18_chapter 10.pdf - Shodhganga
Transcript of 18_chapter 10.pdf - Shodhganga
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 240
10.1. INTRODUCTION
10.1.1 Plant profile of Phyllanthus niruri
Phyllanthus is a large genus of shrubs, trees and rare herbs of the family
Euphorbiaceae, comprising more than 600 species, of which P. accuminatus,
P. amarus, P. pulcher, P. niruroides, P. anisolobus, P. orbiculatus, P.
emblica, P. oxyphyllus, P. flexuosus, P. raticulatus, P. fraternes, P. simplex, P.
mullernus, P. urinaria, P. mytrifolis, P. virgatus, P. niruri and P. watsonii
were investigated for their phytochemical and pharmacological properties.
The genus is found in almost over all warmer parts of the world.
Phyllanthus niruri 331
(a) Classification:
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliophyta
Order: Malpiales
Family: Phyllanthaceae
Genus: Phyllanthus
Species: Niruri
(b) Vernacular names:
Sanskrit: Bhumyamlaki
Hindi : Jar amla or Jangli amla
Kannada : Nela nelli,
Malayalam : Keelar nelli
Tamil : Kizhanelli
Marathi: Bhuiamla
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Other common names: Stonebreaker and Seed-Under-Leaf (English), Chanca
Piedra (Spanish) and Quebra Pedra (Portuguese) 332
.
(c) Part used: Whole plant
(d) Botanical description: Phyllanthus niruri is an erect annual herb, 10 to
50 cm high and is indigenous to the Amazon rainforest and other tropical
areas, including South East Asia, Southern India and China. It has smooth
cylindrical stem 1.5 to 2 mm thick and deciduous horizontal branchlets 4 to
12 cm long and about 0.5 cm thick, with 15 to 30 leaves. The leaves are
alternate, elliptic, oblong or obovate, 5 to 11 mm long and 3 to 6 mm wide,
rounded to slightly pointed at the tip, scarcely oblique on one side at the base,
petioles 0.3 to 0.5 mm long,. It has small off-white-greenish flowers, which
are solitary, auxiliary, pedicellate, apetalous and monoecious.The flowers are
alone or usually one male and one (larger) female are in each leaf axil
together.
The seed capsules on stalks are 1 to 2 mm long, round, smooth, 2 mm wide,
with 6 seeds. When the fruits burst open the seeds are hurled away. Seeds are
triangular (like an orange segment), light brown, 1 mm long, with 5 to 6 ribs
on the back 2.
P. amarus and P. sellowianus are closely related to P. niruri in appearance,
phytochemical contents and history, but they are found in drier regions of
India and Brazil, and even in Florida and Texas. In a recent report, cladistic
analysis indicated that the Phyllanthus genus is paraphyletic and therefore the
two problematic and confusing species, P. niruri and P. amarus, are two
individual species.
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(e) Origin and distribution: Phyllanthus niruri is widely distributed in all
tropical regions of the planet. Paleobotanical studies have not found the exact
geographic origin of this plant. This plant may be indigenous to the tropical
Americas, the Philippines or India.
Plants in the genus Phyllanthus can be found around all tropical regions of the
world: from Africa to Asia, South America and the West Indies. P. niruri can
be found in all the tropical regions of the world: through the roads, valleys, on
the riverbanks and near lakes. This plant is a common arable weed of
disturbed ground in Southern Florida, the Bahamas, the West Indies and
tropical America and is naturalized in the Old World tropics.
(f) Anatomy of the Plant333
:
Leaf : Epidermal walls wavy, stomata anisocytic, which is distributed mainly
on the lower epidermis. Upper epidermis has a thin cuticle. Stomata are
followed by respiratory cavities beneath.There is a single layer of palisade
cells, which occupy nearly half of the space between the two epidermis.
Below the palisade there is a row of broad collecting cells, each of which
occur in relation to 3 or 4 palisade cells. Reduced vascular elements are
clearly seen running on long stretch beneath the collecting cells. The palisade
ratio has been determined to vary between 13 and 17.
Branchlet :- Rounded in transverse section, cortex 6-8 cells thick most of the
cells contain chloroplast and few druses crystals. After 3 -4 rows, there is a
row of cells containing starch grains followed by 2-3 layers of fiber cells
which are interrupted by cortex parenchyma. Phloem 5 - 7 cell thick, xylem
vessels 8 - 33 mm in diameter, pith cells contain chloroplasts.
Stem :- Epidermal cells and some of the cortical cells contain tannin, cortex
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about 15 cells thick, some contain calcium oxalate druse crystals, inner cortex
contains groups of 7 -10 thick walled cells interrupted at regular intervals by
parenchyma cells on the outer side of the group of thick walled cells there is a
row of parenchymatous cells containing starch grains. Phloem 7 - 10 cells
thick, thin walled, without any contents. Xylem vessels 16 -54 mm in
diameter, pith cells thin walled may contain a few druse like crystals.
Root :- Cork cells 6 - 8 cells thick, contain dark brown tannin, cortex 10 - 15
cells thick, some filled with tannin and some with starch, phloem 4 - 6 cells
thick, xylem vessels 12 - 53 mm in diameter.
(g) Traditional uses333
: In many countries around the world, plants in the
genus Phyllanthus are used in folk remedies; therefore this genus is of great
importance in traditional medicine. P. niruri has a long history in herbal
medicine systems such as Indian Ayurveda, Traditional Chinese Medicine and
Indonesian Jamu.
The genus Phyllanthus has a long history of use in the treatment of liver,
kidney and bladder problems, diabetes and intestinal parasites. Some related
species in this region with medicinal significance are P. epiphyllanthus, P.
amarus, P. urinaria, P. acuminatus, P. emblica. P. niruri, P. amarus and P.
urinaria are used in the treatment for kidney/gallstones, other kidney related
problems, appendix inflammation, and prostate problems. The whole plant is
used as remedies for many conditions such as dysentery, influenza, vaginitis,
tumours, diabetes, diuretics, jaundice, kidney stones and dyspepsia. The plant
is also useful for treating hepatotoxicity, hepatitis B, hyperglycaemia and viral
and bacterial diseases.333
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In India, where it is called Pitirishi or Budhatri, it is a common household
remedy for asthma, bronchitis, cough, extreme thirst, anaemia, liver diseases,
jaundice, tuberculosis and cardiovascular problems. P. niruri has been used in
Ayurvedic medicine for over 2000 years and has a wide number of traditional
uses for jaundice, gonorrhoea, frequent menstruation and diabetes.
It is an important medicinal plant in Jamu (traditional medicine in Indonesia),
a well-known Indonesian traditional herbal medicine to treat various diseases.
In Jamu preparations, the plant is used as antiviral and hepatoprotective agent.
