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8.3 Quantitative Methods of Phytochemical Analysis 151
across a diverse array of plants, including fruits, vegetables,
whole grains, tea, coffee, and many medicinal herbs [47].
Determining the total phenolic content in various samples
is of paramount importance in several scientific fields and
industries due to the numerous benefits and applications
associated with these compounds. Phenolic compounds
serve as lead compounds in drug development because of
their phenolic compounds exhibit various pharmacological
properties, including anti-cancer, anti-inflammatory, and
antimicrobial activities [48]. Many herbal medicines derive
their therapeutic properties from phenolic compounds.
Determining phenolic content ensures the quality and effectiveness of these traditional remedies.
8.3.1.1 Folin-Ciocalteu Method Principle of the Folin-Ciocalteu Assay The Folin-Ciocalteu
method is a widely utilized colorimetric technique for
assessing the total phenolic content in samples [49]. It
operates on the principle of a redox reaction, where
phenolic compounds within the sample act as reducing
agents, donating electrons to the Folin-Ciocalteu reagent,
composed of phosphomolybdic and phosphotungstic acids.
This reaction occurs under alkaline conditions, facilitated
by the addition of sodium carbonate, leading to a change in
the reagent’s color from yellow to blue. The strength of the
resulting blue color increases in direct correlation with the
amount of phenolic compounds present. This color change
is quantified using a spectrophotometer at a specific
wavelength, usually around 725 nm [50]. To determine the
phenolic content, the absorbance of the sample’s colored
complex is compared to a standard curve created with
known concentrations of a reference phenolic compound,
such as gallic acid. This comparison allows for the
calculation that the intensity is directly proportional to the
concentration of phenolic compounds in the sample. The
simplicity, sensitivity, and reliability of this method make it
a valuable tool in scientific research, quality control, and
various applications within analytical chemistry and
biology [51].
Procedure for Determining Total Phenolic Content Using the
Folin-Ciocalteu Method The determination of total phenolic
content through the utilization of the Folin-Ciocalteu
method encompasses a methodical process. (doi: 10.12980/
APJTB.4.201414B295). Initially, a sample extract is
prepared by utilizing an appropriate solvent, such as
methanol, ethanol, or water, tailored to the sample’s nature.
If necessary, the extract is concentrated using rotary
evaporation. Subsequently, a reaction mixture is created by
pipetting a specific volume. Transferring the sample extract
into a test tube. An equal volume of Folin-Ciocalteu reagent
is added, and blend the contents completely, and then let
the mixture stand in darkness for 3–5 minutes, turning
blue due to the formation of a colored complex. An alkaline
solution, typically 20% sodium carbonate, is added to the
test tube to alkalize the mixture, ensuring homogeneity
through gentle mixing. The mixture is then left to incubate
in darkness at room temperature for a specific duration,
usually 30 minutes to two hours, facilitating full color
development as phenolic compounds in the sample reduce
the Folin-Ciocalteu reagent [52]. Following incubation, the
reaction mixture’s absorbance is measured at a specific
wavelength. (765 nm) using a spectrophotometer. A blank,
containing only the solvent and reagents without the
sample, is used for calibration. The phenolic content in the
sample is Its concentration is determined by comparing its
absorbance to A calibration curve is created using standard
gallic acid solutions [53]. The outcomes are represented as
milligrams of gallic acid equivalents per gram or milliliter
of the sample (mg GAE/g or mg GAE/mL) using the
calibration curve., providing a quantitative measure of the
sample’s phenolic content [54].
8.3.2 Determination of Total Flavonoid Content
The quantification of flavonoids, a class of polyphenolic
compounds found in plants, has gained attention for its
diverse health benefits. Flavonoids possess antioxidant
[55], anti-inflammatory [46], antidiabetic [56], and anticancer properties [57], making them valuable subjects of
scientific inquiry. The objective of this research was to
assess the total flavonoid content in plant samples using a
precise and validated method based on the aluminum chloride colorimetric assay [58].
Principle
The aluminum chloride colorimetric method operates
on the principle of specific complex formation between
flavonoids present in the sample and aluminum chloride
in the presence of a base [59]. Flavonoids, owing to their
phenolic structure, readily react with aluminum chloride under mildly acidic conditions, resulting in the creation of a distinct colored complex [60]. The intensity of
this color correlates directly with the concentration of
flavonoids in the sample. Measure the absorbance of
this colored complex at a specific wavelength (420 nm)
using a spectrophotometer and quantify the flavonoid
content in the sample. To achieve this, standard solutions of known flavonoid quercetin are utilized in the
formation of a calibration curve. The absorbance of the
sample is then compared to this curve, enabling the calculation of flavonoid concentration in the sample,
152 8 Qualitative and Quantitative Methods of Phytochemical Analysis
expressed in terms of the equivalent amount of the
standard flavonoid per unit of sample weight (mg QE/g
or μg QE/mL). This method provides a reliable and
widely used approach for the accurate determination of
flavonoid content in various natural samples [58].
Method
The total flavonoid content in the sample was assessed
through the aluminum chloride colorimetric method [61].
Quercetin is utilized to establish the standard calibration
curve for the quantification of total flavonoid determination [62]. A solution containing 5.0 mg of quercetin was
prepared by dissolving it in 1.0 mL of methanol [63]. From
this stock solution, standard solutions of quercetin were
generated by serially diluting the compound in methanol,
covering a concentration range from 5 to 200 μg/mL.
