fluids. It entails joining carboxylated latex polymers to
amino lower alkyl ethers of the phenolic hydroxyl group
of poppy alkaloids via a peptide connection. Alkaloids
can be detected in bodily fluids using this technique. A
different method of checking the alkaloid content of
seeds was created utilizing CHCl3 as a solvent and an
indicator called tetrabromophenolphthalein ethyl ester
in an acid-base titration with p-toluenesulphonic acid.
This quick screening method can be used to quickly
assess the alkaloid content of sweet lupin seeds and can
be applied to new sweet lupin varieties [11]. HPLC with
numerous detectors was described as a generic screening approach for alkaloid medicines in meals. In comparison to employing the ultraviolet detector alone, this
approach has lower detection limits and can detect both
acidic and basic alkaloids in food. Amperometric DNA
sensors and immunoenzyme test-systems were used to
develop bioaffine procedures for identifying particular
indole-containing alkaloids. These techniques enable
the efficient concentration and identification of the
alkaloids ajmaline and vincristine [13].
Procedure: The test solution should be mixed with Mayer’s
reagent, a potassium mercuric iodide solution.
Observation: Alkaloids are confirmed to exist when a
creamy or yellowish precipitate forms.
Wagner’s Test: Alkaloids are tested for by Wagner’s test. It
is employed to find opium alkaloids in bodily fluids.
The test involves attaching amino lower alkyl ethers
of the phenolic hydroxyl group of opium alkaloids to
carboxylated latex polymers in order to produce reagents that are sensitive to detecting opium alkaloids.
For the purpose of diagnosing, this test can be used to
find opium alkaloids in bodily fluids [15].
Procedure: Iodine-potassium iodide solution is added as
Wagner’s reagent to the test solution.
Observation: A reddish-brown substance forming means
there are alkaloids present.
Hager’s Test: Hager’s test is a spot examination used to
identify alkaloids. Lead dioxide is used in the test, and it
details how electrons go from the alkaloid to the complicated oxidation product. The test has been upgraded and
has toxicological implications [16].
Procedure: Hager’s reagent needs to be combined with the
test solution (saturated picric acid solution).
Observation: Alkaloids are present when an orange-red or
brown precipitate forms.
Ehrlich’s Test: A straightforward and useful qualitative test
for the presence of alkaloids is Ehrlich’s test.
Di-methylamido-benzaldehyde reacts with urobilinogen
and other urine metabolites in this process. This examination has been used to identify carcinoid, porphyrinopathies, hemolytic processes, common bile duct obstruction,
and liver disorders. Additionally, Ehrlich’s aldehyde reagent has grown in significance in chromatography and can
be employed with the contemporary test-strip approach to
quickly and easily examine urobilinogen and bilirubin [17].
Procedure: Put a few drops of Ehrlich’s reagent, which is
para-dimethylaminobenzaldehyde in alcohol, into the
test solution.
Observation: Alkaloids are present when a violet or purple
coloring develops.
8.2.2 Glycosides
Glycosides can be found in plant extracts or other natural
substances using a process known as phytochemical testing. A sugar molecule (the glycone) is joined to a non-sugar
molecule (the aglycone) by a glycosidic connection to form
a chemical known as a glycoside [18].
Keller-Killiani Test: A technique for finding glycosides
is the Keller-Killiani test. It has been used to quantify
the amounts of digitoxose and digitoxose-containing
glycosides. Another colorimetric approach that makes
use of cyanide’s ability to hinder a coloring process has
been devised to measure cyanide and cyanogenic glycosides. Additionally, the glycoalkaloids -solanine
and -chaconine have been quickly and accurately
detected using cholinesterase-based sensors [19].
8.2 Phytochemical Screening Through Chemical Test 147
Procedure: The test solution containing HCl is brought to
a boil before being cooled. Drops of ferric chloride
(FeCl3) solution should be added.
Observation: The presence of cardiac glycosides is indicated
by a red, violet, or purple tint (e.g. digitalis glycosides).