In Malaysia, P. niruri is known as Dukong anak. It is used internally for
diarrhoea, kidney disorders, gonorrhoea and cough. 331,333
This plant is traditionally used around the world in the treatment of liver
ailments and kidney stones. The Spanish name ‘chanca piedra’ means “stone
breaker or shatter stone.” In South America, ‘chanca piedra’ has been used to
eliminate gall bladder and kidney stones, and to treat gall bladder
infections334
.
In Brazilian herbal medicine, it is called “Quebra Pedra” and is considered an
excellent remedy for hydropsy, urinary and bladder infections. It is also used
to cure kidney disorders, hepatitis and diabetes. 335-337
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(h) Pharmacology and Clinical Studies334-336
:
I. Hepatoprotective Effect –
Hepatitis B is one of the major diseases inflicting human population.
Alternative herbal medicine using extracts of Phyllanthus niruri has been
reported to be effective against hepatitis B and other viral infections. A study
reports quantitative determination of the anti viral effect of these herbs in
well-defined in vitro systems338
.
In one study, 37 patients with chronic viral hepatitis B were treated with a
daily dose of 600 mg of Phyllanthus niruri for 30 days. HBsAg had lost in 59 %
of the patients with in the two weeks after the end of the treatment.
Furthermore, none of the cases when followed for up to 9 months had any
symptoms of HBsAg339-342
.
II. HIV Replication Inhibition–
Aqueous extract of Phyllanthus niruri is reported to have inhibitory effect on
human immunodeficiency virus. The investigation examines the anti-HIV
Fig 10.1: Young plant of Phyllanthus niruri
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effects of the alkaloidal extract of Phyllanthus niruri in human cell lines. The
inhibitory effect on HIV replication was monitored in terms of inhibition of
virus induced cytopathogenecity in MT-4 cells. The alkaloidal extract of
Phyllanthus niruri showed suppressing activity on strains of HIV-1 cells
cultured on MT-4 cell lines. The alkaloidal extract of Phyllanthus niruri was
thus found to exhibit sensitive inhibitory response on cytopathic effects
induced by both the strains of human immunodeficiency virus on human MT-
4 cells in the tested concentrations. 343, 344
III. Lipid Lowering Activity -
Lipid lowering activity of Phyllanthus niruri alcoholic extracts in triton
induced hyperlipidaemia was examined in rats. In an experiment with
cholesterol fed rats, Phyllanthus niruri at a dose of 100 mg/kg lowered the
elevated level of low-density lipoprotein lipids in hyperlipidemic and drug fed
animals.345
IV. Antidiabetic Activity –
An alcoholic extract of Phyllanthus niruri was found to significantly reduce
the blood sugar in normal rats and in alloxan diabetes rats. In normal rats,
administration of Phyllanthus niruri 200 mg/kg body weight reduced the
blood sugar by 34.5 percent and to 47.4 percent at the concentration of 1000
mg/kg by weight within 1 hour. The results indicate potential antidiabetic
action of Phyllanthus niruri.346
V. Antimalarial Activity –
The ethanolic, dichloromethane and lyophilized aqueous extracts of whole
plants of Phyllanthus niruri were evaluated for their antimalarial activity in
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vivo, in 4-day, suppressive assays against Plasmodium berghei ANKA in
mice. No toxic effect or mortality was observed in treated mice, orally, with
any of the extracts as a single dose, of 500 mg/kg body weight, or as the same
dose given twice weekly for 4 weeks (to give a total dose of 4 g/kg). The most
active ethanolic extract, that of Phyllanthus niruri, reduced parasitaemia by
73%347, 348
.
VI. Antispasmodic activity –
Research done in Brazil at the Federal University of Santa Catarina in 1984
on Phyllanthus niruri revealed an alkaloid (phyllanthoside) in the leaves and
stem with strong antispasmodic activity. It served as a relaxing agent for
smooth muscles and they concluded that its spasmolytic action probably
accounted for the efficacy of Phyllanthus niruri in expelling stones.349
VII. Analgesic activity –
Methanol extract of dried callus tissue of P. niruri at a concentration of 10
mg/kg, administered intraperitonially to mice was active vs. acetic acid
induced writhing and vs. formalin – induced pedal edema showed a good
analgesic activity. 350, 351
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(i) Phytochemistry:
Table 10.1: Phytoconstutients of Phyllanthus niruri with their
pharmacological effects352-424
Sr.
No
Chemical
constituents
Parts of plant Pharmacological effects
1.
Flavonoids:
a. Rutin Whole plant Antioxidant
b. Quercetin Leaf, Whole
plant
Anti-aggregant, Anticancer,
Antifungal, Anti-
inflammatory, Antispectic
Antioxidant
c. Quercitrin Leaf Antidiarrhoel activity, Anti
leishmanial,
antinociceptive, Anti
inflammatory
d. Astragalin Leaf Diuretic, Anti inflammatory
e. Catechin Root culture Anti tumor
f.Prenylated
flavanone glycoside
Whole plant Antioxidant
g. Nirurin Whole plant Antioxidant
h. Niruriflavone Antioxidant
2.
Terpenes
a. Limonene Anticarcinogenic
b. p- Cymene Leaf Antioxidant, antimicrobial
c.Lupeol, Lupeol
acetate
Root culture Anti inflammatory, Anti
tumor
3.
Coumarins
a.Ellagic acid Whole plant Anti carcinogenic and Anti
viral
b.Methyl
brevifolincarboxylate
Vaso relaxant effect
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4. Lignans
a.Phyllanthin
Leaf and aerial
parts
Hepatoprotective and Anti
genotoxic , Anti viral
activity
b. Hypophyllanthin Whole plant Hepatoprotective, Anti
genotoxic
c. Niranthin , Hydroxy
niranthin ,
Demethylenedioxy
niranthin
Leaf
Anti-inflammatory,
Hepatoprotective
d. Phylltetralin Leaf Anti inflammatory
e. Nirtetralin Leaf
Anti inflammatory,
Hepatoprotective
f. Isolintetralin and
Lintetralin
Leaf Anti tumor activity
5.
Tannins
a. Repandusinic acid Whole plant Anti- HIV activity
b. Geranin Whole plant Anti-nociceptive activity
c. Corilagin Whole plant Inhibits Plasminogen –
activator- inhibitor-I,
antifungal
6.
Saponins
a. Diosgenin Whole plant Anti fungal and
cardiovascular activity
7.
Alkaloids
a. Norsecurinine Whole plant Strong anti spasmodic
activity
b. Nirurine Aerial
c. Phyllanthine Leaf , root
culture, stem
d. Phyllochrysine Leaf, stem
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8.
Other compounds
a. β – glucogallin Whole plant Effective in haemolytic
disease
b.1-O-galloyl-6-O-
luteoyl-α- D-glucose
Whole plant Effective in haemolytic
disease
Linear and complex
hetero xylans
Whole plant Immunomodulators and
Anti- tissive
a. Niruriside Whole plant Anti-HIV activity
9.