Following this, 0.6 mL of diluted standard quercetin solutions or extracts were combined with 0.6 mL of 2% aluminum chloride. After thorough mixing, the solutions were
incubated for 60 minutes at room temperature. The absorbance of the resulting reaction mixtures was measured
against a blank at a wavelength of 420 nm using a UV–Vis
spectrophotometer [64]. The total flavonoid content in the
test samples was then determined from the calibration plot
and expressed as milligrams of quercetin equivalent (QE)
per gram of dried plant material (mg QE/g) [65]. This
method provides a reliable means to quantify flavonoid content in samples, offering valuable insights into their composition for various research and analytical purposes.
8.3.2.1 Determination of Tannins
Tannins comprising a varied range of polyphenolic compounds discovered within various plant species, are recognized for their distinctive attributes ability to bind and
precipitate proteins, making them essential for processes
like tanning leather and often providing astringent taste in
foods and herbal drugs [66].
Methods like the Folin-Ciocalteu method, the VanillinHCl method, and the Butanol-HCl method are commonly
utilized for assessing the total tannin content in a sample
[67]. These methods involve chemical reactions that result
in the formation of colored complexes, the intensity of
which correlates with the tannin concentration.
8.3.2.2 Estimation of Total Tannin Content
Folin-Ciocalteu Method The determination of tannins was
conducted using the Folin-Ciocalteu method [68]. Initially,
a minute A portion of the sample was taken and transferred
in a calibrated flask. Subsequently, precise volumes of
Folin-Ciocalteu phenol reagent, distilled water, and 35%
sodium carbonate solution were added to the flask,
followed by thorough mixing [68]. This concoction was
permitted to incubate at ambient room temperature for
precisely 30 minutes, permitting the interaction between
the phenolic compounds within the sample and the reagent
[69]. Simultaneously, a series of standard solutions
containing known concentrations of tannic acid were
meticulously prepared using the same procedure as the
sample. These standard solutions served as crucial
reference points for our analysis [70]. Upon completion of
the incubation period, the UV/visible spectrophotometer
was employed to measure the absorbance of both the
sample and the standard solutions at a specific wavelength
of 700 nm [71]. This spectral analysis, conducted against a
blank solution, facilitated the precise analysis of the tannin
content within the sample [71]. The findings were
presented in terms of milligrams of tannic acid equivalents
per gram of the dried sample, providing a quantitative
measure of the tannin concentration [72].
Vanillin-HCl Method The total condensed tannin
contents, or proanthocyanidins, and the outcomes were
indicated within the samples were determined using the
method outlined by Broadhurst et al. in 1978, with slight
modifications. In this method, catechin was employed
as the reference compound [73]. To initiate the analysis,
the sample extract, measuring 400 µL, was blended with
3 mL of a vanillin solution (4% in methanol) and 1.5 mL
of concentrated hydrochloric acid [74]. After a 15-minute
incubation period, the absorbance of the resulting
mixture was measured at 500 nm. The concentration of
condensed tannins was expressed as grams of equivalent
catechin per 100 grams of dry matter (g E.Catechin.
–1
DM) [75]. This methodology allowed for the
100g
precise quantification of condensed tannin content in
the samples, providing valuable insights into their
composition and characteristics [76].
Butanol-HCl Method This method is used for estimating
condensed tannins in leaf, bark, and fruit samples, tannin
extracts are prepared from the samples and subjected to a
series of chemical reactions [77]. Initially, 0.5 mL of these
tannin extracts were mixed, 3.0 mL of butanol was
introduced, and then 0.1 mL was added of hydrochloric
acid and the ferric reagent. The resulting mixture in each
test tube is thoroughly vortexed and then heated, covered
with glass slides, for 60 minutes. Simultaneously, blank
samples are prepared in a similar manner, except for the
omission of the heating step. After cooling to room
temperature, the samples and corresponding blanks are
analyzed for absorbance at 550 nm employing a
spectrophotometer [78]. The obtained absorbance values
8.3 Quantitative Methods of Phytochemical Analysis 153
are then used to calculate the concentration of condensed
tannins, expressed in leucocyanidin equivalents per gram
or milliliter of the sample, through a calibration curve
derived from known standards [79].
Percentage of Condensed
Tannins = A550nm × 78.26 ×
Dilution factor
______________
% of Dry matter
Hydrolyzed tannins are derived by subtracting the concentration of condensed tannins from the total tannins in a
given sample [80].
8.3.2.3 Determination of Total Alkaloid
Alkaloids, a diverse group of natural compounds, have
played a significant role in shaping human history, influencing economic, medical, political, and social spheres.
These compounds possess potent physiological effects on
mammals and other organisms, making them vital therapeutic agents. Notable examples include atropine, morphine, quinine, and vincristine, which have been
instrumental in treating a range of diseases from malaria to
cancer [81]. The principle behind the determination of
alkaloids in plant samples involves specific chemical reactions and measurements. Alkaloids constitute a category of
naturally found organic compounds containing fundamental nitrogen atoms [82]. As a result, the accurate determination of total alkaloids holds great importance, particularly
in evaluating the quality of medicinal plants. This precise
analysis is essential to verify the efficiency and safety of
herbal remedies, underscoring the critical nature of accurate alkaloid quantification methods [83]. The principle
methods for alkaloid determination often include techniques like spectrophotometry and chromatography [84].