Modified Legal’s Test: A redesigned test tube has been
created that can be used without a test tube rack and
can sit on any flat surface. The design consists of a tubular neck portion and a tubular containment piece, the
latter of which has a closed end and a bottom surface
that is flattened. The mouth of the neck section opens
parallel to the bottom surface at an angle of about 45° to
the flattened surface. With this design, the contents are
kept from spilling, and convenient monitoring is possible without having to move the test tube. Additionally,
cyanide and cyanogenic glycosides can now be measured using colorimetric techniques. These techniques
make use of cyanide, which is either added or released
from a cyanogenic glycoside, to hinder a coloring reaction. Test plates coated with films offer a semi-quantitative alternative, whereas the spectrophotometric
approach is quantitative [20].
Procedure: To the test solution, add a few drops of ferric
chloride (FeCl3) solution.
Observation: A bluish-green coloring that develops shows
the presence of cardiac glycosides.
Observation: A reddish-brown hue at the boundary
between the two layers indicates the presence of terpenoid glycosides [23].
Froth Test: The provided abstracts do not particularly ref-
erence the froth test. However, several of the abstracts
make mention of techniques for identifying glycosides.
For the detection of cyanide and cyanogenic glycosides,
Tatsuma et al. developed colorimetric techniques.
Additionally, Korchagina and Petrova talk about using
medications that include cardiac glycosides, a kind of
glycoside. Although these abstracts don’t specifically
discuss the froth test, they do offer information on how
to find and identify glycosides. The froth test could be an
alternative or less often employed technique for evaluating glycosides [24].
Procedure: Shake the test fluid briskly to check for the
development of persistent foam.
Observation: Saponin glycosides, which have surface-
active characteristics, are present when foam forms.
8.2.3 Flavanoids
Flavonoid chemicals can be found in plant extracts or natural products using phytochemical testing for flavonoids. A
subclass of polyphenolic chemicals called flavonoids is
famous for their ability to reduce inflammation and promote good health [25].
Baljet Test:
Procedure: Before adding a drop of ferric chloride (FeCl3)
solution, glacial acetic acid should be added to the test
solution.
Observation: Cardenolides are indicated by a green color-
ing or a blue-green fluorescence under UV light [21].
Bornträger’s Test: Glycosides are discovered using the
Bornträger’s assay. An aqueous solution of the glycoside is
mixed with a colorant based on an aromatic ketone for the
test, after which the level of coloration is measured. This
test is frequently used to identify cyanide and cyanogenic
glycosides like linamarin and amygdalin. HPLC is also utilized for the synthesis of glycosides to check the stereospecificity of the synthesis [22].
Procedure: The test solution should be added to a pyridine
and 1% hydrochloric acid (HCl) solution before being
extracted with chloroform.
Observation: Chloroform layer colorations of pink, red, or
purple indicate the presence of anthraquinone glycosides.
Salkowski Test:
Procedure: Chloroform should be added to the test solu-
tion, and then concentrated sulfuric acid should be carefully added along the test tube’s side.
Shinoda Test:
Procedure: Add a piece of magnesium ribbon after a few
drops of strong hydrochloric acid (HCl) have been added
to the test solution.
Observation: Flavonoids are present when a pink, red,
purple, or violet hue begins to appear (26).
Ferric Chloride Test: Different chemicals have been found
using the ferric chloride test in various circumstances.
However, none of the provided abstracts directly refer to
the ferric chloride test as a method for identifying flavonoids. Nagendran and Bhuvaneswari report a false positive ferric chloride test in a case of phenylketonuria. Grlić
and Tomić’s, written in a different language, discuss the
use of ferric chloride to characterize certain types of
hemp resin [27].
Procedure: The test solution should be diluted with a few
drops of a 10% ferric chloride (FeCl3) solution.
Observation: Flavonoids may be present if a blue, green,
or black color develops.
Zinc-Hydrochloric Acid Reducing Sugar Test:
Procedure: After incorporating a few drops of zinc dust
with the test solution, add a few drops of strong hydrochloric acid (HCl).
148 8 Qualitative and Quantitative Methods of Phytochemical Analysis
Observation: Flavonoids can be identified by their red
coloring or by the development of a red precipitate [28].
Alkaline Reagent Test (NaOH Test):
Procedure: Add a few drops of sodium hydroxide (NaOH)
solution that has been diluted to the test solution.
Observation: When upright, flavonoids are visible as a
bright yellow color that darkens [23].