Hydrocarbons
a. Triacontanal Aerial Hepatoprotective
b. Tricontanol Aerial
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10.1.2 Kaempferol, Rutin and Quercetin as marker compound for
Standardization
Flavonoids are polyphenolic compounds that occur ubiquitously in foods of
plant origin. They are extremely important because of their health effects. It
has been predicted that average intake of all flavonoids is several grams per
day. They occur in foods as O-glycosides with sugars bound at the C3
position.
Flavonoids with a diphenylpropane skeleton (C6–C
3–C
6) are known to be
antimutagenic and anticarcinogenic. They also have antioxidant properties
and inhibit the oxidation of LDL. They also have anti-inflammatory and anti-
allergic effects. Flavonoids consist mainly of flavonols, flavones, catechins
and flavanones.
Kaempferol is an example flavonol. It is a strong antioxidant and helps to
prevent oxidative damage of our cells, lipids and DNA. Kaempferol seems to
prevent arteriosclerosis by inhibiting the oxidation of low density lipoprotein
and the formation of platelets in the blood. Studies have also confirmed that
kaempferol acts as a chemopreventive agent, which means that it inhibits the
formation of cancer cells.
Rutin is a member of bioflavonoids, a large group of phenolic secondary
metabolites of plants that include more than 2,000 different known chemicals.
Rutin has ability to strengthen and modulate the permeability of the walls of
the blood vessels including capillaries. Rutin is known to offer nutritional
support to the circulatory systems including the capillaries in eyes. It has
proved to be especially helpful in preventing recurrent bleeding caused by
weakened blood vessels, and has been used in treatment of hemorrhoids and
Simultaneous estimation of kaempferol, rutin and quercetin in
Standardization of Some Plant
varicose veins, helping to prevent blood vessel walls to become fragile. Rutin
is safe and effective for poor circulation, high blood pressure
Quercetin has a common flavone nucleus composed of two benzene rings
linked through a heterocyclic pyrone ring.
scavenge damaging particles in the body known as free radicals, which
damage cell membranes,
can neutralize free radicals and may reduce or even help to prevent some of
the damage they cause. Quercetin also acts like an antihistamine and an anti
inflammatory.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques
varicose veins, helping to prevent blood vessel walls to become fragile. Rutin
is safe and effective for poor circulation, high blood pressure, chilblains, etc.
Quercetin has a common flavone nucleus composed of two benzene rings
linked through a heterocyclic pyrone ring. Quercetin is antioxidants. They
scavenge damaging particles in the body known as free radicals, which
damage cell membranes, tamper with DNA, and even cause cell death. Thus,
can neutralize free radicals and may reduce or even help to prevent some of
the damage they cause. Quercetin also acts like an antihistamine and an anti
inflammatory.
Fig.10. 2:Structure of Kaempferol
herbal products 10
Based Formulations By Modern Analytical Techniques 252
varicose veins, helping to prevent blood vessel walls to become fragile. Rutin
, chilblains, etc.
Quercetin has a common flavone nucleus composed of two benzene rings
Quercetin is antioxidants. They
scavenge damaging particles in the body known as free radicals, which
tamper with DNA, and even cause cell death. Thus,
can neutralize free radicals and may reduce or even help to prevent some of
the damage they cause. Quercetin also acts like an antihistamine and an anti-
Kaempferol
Simultaneous estimation of kaempferol, rutin and quercetin in
Standardization of Some Plant
These three compounds are pharmacologically and biologically very active.
Thus, they can be considered as biomarkers. These compounds are also found
in many plants together. Hence, a single method which can determine the
content of these markers simultaneo
manufacturer and analysts of plant based formulations.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques
Fig.10. 3: Structure of Rutin
Fig.10. 4: Structure of Quercetin
These three compounds are pharmacologically and biologically very active.
Thus, they can be considered as biomarkers. These compounds are also found
in many plants together. Hence, a single method which can determine the
content of these markers simultaneously will be a very beneficial tool for the
manufacturer and analysts of plant based formulations.
herbal products 10
Based Formulations By Modern Analytical Techniques 253
These three compounds are pharmacologically and biologically very active.
Thus, they can be considered as biomarkers. These compounds are also found
in many plants together. Hence, a single method which can determine the
usly will be a very beneficial tool for the
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
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Thus, the objective of the present work was to develop and validate a
simultaneous HPTLC method for estimation of Kaempferol, Rutin and
Quercetin in various herbal formulations. We even aim at to use this
method for estimation of any of the three markers individually. Thus,
this method will show wider applicability in analysis of many different
plant-based formulations with three different biomarkers.
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10.2 ISOLATION OF MARKER COMPOUNDS
10.2.1 Procurement of dried whole plant of Phyllanthus niruri
The dried whole plant of Phyllanthus niruri was supplied by Amsar Pvt. Ltd.
10.2.2 Preparation of extracts
Approximately 100 g of air-dried and powered whole plant of P. niruri was
successively extracted in a soxhlet extraction apparatus with 1000 ml of each
of hexane, chloroform, butanol and water for 18 hours. The respective
extracts were dried and the nature of the extracts and percentage yield was
recorded (Table 10.2).
Table10. 2: Different extracts of P. niruri by successive extraction
Extracts Nature Yield (g/100 g of
crude drug)
% Yield
Hexane Dark greenish sticky
solid
1.68 1.68
Chloroform Greenish solid 1.59 1.59
Butanol Brownish solid 7.83 7.83
Aqueous Brownish solid 12.07 12.07
10.2.3 Isolation of Kaempferol, Rutin and Quercetin
Kaempferol, rutin and quercetin are some of the major flavonoids present in
P.niruri and these can be used as marker compounds for standardization of
formulations containing P.niruri or any other plant based formulations
containing these compounds.
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10.2.3.1 Isolation of Kaempferol and Quercetin
a) Preparation of column:
500 gm of silica gel (60-120 mesh) was activated for 30 min at 105 ºC. Slurry
of silica gel was prepared in 1000 ml petroleum ether (60-80 °C) and was
transferred to glass column; precaution was taken to avoid air entrapment.
The length of the column was approximately kept to 110 cm.
b) Preparation of sample:
The chloroform extract was adsorbed on silica and loaded on the column in
form of thin band. Petroleum ether was added slowly to the column,
precaution was taken to avoid air entrapment.
c) Elution
Following combinations of solvents were used to elute the column. 100 ml of
each mobile phase was used at a time to run the column.
Table 10.3: Pattern of column chromatographic elution for Kaempferol
and Quercetin
Sr.
No
Solvents composition
(%)
TLC studies of eluents
with mobile phase CHCl3 :
MeOH: Formic acid
(9:1:0.5)
Inference
Pet.
Ether
Chloroform
1. 100 0 No spot --
2. 90 10 No spot --
3. 80 20 No spot ---
4. 70 30 There were three spots very
close to each other with Rf
range from (0.95 to 0.8.1)
with a blackish blue color
after spraying with 10 %
alcoholic sulphuric acid.