Principle In spectrophotometry, alkaloids can often form
colored complexes with certain reagents. These complexes
can absorb light at specific wavelengths, allowing for their
quantification [85]. The formation of a complex between
alkaloids and bromocresol green (BCG) in a chloroform
phase can be measured spectrophotometrically at 470 nm
[86]. Measuring the absorbance of this complex enables the
calculation of alkaloid concentration in the sample, utilizing
a calibration curve established from standard solutions.
Methods for Determination of Alkaloids The method involves
preparing a BCG solution by dissolving BCG in 2N NaOH
and water, which is then diluted [87]. A pH 4.7 phosphate
buffer solution is created by adjusting 2M sodium
phosphate with citric acid. Additionally, an atropine
standard solution is prepared by dissolving pure atropine
in water. To create the standard curve, varying volumes of
atropine solution are mixed with phosphate buffer and
BCG solution, shaken with chloroform, and the resulting
extracts are measured at 470 nm [88]. For sample extraction,
plant material is extracted with methanol, and the extract
is evaporated, dissolved in 2N HCl, and washed with
chloroform [89]. After adjusting the pH, a solution of BCG
and phosphate buffer is combined, and the resulting
mixture is then extracted with chloroform [90]. The
absorbance of the chloroform extracts is determined at
470 nm using a spectrophotometery providing data for
quantifying atropine content in the plant samples [91].
8.3.2.4 Determination of Carbohydrates
Carbohydrates are indeed the most abundant organic
compounds found in nature. They are vital for various living organisms, serving as a crucial source of energy and
playing structural roles in cells [92]. Carbohydrates are
organic compounds composed of carbon, hydrogen, and
oxygen atoms. They exhibit diverse structures, ranging
from straightforward sugars like glucose and fructose to
intricate polysaccharides, such as starch, cellulose, and
glycogen [93]. Polysaccharides are intricate carbohydrates
made up of diverse monosaccharide units linked by glycosidic bonds. Found in plants, they serve crucial roles:
providing structural support, acting as an energy source,
and participating in metabolic processes and cell signaling. Additionally, polysaccharides act as barriers against
pathogens, making them vital for plant defense mechanisms. Their varied properties have led to applications in
pharmaceuticals, agriculture, medicine, and material science. Understanding these complex molecules is essential, as they play pivotal roles in cellular metabolism,
stress responses, hormone signaling, and intricate communication systems within organisms [94].
Principle The phenol-sulfuric acid method is a commonly
employed technique for chemical assays for quantifying
carbohydrates, especially sugars and polysaccharides. This
method is highly sensitive and simple, making it popular in
a multitude of fields, including biochemistry, food science,
and environmental science. Its ability to work in the
presence of salts and protein residues adds to its versatility.
This approach employs concentrated sulfuric acid to
hydrolyze complex carbohydrates (polysaccharides, oligosaccharides, and disaccharides) into monosaccharides
[95]. Under the influence of sulfuric acid, these monosaccharides undergo dehydration, forming furfural derivatives. Subsequent reactions between these derivatives and
phenol lead to the creation of yellow-gold colored complexes [96].
154 8 Qualitative and Quantitative Methods of Phytochemical Analysis
The depth of this color directly corresponds to the
quantity of carbohydrates within the sample. Compounds
generated during this reaction absorb visible light, with
peak absorption occurring at specific wavelengths,
approximately around 490 nm for hexoses (six-carbon
sugars) and 480 nm for pentoses (five-carbon sugars) and
uronic acids. This absorption pattern allows for the quantification of carbohydrates in a given sample by measuring the absorbance at these specific wavelengths using a
spectrophotometer [97].
Method In the standard procedure for this method, a 2 mL
portion of a carbohydrate solution is combined with 1 mL
of a 5% aqueous phenol solution in a test tube. Following
this, 5 mL of concentrated sulfuric acid is rapidly added to
the mixture [98]. After allowing the test tubes to stand for
10 minutes, they are vortexed for 30 seconds and then
placed in a water bath at room temperature for 20 minutes
to facilitate color development [99]. Subsequently, light
absorption at 490 nm is recorded using a spectrophotometer.
Comparable reference solutions are prepared, replacing
the 2 mL aliquot of carbohydrate with DDI water [99]. It is
crucial to note that the phenol used is redistilled, and a 5%
phenol solution in water (w/w) is freshly prepared just
before the measurements are taken [97].
8.3.2.5 Determination of Protein
Proteins, intricate macromolecules, are composed of long
chains of amino acid residues [100]. Their significance lies
in shaping, regulating, and functioning within the body’s
cells, tissues, and organs. These biomolecules play diverse
roles in living organisms, such as catalyzing metabolic
reactions, DNA replication, responding to stimuli, and
facilitating the transportation of molecules between different locations [101].