Lead Acetate Test: A simple, affordable, and reliable
approach for evaluating low-yield H
lead acetate test paper. When compared to AlCl
S yeast strains is the
2
, it
3
reacts more quickly and causes a more noticeable color
shift when used to detect anthocyanins that include a
catechol group. The test paper has also been used as a
lead intoxication screening test, demonstrating a direct
correlation between blood lead levels and free erythrocyte protoporphyrin (FEP) fluorescence. Additionally,
even in patients with pre-existing renal illness, the leadmobilization test employing CaNa
EDTA has been
2
shown to be non-nephrotoxic [29].
Procedure: A couple of drops of lead acetate solution have
been added to the test solution to dilute it (lead sugar).
Observation: The precipitate turns yellow; it shows that
flavonoids are present.
Ammonia Test:
Procedure: Add a few drops of a concentrated ammonia
solution to the test solution.
Observation: An acid-induced color change from yellow
to colorless is a sign that flavonoids are present [30].
Sodium Nitrite Test: Nitrite content in a sample can be
determined using sodium nitrite. In order to create a
solution of diazo salt, one method calls for passing a
water sample into a colorimetric pipe, where potassium
bromide, sulfanilic acid, and hydrochloric acid are then
added. When this solution is combined with sodium
carbonate and 2-N-ethyl-5-naphthol-7-sulfonic acid in a
volumetric flask, the resulting orange diazo compound
solution’s absorbance is measured at 480 nm. Using a
curve regression equation, it is possible to determine
the sample’s sodium nitrite concentration. Using nitrite
test paper soaked in a mixture of sulfonic acid, tartaric
acid, alpha-naphthylamine, sulfanilamide, and N-1naphthylethylenediamine dihydrochloride is an alternative approach. After making contact with the sample
with the test paper, the nitrite content is ascertained by
contrasting the color development with a reference
color card [30].
Procedure: Add some sodium nitrite and a few drops of
diluted hydrochloric acid (HCl) to the test solution
(NaNO2).
Observation: Flavonoids are present when an object has
an orange or red coloring.
8.2.4 Tannins
Tannins are polyphenolic substances with astringent
qualities that can be found in many plant components.
The presence of tannins in plant extracts can be determined chemically using a variety of methods [31].
Ferric Chloride Test for Tannins: Different fields have
used the ferric chloride test for different things. A ferric
test was created by Poulsen et al. to identify vegetable
tannins. Ferric chloride was used by Prigal to create a
chemical spot test for standardizing and checking waterin-oil emulsions. Hardin discovered that when exposed
to a ferric chloride solution, mechanically damaged portions of legume seeds turn black. In an instance of accidental ferric chloride consumption, Pucci et al. described
significant gastrointestinal ulceration and inflammation. Leaching with ferric chloride has been successfully
used by Murphy et al. to remove lead from galena concentrations. In a test tube or other tiny container, place a
small amount of the plant extract or sample that will be
evaluated [31].
1. Add a few drops of a 1% ferric chloride (FeCl3)
solution to the test sample.
2. Observe the color change in the mixture.
Observation:
• The development of a bluish-black or greenish-black
color in the mixture indicates the presence of tannins.
8.2.5 Saponins
Saponins are chemical substances that are present in a
variety of plant species and are well-known for their distinctive ability to create a soapy lather when mixed with
water. The presence of saponins in plant extracts can be
determined chemically using a variety of methods. The
Froth Test is one typical test [32].
Froth Test for Saponins: The froth test is a technique for
detecting saponins in a variety of substances. Plants contain substances called saponins, secondary metabolites
with foaming characteristics. They can be found in many
angiosperm plants, tiny aquatic animals, and even certain
microbes. Saponins’ foaming and hemolytic characteristics are caused by steroid saponins and glycoalkaloids, particularly furostanol glycosides [33]. The saponins from
Sapindusmukorossi have been deemed safe for use in cosmetics after being examined for acute oral and dermal toxicity, as well as for skin irritation. Soybean saponins have
been found to impede the growth of human carcinoma
cells, suggesting that they may have anticancer properties.
The presence of saponins can be determined using the
froth test on a variety of medicinal plants and marine species [34].