The spots
where difficult
to separate.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 257
5. 60 40 No spot ---
6. 50 50 One single spot was
observed with Rf value 0.81
with a blackish blue color
after spraying with 10 %
alcoholic sulphuric acid.
The amount of
obtained
compound was
too less to be
considered.
7. 40 60 No spot ---
8. 30 70 No spot ---
9. 20 80 One single spot detected at
254 nm with Rf 0.77
Sticky dark
yellow
compound
was obtained
(Component
V)
10. 10 90
11. 0 100 No spot ---
Chlor
oform
Methanol
12. 90 10 No spot ---
13. 80 20 No spot ---
14. 70. 30 One Spot detected at 254
nm with Rf 0.70
The compound
was oily in
nature.
15. 60 40
16. 50 50 One single spot detected at
254 nm with Rf 0.64
Sticky yellow
compound
was obtained
(Component
VI)
17. 40 60 The same compound was
detected
The compound
was very less
quantity.
50 ml aliquots were collected, concentrated and subjected to TLC studies. 9th
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Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 258
and 10th
fractions [eluted with Petroleum ether: Chloroform (60:40), (50:50)]
gave an intense spot at a dark brown sticky mass Rf 0.81 under UV lamp
under 254 nm (Fig.10.5) with mobile phase chloroform: methanol: formic
acid (9:1:0.5) (v/v). 15th
and 16th
fractions [eluted with chloroform: methanol
(60:40), (50:50)] gave an intense spot at a dark brown sticky mass Rf 0.64
under UV lamp under 254 nm (Fig.10.5) with same mobile phase. Both the
fractions were washed successively with excess amount of petroleum ether
(60 – 80°C) to remove the sticky matter and recrystallized from diluted
alcohol to obtain a dark yellow crystalline powder (component V) and light
yellow crystalline powder (component VI). The yield of component V and VI
obtained was 0.170% w/w and 0.151%w/w.
10.2.3.2 Isolation of Rutin
Butanol extract of P.niruri was used for the isolation of further marker
compounds. Column chromatographic technique was used. The above
mentioned procedure was followed. The butanol extract (700 mg) was
dissolved in methanol and adsorbed on silica gel for loading on column. The
column and extract slurry was prepared in chloroform. The column was eluted
successively with chloroform and methanol in order of increment of polarity.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 259
Table 10.4: Pattern of column chromatographic elution for Rutin
Sr.
No
Solvents composition
(%)
TLC studies of
eluents
with mobile phase
CHCl3 : MeOH:
Formic acid (8:2:0.5)
Inference
Chloroform Methanol
1. 100 0 No spot --
2. 90 10 There were two spots
with Rf values 0.83 and
0.78 with blue color
after spraying with 10
% alcoholic sulphuric
acid.
One more spot at Rf
0.75 gave a blackish
blue color with 10 %
alcoholic sulphuric
acid.
The spots
were very
light in color
and total
amount was
too less to
be
considered.
3. 80 20 One Spot detected
under UV lamp at 254
nm with Rf 0.71 and
another been was
observed at Rf 0.68 at
366 nm.
This fraction
was very
sticky and in
less amount.
4. 70 30 There was a violet
colored spot at Rf 0.62
detected under UV
lamp at 254 nm. Other
spot with fluorescent
white color was
detected under UV
lamp at 366 nm 0.59
The fraction
had many
spots with
closely
related Rf.
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Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 260
and two more bands of
Rf 0.54 and 0.50 with a
blackish blue color
after spraying with 10
% alcoholic sulphuric
acid.
5. 60 40 No spot ---
6. 50 50 No spot ---
7. 40 60 One single spot was
observed with Rf
value 0.34 with a
blackish blue color
after spraying with
10 % alcoholic
sulphuric acid.
Yellowish
compound
was
obtained.
(Componen
t VII)
8. 30 70
9. 20 80 No spot ---
10. 10 90 No spot ---
Simultaneous estimation of kaempferol, rutin and quercetin in
Standardization of Some Plant
7th
and 8th
fractions [eluted with chloroform: methanol (30:70)] gave an
intense spot under UV at R
(Fig.10.5) with mobile phase chloroform: methanol: formic acid (8:2:0.5) and
recrystallized from diluted alcohol to obtain a yellow crystalline powder
(component VII)
yield of component VII obtained was 0.214%
Fig.10.5 Video
Track 1: Component V
Track 2: Component VI
Track 3: Component VII
Component V, VI and VII showed single violet colored spots on TLC plate
under UV lamp.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques
fractions [eluted with chloroform: methanol (30:70)] gave an
intense spot under UV at Rf 0.34 (yellow colored compound
with mobile phase chloroform: methanol: formic acid (8:2:0.5) and
recrystallized from diluted alcohol to obtain a yellow crystalline powder
component VII) and light yellow crystalline powder (component VII)
yield of component VII obtained was 0.214% w/w.
5 Video-images of TLC plates showing Component V, VI and VII
Track 1: Component V
Track 2: Component VI
Track 3: Component VII
Component V, VI and VII showed single violet colored spots on TLC plate
under UV lamp.
1 2 3
herbal products 10
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fractions [eluted with chloroform: methanol (30:70)] gave an
0.34 (yellow colored compound) on TLC plate
with mobile phase chloroform: methanol: formic acid (8:2:0.5) and
recrystallized from diluted alcohol to obtain a yellow crystalline powder
component VII). The
images of TLC plates showing Component V, VI and VII
Component V, VI and VII showed single violet colored spots on TLC plate
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 262
All three components were evaluated for physicochemical parameters such as
melting point, solubility, elemental analysis. The parameters were compared
with those of the reference standard.
10.2.4 Physicochemical of component V
The melting point of component V was found to be 277 °C which was
matching with the standard (277 °C). Solubility was tested in different
solvents such as chloroform, methanol and water. It was observed that
component V was readily soluble in methanol and chloroform. The
Lassaigne’s sodium fusion test was carried out for detection of elements.
Carbon, hydrogen is present and nitrogen, halogen and sulphur were found to
be absent. The yield obtained was 0.170% w/w from chloroform extract by
column chromatography. The summarized data is mentioned in table 10.5.
Table 10.5: Physicochemical analysis of component V
Sr. No Parameters Component V Standard
1. Color Dark yellow Dark yellow
2. Melting point 277°C 277 °C
3. Solubility
Methanol,
Chloroform
Methanol,
Chloroform
4. Element present C, H,(O) C,H, (O)
5. Yield (%w/w) 0.170% -
.