Proteins play a fundamental role in the growth, maintenance, and overall functioning of the human body. They
serve as the building blocks for tissues, aiding in the repair
and growth of muscles, skin, and organs. Additionally, proteins act as enzymes, catalyzing vital biochemical reactions,
and function as hormones, regulating various physiological
processes. While proteins do provide energy, carbohydrates,
and fats are the primary energy sources in the body. Beyond
their energy-giving role, proteins have diverse functions,
including facilitating the transport of molecules and ions
across cellular membranes, regulating processes like the cell
cycle and gene expression, and maintaining the body’s pH
balance as buffers. A balanced diet rich in proteins from both
animal and plant sources is essential to ensure the body
receives the necessary amino acids and nutrients for optimal
health and well-being [102].
Estimating protein content in various substances is
crucial in fields such as nutrition, biochemistry, and food
science. There are several methods employed for the estimation of protein [103], each with its own advantages
and limitations. There are many methods for estimating
protein content, like Kjeldahl Method, Lowry Method,
and Bradford Assay [104].
Determination of Protein by Kjeldahl Method
Principle This method involves the digestion of the sam-
ple with concentrated sulfuric acid, converting nitrogen to
ammonium sulfate. The ammonia released is then distilled
and titrated [105].
Method [106] In the ammonia determination process,
a 2 g sample underwent digestion in a Kjeldahl digestion
flask. Modify it by boiling with 20 mL of concentrated
and a Kjeldahl digestion tablet (catalyst) continued
H
2
4
until the solution clarified. Following digestion after filtration, the mixture was diluted with distilled water to reach
the 250 mL mark in a volumetric flask. Next, the diluted
solution was subjected to distillation. Ammonia was steam
distilled by introducing 50 ml of 45% sodium hydroxide
solution. A 150 mL of the distillate were gathered in a conical flask, along with 100 mL of 0.1 N HCl and methyl red
indicator. The ammonia in the distillate reacted with the
acid, and any surplus acid was titrated back with a 2.0M
NaOH solution until reaching the endpoint indicated by a
color change from red to yellow, was reached. To ensure
precision, blank determinations were performed on all reagents individually. This meticulous method allowed for
accurate quantification of ammonia content in the original
sample, achieved through a series of chemical reactions
and titrations.
The nitrogen percentage was calculated through the following method:
[(ml standard acid × N of acid) − (ml blank × N
of base)] − (ml std base × N of base) × 1.4007 /
Weight of sample in grams
Where N = normality
Determination of Protein by Lowry Method
Principle In an alkaline solution, proteins undergo a reac-
tion with copper ions and Folin-Ciocalteu reagent forming a
blue color that can be measured spectrophotometrically [107].
Method Initially, the extracts underwent dilution to a vol-
ume of 1 mL with water. Subsequently, 0.9 mL of solution A
was incorporated into this mixture, comprising 2 g/L potassium sodium tartrate and 100 g/L sodium carbonate in
8.3 Quantitative Methods of Phytochemical Analysis 155
0.5 M NaOH. The concoction was then subjected to an incubation period at 50 °C for 10 minutes. Following this incubation, the samples were cooled to room temperature, and 1 mL
of solution B was introduced. Solution B consisted of 0.2 g/L
potassium sodium tartrate and 10.1 g/L copper sulfate pentahydrate in 0.1 M NaOH. Another incubation period of 10
minutes ensued after the addition of solution B.
In the concluding stage, 3 mL of solution C, a blend of
Folin-Ciocalteu phenol reagent in water at a ratio of
1 : 16 v/v, was introduced to the samples. The samples
underwent another incubation at 50 °C for 10 minutes.
For the creation of a calibration standard curve, diverse
concentrations of bovine serum albumin (BSA) spanning
from 0 to 1 g/L were readied. Following this, the absorbance of both the samples and the standards was recorded
at 650 nm. This measurement allowed for the determination of protein concentrations based on the absorbance
values obtained from the standard curve. This method
provided a reliable approach for quantifying protein content in the given samples [108].
Determination of Protein by Bradford Assay
Principle The Coomassie Brilliant Blue G-250 dye changes
color when it binds to proteins, and the absorbance of this
dye-protein complex is measured at a specific wavelength
[109].
Method Quantifying the entire extracellular protein con-
tent in medicinal plants was carried out using the Bradford
test method [110]. In this method, researchers utilized the
capacity of proteins to interact with the Coomassie Brilliant
Blue G250 dye. Bovine serum albumin served as the standard protein. Various volumes of the standard solution and
supernatant from the sample were pipetted into separate
tubes and diluted to 1 mL with sterilized distilled water.
Bradford reagent was added to each tube, and the contents
were thoroughly mixed. A blank solution containing distilled water and Bradford reagent was used for reference.
The absorbance of the samples was measured at 595 nm
against the blank. By comparing these absorbance values
to a standard curve generated by employing known concentrations of bovine serum albumin, the extracellular protein content per milliliter of the test samples was
determined. This process was repeated at different time
points (up to 72 hours) to analyze the production of extracellular proteins in the sample [111].
8.3.3 Analytical Parameters for Fixed Oils and Waxes
The physicochemical parameters IV, saponification value,
acid value, and peroxide value are essential measures used
in the analysis of fats and oils, providing crucial insights
into their composition and quality. These analytical methods provide vital insights into the composition, quality, and
stability of these natural substances. These parameters are
essential in quality control and formulation processes for
various industries, including food, cosmetics, and pharmaceuticals. By analyzing these values, manufacturers can
ensure the integrity and suitability of fixed oils and waxes
for specific applications [112].