8.2 Phytochemical Screening Through Chemical Test 149
1. Take a small amount of the plant extract or sample
to be tested in a test tube or a small container.
2. Add distilled water to the test sample, filling it
about two-thirds full.
3. Shake the mixture vigorously for a few minutes.
You can do this manually or by using a mechanical
shaker.
4. Observe the formation of a frothy lather.
Observation:
• The presence of saponins is confirmed if a stable, per-
sistent froth or lather is produced upon shaking.
8.2.6 Terpenoids
A complex class of naturally occurring chemical molecules
found in plants and some animals are terpenoids, commonly referred to as terpenes. The isoprene (C5H8) molecules that make up the structural backbone of terpenoids
give them their distinctive properties. Depending on how
many isoprene units a terpene has, there are distinct types
of terpenoids, which result in varied structures and functions [35].
Salkowski Test for Terpenoids: The Salkowski test is a
chemical assay designed for the identification of terpenoids. This procedure entails the interaction of terpenoids with concentrated sulfuric acid, leading to the
development of a red color.
1. Take a small quantity of the plant extract or sample
and place it into a test tube.
2. Add two milliliters of chloroform to the test tube
containing the sample.
3. Carefully layer concentrated sulfuric acid below the
chloroform layer by gently pouring it along the test
tube’s side to create two separate layers.
4. Allow the test tube to stand undisturbed for a few
minutes.
Observation:
• The existence of terpenoids is confirmed if a reddish-
brown coloration emerges at the junction of the chloroform and sulfuric acid layers. The intensity of the
color change can vary depending on the concentration
of terpenoids [17].
and sodium citrate in an alkaline solution) changes
from blue to a reddish-orange or brick-red color upon
heating [16].
Fehling’s Test: Similar to Benedict’s test, Fehling’s test
serves the purpose of detecting reducing sugars.
Fehling’s reagent is a two-part solution, which is mixed
with the test sample and heated. A positive outcome is
signaled by the development of a reddish-brown precipitate [17].
Barfoed’s Test: This test is specific for monosaccharides,
especially pentoses like ribose and deoxyribose. Barfoed’s
reagent, which is copper acetate in acetic acid, forms a
reddish-brown precipitate upon heating with a positive
sample [36].
Seliwanoff’s Test: Seliwanoff’s reagent is employed to dif-
ferentiate between aldoses and ketoses. It forms a red
color upon heating with ketoses but has a very faint reaction with aldoses [37].
Molisch’s Test: This assay, as previously stated, is a broad
test for the existence of carbohydrates. It includes the
addition of Molisch’s reagent (alpha-naphthol in ethanol) followed by concentrated sulfuric acid to the sample. The appearance of a purple-to-violet ring at the
interface between the two layers signifies the presence
of carbohydrates [38].
Iodine Test: Iodine solution is used to test for the presence
of starch. It changes from brown to blue-black or purple
when it reacts with starch [39].
Osazone Test: This test is used to identify and characterize
specific carbohydrates based on the crystalline structure
of their osazone derivatives. It is particularly useful for
distinguishing between different types of reducing sugars [40].
Tollen’s Test: Commonly referred to as the silver mirror
test, this method is employed to recognize reducing sugars through the creation of a silver mirror on the inner
surface of a test tube [39].
Seliwanoff’s Test: This test is specific for ketohexoses,
such as fructose. Seliwanoff’s reagent produces a deep
cherry-red color when ketohexoses are present [41].
Acetoin Test: This test is used to identify the existence of
acetoin, which is a product of carbohydrate fermentation. It involves the Voges-Proskauer reaction and is
commonly used in microbiology [42].
8.2.7 Carbohydrates
Benedict’s Test: This test is used to detect reducing sugars,
such as glucose and fructose. When reducing sugars are
present, Benedict’s reagent (containing copper sulfate
8.2.8 Lipids [43]
Sudan III or Sudan IV Test: Sudan dyes are used to stain
lipids and fats. The sample is mixed with Sudan III or
Sudan IV, and if lipids are present, a red or orange color
will develop.
150 8 Qualitative and Quantitative Methods of Phytochemical Analysis
Grease Spot Test: In this test, place a drop of the sample
onto a piece of filter paper. If the spot becomes translucent or leaves a grease mark, it shows the presence of
lipids.