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Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 263
10.2.5 Chromatographic and Spectral Studies of Component V:
� HPTLC Studies: Standard Kaempferol and the isolated component V
were dissolved in chloroform and the HPTLC analysis was carried out using
the following densitometric conditions:
� HPLC Studies: Standard kaempferol and component V were analyzed
by HPLC technique using the following conditions:
• Column: C18 Phenomenex (250 x 4.60mm) - 5µ
• Mobile phase : Acetonitrile (70) : Water (30)
• Flow rate: 1.0 ml/min
• Wavelength : 254nm
• Injection loop capacity: 20µl
• Concentration of Samples: 1mg/ml of standard and component
V
� Stationary phase : Precoated plates of Silica Gel 60 GF254 (Merck)
� Mobile phase : Chloroform : methanol: formic acid (9:1:0.5)
� Saturation time :15 min
� Development time :15 min
� Band width :7 mm
� Solvent front : 8 mm
� Wavelength :254 nm
� Lamp : Deuterium
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 264
Fig. 10.6: HPTLC chromatogram of standard
Kaempferol
Fig. 10.7: HPTLC chromatogram of Component V
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 265
Fig. 10.8: HPLC profile of reference standard Kaempferol:
RT=12.099 min
Fig. 10.9: HPLC profile of Component V: RT=12. 107 min
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 266
The HPTLC fingerprints (Fig.10.6 and Fig.10.7) and HPLC profile (Fig. 10.8,
Fig. 10.9) of component V matched with reference standard of kaempferol (Rf
=0.77 and RT=12.099). Thus component V was confirmed to be kaempferol.
� UV Spectroscopy: The UV spectrum was recorded in methanol for the
standard kaempferol and isolated component V.
The UV λ max of standard kaempferol was recorded to be 254 nm and 368 nm.
The component V also gave 254 nm and 368 nm of λ max.
� NMR Spectroscopy: The NMR (proton and 13
C) spectra were recorded
by dissolving the component V in deuterated methanol.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 267
Fig. 10.10: Proton NMR spectrum of Component V
Fig. 10.11: 13
C NMR spectrum of Component V
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 268
NMR INTERPRETATION
Compound V was analyzed by 1H and
13C NMR and the data was compared
with the reported literature.
PMR spectrum (400 MHz, MeOD): 8.08 (d, J=7.8Hz, 2H, H3’/5’), 8.10 (d,
J=7.8Hz, 2H,H2’/6’), 6.41 (s,1H,H6), 6.19 (s,1H,H8)
13C spectrum (100 MHz, MeOD): 176.4 (C=O, C-4), 161.0 (C-5), 159.1(C-
9), 156.8 (C-7), 147.0 (C-4’), 146.6 (C-2), 135.0 (C-3), 129.2 (C-2’/6’), 114.9
(C-3’/5’), 122.3 (C-1’), 97.8 (C-6), 93.0 (C-8).The 1H- NMR (400 MHz,
MeOD) spectrum of V shows five proton signals, shifted at 6.00 – 8.50 ppm
which represents a polyphenol skeleton. The doublet signals, 8.08 (d, 2H,
J=7.8Hz, H3’/5’), 8.10 (d,2H,J=7.8Hz, H2’/6’), were integrated of two
protons for each, so it proves the symmetry positions in compound structure.
13C- NMR exhibit fifteen carbon signals, including two signal of symmetry
carbons 129.2 (C-2’/6’), 114.9 (C-3’/5’), six oxygenated carbons 161.0 (C-5),
159.1(C-9), 156.8 (C-7), 161.0 (C-4’), 146.6 (C-2), 135.0 (C-3) and a
carbonyl 176.4(C=O, C-4). Thus, compound V was elucidated as
Kaempferol.
Thus from the comparison with reference standard of kaempferol and from
chemical and spectral studies, component V was confirmed to be kaempferol.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 269
10.2.6 Physicochemical analysis of component VI
The melting point of component VI was found to be 314 °C which was
matching with the standard (315 °C). Solubility was tested in different
solvents such as chloroform, methanol and water. It was observed that
component VI was readily soluble in methanol and chloroform. The
Lassaigne’s sodium fusion test was carried out for detection of elements.
Carbon, hydrogen is present and nitrogen, halogen and sulphur were found to
be absent. The yield obtained was 0.151%w/w from chloroform extract by
column chromatography. The summarized data is mentioned in table 10.8.
Table 10.8: Physicochemical analysis of component VI
Sr. Parameters Component VI Standard
1. Color Yellow Yellow
2. Melting point 314 °C 315 °C
3. Solubility
Methanol,
Chloroform
Methanol,
Chloroform
4. Elements present C, H,(O) C,H,(O)
5. Yield (%w/w) 0.151% w/w -
.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 270
10.2.7 Chromatographic and Spectral Studies of Component VI:
� HPTLC Studies: Standard Quercetin and the isolated component VI
was dissolved in chloroform and the HPTLC analysis was carried out using
the following densitometric conditions:
� HPLC Studies: Standard quercetin and component VI were analyzed
by HPLC technique using the following conditions:
• Column: C18 Phenomenex (250 x 4.60mm) - 5µ
• Mobile phase : Acetonitrile (70) : Water (30)
• Flow rate: 1.0 ml/min
• Wavelength : 254nm
• Injection loop capacity: 20µl
• Concentration of Samples: 1mg/ml of standard and component
VI
• Stationary phase : Precoated plates of Silica Gel 60 GF254 (Merck)
• Mobile phase : Chloroform : methanol: formic acid (9:1:0.5)
• Saturation time :15 min
• Development time :15 min
• Band width :7 mm
• Solvent front : 8 mm
• Wavelength :254 nm
• Lamp :Deuterium
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 271
Fig.10.13: HPTLC chromatogram of Component VI
Fig. 10.12: HPTLC chromatogram of standard Quercetin
Simultaneous estimation of kaempferol, rutin and quercetin in
Standardization of Some Plant
Fig. 10.14: HPLC profile of reference standard
min
Fig. 10.15
Simultaneous estimation of kaempferol, rutin and quercetin in herbal
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques
: HPLC profile of reference standard Quercetin
15: HPLC profile of Component VI : RT=8.134
herbal products 10
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Quercetin : RT=8.107
8.134 min
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 273
The HPTLC fingerprints (Fig.10.12 and Fig.10.13) and HPLC profile
(Fig. 10.14, Fig. 10.15) of component VI matched with reference standard of
quercetin (Rf =0.64 and RT=8.107). Thus component VI was confirmed to be
quercetin
� UV Spectroscopy: The UV spectrum was recorded in methanol for the
standard quercetin and isolated component VI.
The UV λ max of standard quercetin was recorded to be 257 nm and 369 nm.
The component VI also gave 257 nm and 369 nm of λ max.
� NMR Spectroscopy: The NMR (proton and 13
C) spectra were recorded
by dissolving the component VI in deuterated chloroform.
Simultaneous estimation of kaempferol, rutin and quercetin in
Standardization of Some Plant
Fig.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques
Fig. 10.16: Proton NMR spectrum (2) of Component VI
Fig. 10.17: 13
C NMR spectrum of Component VI
herbal products 10
Based Formulations By Modern Analytical Techniques 274
Component VI
Component VI
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 275
NMR INTERPRETATION
PMR spectrum (400 MHz, MeOD): 7.81 (s, 1H, H- 2’), 7.70 (d, J= 8.0, 1H,
H-6’), 6.99 (d, J=8.0, 1H, H-5’), 6.53 (s, 1H, H-8), 6.27 (s, 1H, H-6).