Iodine Value
Principle The IV quantifies the grams of iodine that can
be absorbed by 100 grams of a substance. It indicates the
degree of unsaturation in oils and waxes. Oils with high IV
have more unsaturated fatty acids, making them easily
prone to oxidation. Waxes generally have lower IV due to
their higher saturation levels [113].
Method 1 gram sample was dissolved in 10 mL of chloro-
form and labeled as “test.” Then, 20 mL of iodine monochloride reagent was added to the test solution with
thorough mixing. Simultaneously, another sample, labeled
as “blank,” was prepared using the same procedure, excluding the fat sample. Both samples were kept in darkness for
about 30 minutes. After the incubation, 10 mL of potassium iodide solution was added to the test sample, and the
beaker sides were rinsed with 50 mL of distilled water. The
samples were titrated using a 0.1 normality aqueous solution of sodium thiosulfate (Na
) until the color
2S2O3
changed to pale straw. Subsequently, 1 mL of starch indicator was added, turning the solution purple. The titration
continued until the solution became colorless. The IV of
the fat sample was estimated using a specific formula,
applying the obtained titration data from this procedure
[114].
Iodine value = M
×N × (V
W
Blank−VTest
) × 100 × 10–3/W
Where,
MW = molecular weight of Na2S2O3, g/mol
= volume of Na2S2O3 for Blank sample, mL
V
Blank
= volume of Na2S2O3 for the Test sample, mL
V
Test
N = normality of Na
2S2O3
, mol/mL
WS = weight of sample, g
Saponification Value
Principle The saponification value indicates the quantity
of potassium hydroxide (in milligrams) needed to saponify
one gram of a substance. This value is utilized to ascertain
the average molecular weight of the fatty acids contained
in oils and waxes. Different oils and waxes have distinct
saponification values based on their fatty acid composition
[113].
S
156 8 Qualitative and Quantitative Methods of Phytochemical Analysis
Method A 2 g sample was precisely weighed and placed in
a volumetric flask. Subsequently, 25 mL of 1.0 N alcoholic
KOH was pipetted and allowed to drain for approximately
1 minute into the mixture. The flask was then attached to a
condenser, and the sample was gently boiled for 45
minutes to ensure complete saponification. After boiling,
the flask and condenser were cooled, taking care to
prevent gel formation. The inside of the condenser was
rinsed with approximately 1 mL of distilled water.
Following the condenser disconnection, 1 mL of
phenolphthalein indicator was added. The solution was
titrated with 0.5 N hydrochloric acid (HCl) until the
pink color just disappeared. Simultaneously, a blank
determination was conducted without the sample [114].
Saponification Value: 28.05
b − a)/w
(
Where, [115]
w = weight in grams of the solution b = burette reading
with sample a = burette reading without sample
Acid Value
Principle The acid value measures the quantity of potassium
hydroxide (in milligrams) required to neutralize the free
fatty acids in one gram of a substance. It indicates the extent
of hydrolysis and rancidity in oils and waxes. Higher acid
values suggest higher levels of free fatty acids, which can
result from improper storage or processing [113].
Method: In this experimental procedure, a solution was
prepared by combining 1.0 mL of 0.1 mM hydrogen peroxide (H2O2), 1.0 mL of a sample solution with a concentration of 10–100 µg/mL, 2 drops of 3% ammonium molybdate,
10 mL of 2 M sulfuric acid (H
), and 7.0 mL of 1.8 M
2
4
potassium iodide (KI) [117]. The resulting mixture was
then titrated with 5.09 mM sodium thiosulfate (Na
2S2O3
until the yellow color, indicative of iodine presence resulting from the reaction between H
and KI, completely
2O2
disappeared. This titration aimed to determine the quantity
of sodium thiosulfate required to neutralize the hydrogen
peroxide in the sample solution, a crucial step in evaluating
the sample’s properties or reactivity in the given experimental context. The hydrogen peroxide value was calculated as:
% Inhibition = V
− V 1/V 0 × 100
0
where,
= volume of sodium thiosulfate solution used to
V
0
titrate the control sample in the presence of hydrogen peroxide (without sample)
= volume of sodium thiosulfate solution used in the
V
1
presence of the sample
8.4 Analytical Techniques In Phytochemical Analysis
)
Method A mixture was prepared by combining 10 mL of
sample with 25 mL of ethanol and 25 mL of ether.
Phenolphthalein was introduced as an indicator, and the
mixture was titrated with a 0.1 M potassium hydroxide
solution. This process likely aimed to determine the
acidic components present in the oil by measuring the
volume of potassium hydroxide needed to neutralize the
mixture, as indicated by the phenolphthalein color
change [115].
Acid value = 5.61 n/w
Where,
n = Number of ml of 0.1M KOH w = Weight of sample
Peroxide Value
Principle Peroxide value quantifies the amount of peroxides
in a substance, indicating its level of oxidation. It is expressed
in milliequivalents of active oxygen per kilogram of the
substance. Elevated peroxide values suggest that the oil or
wax has undergone oxidation, which can lead to offflavors
and decreased shelf life [116].