Solubility Test: Fats and oils are generally soluble in non-
polar solvents like ether or chloroform. When the sample is mixed with these solvents, lipids will dissolve.
Emulsion Test: Mix the sample with water and shake it. If
a milky or cloudy emulsion forms, it shows lipids are
present. This test is commonly used to find out if there
are lipids in food.
Acrolein Test: When lipids are heated with glycerol and
potassium bisulfate, they produce acrolein. The characteristic smell of acrolein indicates the presence of lipids.
Halphen Test: This test is employed to identify unsatu-
rated lipids. When the sample is mixed with bromine
water, it turns colorless due to the addition reaction of
bromine to the carbon-carbon double bonds in unsaturated fats.
Iodine Value Test: The iodine value (IV) is a way to meas-
ure how unsaturated lipids are. It includes titration with
iodine to figure out the count of double bonds in the
lipids.
Kovacs Test: This test is used to detect the presence of ter-
penoids in lipids. It involves mixing the sample with glacial acetic acid and sulfuric acid. A red-to-violet color
indicates terpenoids.
Transmittance Test: This test measures the turbidity or
cloudiness of a lipid solution when light passes through
it. More cloudiness indicates a higher lipid content.
Flame Test: When lipids are burned, they produce a bright,
sooty flame with a distinct odor. The flame test is not as
specific as other tests but can indicate the presence of
lipids.
Ninhydrin Test: This test can be used to detect lipids that
contain amino acids. When heated with the ninhydrin
reagent, the sample forms a purple or blue color.
Molisch’s Test: This test, as mentioned earlier, is a gen-
eral test for the presence of lipids or any other noncarbohydrate organic compound. It involves the
addition of Molisch’s reagent (alpha-naphthol in ethanol) followed by concentrated sulfuric acid. The formation of a purple-to-violet ring at the junction of the
two layers signals the presence of lipids.
8.2.9 Protiens [44]
Biuret Test: The material is mixed with the biuret reagent.
Proteins are present when the hue changes to purple.
The protein content is correlated with the intensity of
the color change.
Ninhydrin Test: Free amino acids are the basic components
or building blocks of proteins are found using this assay.
The sample takes on a purple or blue hue when heated in
the presence of the ninhydrin reagent.
Millon’s Test: The sample is heated after Millon’s reagent
has been introduced. The presence of phenolic chemicals, which are frequently found in proteins, is shown by
a crimson solution or precipitate.
Xanthoproteic Test: In this test, the material is treated with
strong nitric acid. A yellow color shift denotes the presence of aromatic amino acids, which are frequently found
in proteins and include tyrosine and phenylalanine.
Biuret Test: Proteins can be found using the biuret reagent.
When a biuret reagent is added to the sample, proteins
cause it to turn purple.
Bradford Protein Assay: This is a Coomassie Brilliant
Blue G-250 colorimetric assay. Proteins that the blue dye
attaches to change the color of the material from brown
to blue. There is a linear relationship between the protein concentration and the color change.
Lowry Protein Assay: A series of chemical processes are
used in this assay to create the blue color. The protein
concentration is inversely correlated with the blue
color’s intensity.
Bicinchoninic Acid (BCA) Assay: The BCA assay uses a
copper-based reagent that forms a purple complex with
proteins. The change in color is assessed at a particular
wavelength to determine protein concentration.
UV Absorbance Test: Proteins absorb UV light at 280 nm
due to the presence of aromatic amino acids. The protein
concentration can be determined by measuring the
absorbance at this wavelength.
Sakaguchi Test: Arginine may be found in proteins using
the Sakaguchi reagent. A crimson or pink hue denotes a
successful outcome.
8.3 Quantitative Methods of Phytochemical Analysis
8.3.1 Determination of total phenolic content
Phenolic compounds, or simply phenols, are a type of
chemical that has a phenol ring – a carbon ring with six
members and a hydroxyl group attached. (−OH) group
[45]. These compounds are characterized by their aromatic
nature, and these organisms are abundantly present across
various plant species, where they play various important
roles in the growth, development, and defense mechanisms of plants [46]. Phenolic compounds can be located