13C spectrum (100 MHz, MeOD): 177.3 (C=O, C-4), 163.8 (C-7), 160.8 (C-
5), 156.1 (C-9), 155.3 (C-2), 148.2 (C-4’), 144.6 (C-3’), 133.0 (C-3), 122.1
(C-6’), 121.0 (C-1’), 115.7 (C-5’), 115.2 (C-2’), 103.7 (C-10), 98.4 (C-6),
93.4 (C-8).
The 1H- NMR spectrum of VI showed five proton signals, shifted at 6.00 –
8.0 ppm which represents a polyphenol skeleton. Two doublet signals 7.70 (d,
J= 8.0, 1H, H-6’), 6.99 (d, J=8.0, 1H, H-5’), and three singlet 7.81 (s, 1H, H-
2’), 6.53 (s, 1H, H-8), 6.27 (s, 1H, H-6), correspond to flavonol skeleton. The
fifteen signals in 13
C- NMR spectra including seven oxygenated carbons
{163.8 (C-7), 160.8 (C-5), 156.1 (C-9), 155.3 (C-2), 148.2 (C-4’), 144.6 (C-
3’), 133.0 (C-3)} and carbonyl 177.3 (C=O, C-4) signify the quercetin. Thus
from the analysis of NMR data verify that compound VI is Quercetin.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 276
The NMR Spectra revealed presence of aromatic, hetrocyclic and protons of
phenolic group it was found to be almost identical to the NMR spectra of
Standard Quercetin (Fig. 10.16.1, Fig. 10.16.2, Fig. 10.17, Table 10.9, Table
10.10). Thus from the comparison with reference standard quercetin and from
chemical and spectral studies component VI was confirmed to be quercetin.
10.2.8 Physicochemical analysis of component VII
The melting point of component VII was found to be 212 °C which was
matching with the standard (211°C). Solubility was tested in different
solvents such as chloroform, methanol and water. It was observed that
component VII was readily soluble in methanol. The Lassaigne’s sodium
fusion test was carried out for detection of elements. Carbon, hydrogen is
present and nitrogen, halogen and sulphur were found to be absent. The yield
obtained was 0.214%w/w from chloroform extract by column
chromatography. The summarized data is mentioned in table 10.11.
Table 10.11: Physicochemical analysis of component VII
Sr. Parameters Component VII Standard
1. Color Yellow Yellow
2. Melting point 211°C 315 °C
3. Solubility
Methanol,
Chloroform
Methanol,
Chloroform
4. Element present C, H,(O) C,H,(O)
5. Yield (%w/w) 0.214% w/w -
.
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Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 278
10.2.9 Chromatographic and Spectral Studies of Component VII:
� HPTLC Studies: Standard Rutin and the isolated component VII was
dissolved in methanol and the HPTLC analysis was carried out using the
following densitometric conditions:
� HPLC Studies: Standard rutin and component VII were analyzed by
HPLC technique using the following conditions:
• Column: C18 Phenomenex (250 x 4.60mm) - 5µ
• Mobile phase : Acetonitrile (70) : Water (30)
• Flow rate: 1.0 ml/min
• Wavelength : 254nm
• Injection loop capacity: 20µl
• Concentration of Samples: 1mg/ml of standard and component
VII
• Stationary phase : Precoated plates of Silica Gel 60 GF254 (Merck)
• Mobile phase : Chloroform : methanol: formic acid (8:2:0.5)
• Saturation time :15 min
• Development time :15 min
• Band width :7 mm
• Solvent front : 8 mm
• Wavelength :254 nm
• Lamp : Deuterium
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 279
Fig.10.18: HPTLC chromatogram of standard Rutin
Fig. 10.19: HPTLC chromatogram of component VII
Simultaneous estimation of kaempferol, rutin and quercetin in
Standardization of Some Plant
Fig. 10.20: HPLC profile of reference standard
Fig. 10.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques
: HPLC profile of reference standard rutin : RT=
Fig. 10.21: HPLC profile of Component VII: RT=4.419
herbal products 10
Based Formulations By Modern Analytical Techniques 280
: RT=4.422 min
4.419 min
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 281
The HPTLC fingerprints (Fig.10.18 and Fig.10.19 ) and HPLC profile
(Fig. 10.20 and Fig. 10.21) of component VII matched with reference
standard of rutin (Rf =0.34 and RT=4.422). Thus component VII was
confirmed to be rutin.
• UV Spectroscopy: The UV spectrum was recorded in methanol for the
standard rutin and isolated component VII.
The UV λ max of standard quercetin was recorded to be 257 nm and 369 nm.
The component VII also gave 257 nm and 369 nm of λ max.
� NMR Spectroscopy: The NMR (proton and 13
C) spectra were recorded
by dissolving the component VII in deuterated methanol.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 282
Fig. 10.22: Proton NMR spectrum of Component VII
Fig. 10.23: 13
C NMR spectrum of Component VII
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 283
NMR INTERPRETATION
PMR spectrum (300 MHz, MeOD): 7.57 (s, 1H, H- 2’), 7.554 (d, J= 6.2, 1H,
H-6’), 6.80 (d, J=6.1, 1H, H-5’), 6.31 (s, 1H, H-8), 6.21 (s, 1H, H-6), 4.78
(overlap H-1”), 4.43 (H-1”’), 3.54 (m, H-3”), 3.70 (d, J=8.1, H-6”a), 3.45 (d,
J=7.08, H-6”b), 3.38 (m, H-2”/4”/ 5”), 3.32 (m, overlap, H-2”’/3”’/4”’), 1.03
(d, J=4.3, 3H, H-6”’).
13C spectrum (100 MHz, MeOD): 177.9 (C=O, C-4), 164.6 (C-7), 161.5 (C-
5), 157.9 (C-2), 157.0 (C-9), 148.4 (C-4’), 144.4 (C-3’ ), 134.1 (C-3), 122.1
(C-1’), 121.6 (C-6’), 116.2 (C-5’), 114.6 (C-2’), 104.1 (C-1”’), 103.2 (C-10),
100.9 (C-1”), 98.57 (C-6), 93.5 (C-8), 76.7 (C-3”), 75.7 (C-5”), 74.3 (C-2”),
74.2 (C-4”’), 72.4 (C-3’’’), 70.8 (C-4”), 70.6 (C-2”’), 69.9 (C-4”’) 68.3 (C-
5’’’),67.1 (C-6”), 16.4 (C-6”’).