Various analytical tools are used to determine the quantity
of phytoconstituents (plant-derived compounds) in plant
extracts or herbal products. These tools are essential for
assessing the composition and concentration of specific
phytoconstituents, which can have various applications in
fields such as pharmaceuticals, food, and herbal medicine
[118]. Some of the common analytical techniques used for
phytoconstituent quantification are highlighted in Table 8.2
and described below:
Chromatography: Chromatography is a common
method for analyzing phytochemicals. It makes it possible to recognize and separate the many compounds
present in plant extracts. For instance, Razgonova
et al. conducted a comparative metabolomic analysis
of wheat grains using HPLC and MS. Saha and Saha
used tandem MS, electrospray ionization, and liquid
chromatography to analyze the chemical components
of the pericarp of Acacia auriculiformis [119]. Al
Bratty et al. investigated the phytochemical composition of Eruca sativa leaves using gas chromatographymass spectrometry (GC-MS). For the simultaneous
quantification of marker analytes in the traditional
herbal remedy Sogunjung-tang, Seo and Shin created
8.4 Analytical Techniques In Phytochemical Analysis 157
Table 8.2 Analytical techniques in the phytochemical analysis of herbal drugs.
Analytical technique Principle / Application Examples of phytochemical analysis References
Chromatography Separation based on
Mass spectrometry (MS) Identifies compounds by
Spectroscopy Measures the interaction
Mass spectrometry
imaging (MSI)
Electrophoresis Separates charged
Spectrophotometry Measures the absorbance
Atomic absorption
Spectroscopy (AAS)
Nuclear magnetic
resonance imaging (MRI)
Enzyme-linked
immunosorbent assay
(ELISA)
X-ray diffraction (XRD) Identifies the crystalline
chemical properties.
their mass-to-charge ratio.
of molecules with
electromagnetic radiation.
Maps the spatial
distribution of
compounds within
tissues.
molecules based on
electrophoretic mobility.
or emission of light by
compounds.
Quantifies metal content
in samples.
Visualizes internal plant
structures.
Quantifies specific
proteins or enzymes.
structure of compounds.
High-performance Liquid Chromatography
(HPLC): Quantification of polyphenols in green tea.
Gas Chromatography (GC): Analysis of essential oil
constituents in lavender.
Thin-Layer Chromatography (TLC): Separation of
flavonoids in citrus fruits.
Gas Chromatography-Mass Spectrometry
(GC-MS): Identification of terpenoids in cannabis.
Liquid Chromatography-Mass Spectrometry
(LC-MS): Quantification of alkaloids in medicinal
plants.
UV-Visible Spectroscopy: Determination of
chlorophyll content in plant leaves.
Infrared Spectroscopy (IR): Identification of
functional groups in organic compounds.
Nuclear Magnetic Resonance (NMR)
Spectroscopy: Structural elucidation of plant
alkaloids.
Mapping the distribution of flavonoids in plant leaves.
Localization of alkaloids in plant roots.
Capillary Electrophoresis (CE): Analysis of ions in
plant extracts.
Polyacrylamide Gel Electrophoresis (PAGE):
Separation of plant proteins.
Quantification of anthocyanins in berries using UV-Vis
spectroscopy.
Quantifying the total phenolic content in plant extracts
Measurement of iron content in spinach leaves.
Assessment of mineral content in herbal supplements.
Imaging the water distribution in plant roots.
Visualization of phytochemical distribution in plant
tissues.
Detection and quantification of plant allergens in food
products.
Measurement of cytokine levels in plant-based
medicinal extracts.
Determination of mineral composition in plant
samples.
Analysis of crystallographic properties of plant-derived
compounds.
[136]
[137]
[138]
[139]
[140]
[141]
[142]
[143]
[144]
[145]
an HPLC approach [120]. Nayaka et al. used GC-MS
analysis to identify bioactive compounds in
Caulerpapeltata seaweed. These illustrations show
the value of chromatography for the identification
and quantification of chemicals in plant extracts
during phytochemical analysis [121].
High-performance Liquid Chromatography: One of
the most used methods for phytochemical analysis is
this one. It allows for the measurement of individual
molecules within a complicated mixture and separates
compounds according to their chemical properties
[122].
Gas Chromatography: Essential oils and other volatile
phytochemicals can be examined using GC, which separates substances depending on their vapor pressure and
volatility [123].
158 8 Qualitative and Quantitative Methods of Phytochemical Analysis
Thin-layer Chromatography: On a thin layer of adsor-
bent material, thin-layer chromatography (TLC) is an
easy and affordable approach for separating and visual-
izing phytochemical substances [124].
Mass Spectrometry: In phytochemical analysis, MS is a
potent tool for identifying and characterizing the chemi-
cal components of plant extracts. Among the many sub-
stances that can be detected and quantified using this
method are phenolics, alkaloids, flavonoids, and terpe-
noids. Numerous bioactive chemicals have been found
in numerous plant species thanks to the use of MS in
phytochemical investigation. This method helps to
understand the chemical makeup of plant extracts,
revealing significant information about their prospective
health benefits and therapeutic qualities [125]. HPLC
and other analytical techniques like MS can be employed
to separate and identify specific substances in complex
mixtures. MS can analyze molecules’ mass-to-charge
ratios and fragmentation patterns to reveal extensive
structural details about the chemicals found in plant
extracts. Overall, MS is extremely important in the field
of phytochemical analysis since it makes it possible to
identify and find bioactive chemicals in plants [126].
Gas Chromatography-Mass Spectrometry: The identifi-
cation of volatile substances in complicated combina-
tions is possible when GC and MS are used together.
Liquid Chromatography-Mass Spectrometry: Numerous
phytochemicals, including non-volatile substances, are
identified and quantified using LC-MS [127].
Spectroscopy: A useful tool for analyzing phytochemicals
is spectroscopy. It makes it possible to recognize and
classify the numerous phytochemicals found in plant
extracts. In order to identify the presence of particular
substances based on their distinctive absorption peaks,
UV-VIS spectroscopy is frequently performed. Further
evidence of the presence of phytochemicals is provided
by the identification of functional groups in the extract
using FTIR spectroscopy. It is also done using GC-MS
analysis and X-ray diffraction (XRD) spectroscopy to
study the phytochemical profile of plants. These tech-
niques help in the identification of bioactive chemicals
such as alkaloids, flavonoids, tannins, saponins, and ter-
penoids that have a variety of medical effects. In addi-
tion, spectroscopic techniques like GC-MS can reveal
details about the chemical makeup of plant extracts,
assisting in the creation of new pharmaceuticals.
Overall, spectroscopy is essential for analyzing and com-
prehending plant phytochemicals, advancing the fields
of herbal medicine and medication discovery [128].
UV-visible Spectroscopy: When measuring phytochemi-
cals, chromophores that absorb UV or visible light are
helpful.
Infrared Spectroscopy: Information on the functional
groups of phytochemical substances is available from IR
spectroscopy.
Nuclear Magnetic Resonance Spectroscopy: Under-
standing a compound’s chemical structure requires the
use of NMR spectroscopy.
Mass Spectrometry Imaging (MSI): The measurement of
phytochemicals in plants is done with the help of the
potent technique known as mass spectrometry imaging
(MSI). Without the requirement for intricate chemical
labeling, it enables the mapping of the spatial distribution
of metabolites within plant tissues. Different sample introduction methods are available for MSI; however, the two
that are most frequently used are matrix-assisted laser desorption/ionization mass spectrometry (MALDI) and desorption electrospray ionization mass spectrometry (DESI).
MALDI In order to find substances with potential uses in
cosmetics, such as polyphenols, tannins, kaempferol, isorhamnetin, and fatty acids, MSI was used to examine the
phytochemical content and chemical spatial distribution
of Gliricidiasepium leaves. Additionally, MSI has been
applied to examine fructooligosaccharide (FOS) content
and location in various barley lines, revealing that the
absence of a crucial enzyme results in an increase in
fructan content in the vacuole. Overall, MSI is a useful
technique for figuring out how phytochemicals are distributed in plants and how they might be used. MSI makes it
possible to map the spatial distribution of phytochemicals
within plant tissues, giving information about where bioactive substances are found [129].
Electrophoresis: CE, or capillary electrophoresis, is a
method for analyzing phytochemicals. It has been used
in many different aspects of phytochemical analysis,
such as sample pretreatment, technique development,
and medicinal plant quality assessment. Different pharmaceutical forms and plant materials have been subjected to CE for the separation and detection of substances
including tropane alkaloids, hyoscyamine, and scopolamine. Additionally, CE has been utilized to isolate and
recognize active substances from plants and other natural resources, advancing phytochemistry and phytopharmacology. Additionally, the phytochemical profile of
sawdust from Albizia falcataria was analyzed using CE,
which allowed for the detection of a number of substances, including alkaloids, anthraquinones, flavonoids,
saponins, steroids, tannins, and triterpenoids. In conclusion, CE offers a useful technique for the separation,
identification, and characterization of phytochemicals,
playing a vital role in the analysis of such substances
[130].
Capillary Electrophoresis: Based on their electrophoretic
mobility, charged phytochemicals are separated by CE,
8.5 Conclusion 159
which is frequently employed for the study of tiny ions
and polar substances.
Polyacrylamide Gel Electrophoresis: Proteins and
enzymes in plant extracts are separated and analyzed
using polyacrylamide gel electrophoresis (PAGE).
Atomic Absorption Spectroscopy: For the purpose of
evaluating the nutritional value of plant-based foods,
AAS is used to quantify the metal and mineral content in
plants. To find the presence and concentration of trace
elements in plant samples, phytochemical analysis uses
the technique of atomic absorption spectroscopy (AAS).
Using a spectrophotometer, this method measures how
much light at particular wavelengths is absorbed by the
target substances. AAS has been used in several studies
to examine the elemental makeup of different plant
components, including leaves, stems, and roots. For
example, Ajiwe Vincent et al. analyzed Amaranthus spinosus and detected elements like cobalt, lead, manganese, nickel, copper, cadmium, magnesium, iron,
chromium, and calcium. In a similar vein, alkaloids, flavonoids, saponins, tannins, terpenoids, proteins, acidic
chemicals, reducing sugar, and carbohydrates were all
discovered to be present in Acanthus montanus by Ikezu
et al. 2022 [131]. Other studies have also used AAS to
assess the safety of plant-derived feed for animals, ensuring that the levels of toxic elements like mercury, cadmium, lead, arsenic, and selenium are within acceptable
limits. Overall, AAS is a valuable tool in phytochemical
analysis for determining the elemental composition of
plant samples [132].