The 1H- NMR spectrum of VII demonstrates five proton signals, shifted at
6.00 – 8.0 ppm which represents a polyphenol skeleton. Two doublet signals
7.554 (d, J= 6.2 , 1H, H-6’), 6.80 (d, J=6.1, 1H, H-5’), and three singlet 7.57
(s, 1H, H- 2’), 6.31 (s, 1H, H-8), 6.21 (s, 1H, H-6) correspond to flavonol
skeleton. The fifteen signals in 13
C- NMR spectra including seven oxygenated
carbons {164.6 (C-7), 161.5 (C-5), 157.9 (C-2), 157.0 (C-9), 148.4 (C-4’),
144.4 (C-3’), 134.1 (C-3)} and carbonyl 177.9 (C=O, C-4) signify the
quercetin aglycone.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 284
The 1H- NMR and
13C- NMR spectra illustrate that compound VII posses two
sugar moieties with enomeric positions shift at {4.78 (overlap H-1”), 100.9
(C-1”)}, { 4.43 (H-1”’), 104.1 (C-1”’)}. Among two sugars, one is glucose
{3.70 (d, J=8.1, H-6”a), 3.45 (d, J=7.08, H-6”b) 67.1 (C-6”)}, and other is
rhamnose {1.03 (d, J=4.3, 3H, H-6”’), 16.4 (C-6”’)}. Based on PMR and 13
C-
NMR data compound VII was confirmed as Rutin.
The NMR spectra revealed presence of aromatic, hetrocyclic and protons of
phenolic group it was found to be almost identical to the NMR spectra of
standard rutin. (Fig. 10.22 and Fig. 10.23, Table 10.12 and Table 10.13).
Thus from the comparison with reference standard of rutin and from chemical
and spectral studies, component VII was confirmed to be rutin.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 285
10.3 METHOD DEVELOPMENT
10.3.1 Preparation of standard solutions
Standard stock solutions of Kaempferol, standard were prepared by dissolving
2.1 mg of Kaempferol in methanol, yielding 10 ml of a concentration stock =
2.1 mg ml-1
(210.0 µgml-1
). From this 0.1, 0.3, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8,
3.2, 3.6, 4.0 µL of this solution were applied using LINOMAT 5 applicator
with the band width of 8 mm, which gave different concentration ranging 21-
840 ng / spot.
Similarly, working standard concentration (280 µgml-1
) of rutin and (200
µgml-1
) of quercetin was prepared by dissolving 2.8mg of rutin in and 2.0 mg
of quercetin in 10 ml of methanol. From this different volumes were applied
using LINOMAT 5 applicator in the form of band of width of 8 mm, in such a
manner to get different concentrations ranging from 56 to 840 ng/spot and 60-
1800 ng/ spot of rutin and quercetin respectively.
10.3.2 The different parameters of HPTLC such as mobile phase, band width
and detection wavelength were tried and then were optimized.
Stationary phase: The commonly used stationary phase i.e, Silica Gel 60
GF254 was used. The plates were pre-washed with methanol and activated at
60°C for 5 min prior to chromatography. As all three marker compounds were
well separated this stationary phase was optimized.
Mobile phase: Number of mobile phases combinations were tried. Such as
chloroform: ethyl acetate: formic acid, chloroform: methanol: formic acid
with different combinations. Mobile phase comprising of chloroform:
methanol : formic acid (8.2:1.5:1) gave better resolution as compared to other
mobile phases. All three compounds gave well separated and resolved peaks
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 286
with Rf 0.12, Rf 0.53 and Rf 0.69 of rutin, quercetin and kaempferol
respectively.
Band width: Band width of three different sizes 6 mm, 7 mm and 8mm were
tried. Band width of size 7 mm gave good separation without the over
saturation of the spot with sample and with maximum number of tracks were
spotted in one 20 x 10 cm TLC plate.
Detection: The TLC plate was scanned at 254nm using deuterium lamp.
The sample solutions were applied with a Camag microlitre syringe using
Camag Linomat V (Switzerland) applicator. The plates were pre-washed with
methanol and activated at 60°C for 5 min prior to chromatography. The slit
dimension was kept at 5 mm × 0.45 mm and scanning speed was 10 mm/s.
The slit bandwidth was set at 20 nm, each track was scanned thrice and
baseline correction was done.
The following densitometric conditions were used for HPTLC studies:
Stationary phase : : Precoated plates of Silica Gel 60 GF254 (Merck)
Mobile phase : Chloroform: methanol: formic acid (8.2:1.5:1)
Saturation time : 15 min
Development time :15 min
Wavelength : 254 nm
Lamp : Deuterium
Band width : 7 mm
Length of
chromatogram
: 8 cm
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 287
Linear ascending development was carried out in 20 cm x 10 cm twin trough
glass chamber (Camag, Muttenz, Switzerland) saturated with the mobile
phase. Densitometric scanning was performed with Camag TLC scanner III in
the reflectance-absorbance mode and operated by Win CATS software (1.3.0
Camag). Concentrations of the compound chromatographed were determined
from the intensity of diffusely reflected light. Evaluation was carried out by
comparing peak areas with linear regression.
The proposed method gave very good separation and resolution of all the
three markers as indicated in Table 10.14 and Fig. 10.24.
Table10.14. Marker compounds and respective Rf Values.
Sr. No Marker compounds Rf value
1. Kaempferol 0.69
2. Quercetin 0.53
3. Rutin 0.12
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 288
Thus, the optimized HPTLC method gives a very well resolved and separated
the three marker compound’s chromatogram.
Fig 10.24: HPTLC chromatogram of Kaempferol, quercetin and
rutin using optimised parameters
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 289
10.4 METHOD VALIDATION
Validation of HPTLC method as per ICH guidelines
Analytical method validation is a process of performing several tests designed
to verify that an analytical test system is suitable for its intended purpose and
is capable of providing useful and valid analytical data. The developed
method was validated for various parameters like linearity, limit of detection
(LOD), limit of quantitation (LOQ), accuracy, precision, robustness and
system suitability as per ICH guidelines.
10.4.1 Linearity studies
Different concentrations of markers were spotted and analyzed. The analysis
was done in triplicate and the concentration range showing regression
coefficient (r2) near to one with précised value of r
2 in all triplicate analysis
was chosen.
Linearity was evaluated in the range of 21-840 ng / spot, 56-1008 ng / spot
and 60-1800 ng / spot for kaempferol, rutin and quercetin respectively. Peak
area versus concentration was subjected to least square linear regression
analysis and the slope, intercept and correlation coefficient for the calibration
curve were determined.
The concentration was found to occur in the concentration range of 84-504 ng
ml-1
for Kaempferol, 168-1008 ng ml-1
for Rutin, and 800 -1800 ng ml-1
for
Quercetin. The correlation coefficient of Kaempferol, Rutin and Quercetin
was found to be 0.9997, 0.9998 and 0.9998 respectively. The peak area (y) is
proportional to the concentration of respective marker and the regression
equations as following:
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 290
y = 12.047x + 1489.3
R² = 0.9997
0
1000
2000
3000
4000
5000
6000
7000
8000
0 100 200 300 400 500 600
y = 3.59x + 170.22
R² = 0.9998
0
500
1000
1500
2000
2500
3000
3500
4000
0 200 400 600 800 1000 1200
For Kaempferol, (x1) y = 12.047x1 + 1489.3 (Fig.10.25)
For Rutin(x2) y = 3.59 x2 + 170.22 (Fig.10.26)
For Quercetin (x3) y = 14.255x3 - 4219.3 (Fig.10.27).