Nuclear Magnetic Resonance Imaging: Magnetic
Resonance Imaging (MRI) can be used to visualize the
internal structure and composition of plant tissues,
which can provide insights into the distribution of
phytochemicals. NMR spectroscopy has been widely
used in the phytochemical analysis, particularly in the
identification and structural elucidation of isolated
phytochemicals. NMR spectroscopy provides a fast,
reproducible, and nondestructive technique for analyzing compounds in biological samples, including
plants and their derived medicines. It has also been
applied in the analysis of ginseng extracts, providing
novel data in the context of metabolomics. NMR spectroscopy is still largely underutilized in the field of
food research, mostly because it is expensive, sensitive, and requires specialized knowledge. NMR
approaches have nevertheless been developed for food
analysis, including quality monitoring, authentication, and differentiation of meals based on distinct raw
components and origin. Furthermore, the application
of NMR in conjunction with multivariate statistical
analysis has been demonstrated to be a potent strategy
for overcoming contemporary issues in food quality
assurance and authentication [133].
Enzyme-linked Immunosorbent Assay: Enzyme-
linked immunosorbent assay (ELISA) is used for the
quantification of specific proteins or enzymes within
plant extracts. The ELISA is a popular method for analyzing phytochemicals. It is a specific and straightforward assay for detecting biomolecules in plants. ELISA
has been optimized to improve its sensitivity, and different types of ELISA assays are available for detecting various plant secondary metabolites. ELISA is an important
analytical tool for food authentication and control, as it
offers sensitivity, selectivity, and working capacity. It is
commonly used for species authentication and the
detection of quality-related substances in raw and processed food. ELISA is also used for the quantification of
peptides, proteins, antibodies, and hormones in plants.
It is based on the principle of antigen-antibody binding
and allows for the quantification of these biomolecules
through the formation of an antigen-antibody bond
complex. Additionally, ELISA kits have been developed
for phytochemical analysis, providing a convenient and
accurate method for researchers [134].
X-ray Diffraction: XRD helps in identifying the crystal-
line structures of compounds in plants, particularly
minerals and crystalline phytochemicals. XRDis a
method used in phytochemical analysis to determine the
crystal structure of compounds present in plant samples.
It involves irradiating the sample with X-rays and analyzing the diffraction pattern produced. XRD spectroscopy (XRD) and GC-MS analysis are commonly used
techniques for phytochemical profiling of plants. XRD
analysis can be used to identify the crystal phase contained in a sample based on the diffraction angle of the
diffraction spots. Transmission mode XRD analysis is a
specific method that involves placing the sample on a
substrate and detecting X-ray radiation transmitted
through and diffracted by the sample. XRD has been
instrumental in determining the structures of biomolecules such as myoglobin and hemoglobin, leading to
major advances in biology and biological physics [135].
8.5 Conclusion
In the realm of phytochemical analysis, both qualitative and
quantitative methods play indispensable roles in uncovering
the intricate chemical compositions of plant materials.
Through qualitative methods, researchers can identify the
presence or absence of various phytochemical constituents,
offering valuable insights into the diversity of compounds
160 8 Qualitative and Quantitative Methods of Phytochemical Analysis
within plant samples. Techniques such as chromatography
and spectrophotometry empower scientists to explore the
richness of secondary metabolites, including alkaloids, flavonoids, terpenoids, and phenolic compounds. These methods serve as the initial gateway, unveiling the hidden
treasures of plant chemistry and paving the way for in-depth
investigations. Qualitative tests, such as chromatographic
techniques, including thin layer chromatography (TLC) and
GC-MS, unveil the presence of compounds like alkaloids
and flavonoids. Spectrophotometric analysis, especially
through UV-visible spectroscopy, aids in identifying bioactive components like phenolic acids. On the other hand,
quantitative methods provide the essential numerical data
required for a deeper understanding of phytochemical profiles. Through meticulous calibration and precise measurements, quantitative analyses quantify the specific
concentrations of these phytochemicals, allowing for comparisons across different species, varieties, or environmental
conditions. These quantitative methods, often involving
titrations, MS, or HPLC, are invaluable for evaluating the
therapeutic potential, nutritional value, and ecological significance of plant materials. Additionally, they enable scientists to monitor variations in phytochemical content due to
factors like cultivation techniques, geographical origins, or
climate changes. Additionally, colorimetric assays like the
Folin-Ciocalteu method offer quantitative estimations of
total phenolic content. By integrating these qualitative and
quantitative methodologies, scientists gain a profound
understanding of plant biochemistry, enabling the exploration of plant-derived compounds for diverse scientific applications. This harmonious amalgamation of techniques
forms the cornerstone of phytochemical analysis, enriching
our knowledge and unlocking the potential of nature and
bioactive treasures.
In conclusion, the synergy between qualitative and
quantitative methods in phytochemical analysis forms the
foundation of our knowledge about the bioactive compounds present in plants. Qualitative methods provide a
qualitative understanding, allowing researchers to identify
the diverse array of phytochemicals, while quantitative
methods offer precision, enabling the quantification of
these compounds. This comprehensive approach not only
enriches our understanding of plant biology but also holds
immense implications for fields ranging from pharmacology and medicine to agriculture and environmental
science.
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