Fig 10.25: Calibration curve of Kaempferol
Fig 10.26: Calibration curve of Rutin
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 291
y = 14.255x - 4219.3
R² = 0.9998
0
5000
10000
15000
20000
25000
0 500 1000 1500 2000
Fig 10.27: Calibration curve of Quercetin
10.4.2 Limit of detection (LOD) and limit of quantitation (LOQ):
LOD and LOQ were determined by using standard deviation method. A
calibration curve was prepared using concentrations in the range of 3.45-
10.35 µg/spot which is below the linearity range. Standard deviation of
residuals was measured and kept in the following equation for determination
of detection limit and quantitation limit. Detection limit =3.3σ /S and
quantitation limit=10 σ /S where σ is the residual standard deviation of a
regression line and S is the slope of the calibration curve.
The experimentally derived LOD and LOQ for all three markers were
determined as mentioned in Table 10.15.
Table 10.15: LOD and LOQ of all the three markers.
Markers LOD (ng) LOQ (ng)
Kaempferol 21 63
Rutin 56 168
Quercetin 300 600
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 292
10.4.3 Precision
Precision of the method was evaluated by repeatability (intra-day) and
instrumental precision. Each level of precision was investigated by three
sequential replicates of injections of kaempferol, rutin and quercetin at
concentrations of 168, 252 and 336 ng/spot, 336, 504 and 672 ng/spot and
1200, 1600 and 1800 ng/spot respectively.
Precision data on repeatability (intra-day) and instrumental variation for three
different concentration levels are summarized in Table 10.16.1, Table
10.16.2 and Table 10.16.3. Precision studies showed R.S.D. less than 1%,
indicating a sufficient precision
Table 10.16.1: Results of Precision Studies of Kaempferol
Type of
Precision
Intra-day Inter-day
AUC for concentration of
Kaempferol (ng/µl)
AUC for concentration of
Kaempferol (ng/µl)
Sr. No 168 252 504 168 252 504
1 3515.41 4548.99 5584.16 3518.9 4556.7 5562.69
2 3527.69 4584.17 5545.18 3499.15 4547.89 5579.09
3 3536.12 4571.02 5512.22 3521.45 4569.12 5548.17
Mean 3526.40 4568.06 5547.18 3513.16 4557.90 5563.31
% RSD 0.29 0.38 0.64 0.34 0.23 0.27
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 293
Table 10.16.2: Results of Precision Studies of Rutin
Type of
Precision
Intra-day Inter-day
AUC for concentration of
Rutin (ng/µl)
AUC for concentration of
Rutin (ng/µl)
Sr. No 336 504 672 336 504 672
1 1409.78 1997.14 2598.16 1391.48 1987.15 2579.18
2 1387.46 1975.13 2587.78 1378.09 1974.15 2544.15
3 1389.02 1968.18 2611.1 1370.11 1990.12 2569.18
Mean 1395.42 1980.15 2599.01 1379.89 1983.80 2564.17
% RSD 0.89 0.76 0.44 0.78 0.42 0.70
Table 10.16.3: Results of Precision Studies of Quercetin
Type of
Precision
Intra-day Inter-day
AUC for concentration of
Quercetin (ng/µl)
AUC for concentration of
Quercetin (ng/µl)
Sr. No 1200 1600 1800 1200 1600 1800
1 15812.15 18601.2 21666.45 15845.12 18599.15 21645.16
2 15789.02 18701.11 21412.99 15877.12 18545.16 21745.18
3 15834.24 18540.33 21515.24 15909.54 18478.16 21500.69
Mean 15811.8 18614.21 21531.56 15877.26 18540.82 21630.34
% RSD 0.14 0.43 0.59 0.20 0.32 0.56
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 294
10.4.4 Accuracy
In order to evaluate the validity of the proposed method, accuracy of the
method was determined the percentage recoveries of known amounts of
mixture of kaempferol, rutin and quercetin added to sample containing a
mixture of all three standards. The analyzed samples were spiked with 80, 100
and 120 % of median concentrations (336 ng kaempferol, 672 ng rutin and
1400 ng quercetin) of standard solution in a solution containing the 100 % of
respective concentration of three standards. Accuracy was calculated from the
following equation:
[(spiked concentration − mean concentration)/spiked concentration] × 100.
The sample containing 336 ng of kaempferol, 672 ng of rutin and 1400 ng of
quercetin were spiked with the known amount of standard, and the percent
ratios between the recovered and expected concentrations were calculated.
Recoveries were obtained in the range of 81.80-118.97 %, depicting the
HPTLC proposed method for simultaneous estimation is accurate for the
quantification of all three marker compounds. (Table 10.17.1, Table 10.17.2,
Table 10.17.3)
Table 10.17.1: Recovery studies of Kaempferol
In 336 ng
Kaempferol
AUC of kaempferol
Recovery ±
S.D. (%) In sample
In
standard
In spiked
samples
268.8 (80%) 5537.09 4727.53 10729.60 104.53
336 (100%) 5537.09 5562.6 13001.06 117.13
403.2 (120%) 5537.09 7556.25 14980.09 114.41
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 295
Table 10.17.2: Recovery studies of Rutin
In 672 ng
Rutin
AUC of rutin
Recovery ±
S.D. (%) In sample
In
standard
In spiked
samples
537.6 (80%) 2579.01 2104.41 3841.80 82.03
672 (100%) 2579.01 2590.80 4195.81 81.16
806.4 (120%) 2579.01 3077.20 4626.77 81.80
Table 10.17.3: Recovery studies of Quercetin
In 2000 ng
Quercetin
AUC of Quercetin
Recovery ±
S.D. (%) In sample
In
standard
In spiked
samples
1120 (80%) 15789.01 11746.3 30299.85 110.04
1400 (100%) 15789.0 15737.7 30930.84 98.11
1680 (120%) 15789.0 19729.1 42255.88 118.97
10.4.5 Robustness
For the determination of the robustness of method, chromatographic
parameters, such as mobile phase composition and detection wavelength,
were intentionally varied to determine their influence on the retention factor
and quantitative analysis.
The mobile phase composition was altered by ± 5 % changes in the
composition of methanol. The two composition of methanol were tried 1.625
(+5% of 1.5) and 1.375 (-5% of 1.5) were tried. The chamber saturation time
was altered from 15 min to 30 min.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
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No changes were observed in retention time and peak shape of all three
markers with the changes made with mobile phase and chamber saturation
time (Table 10.18.1, Table 10.18.2, Table 10.18.3, Table 10.18.4, Table
10.18.5 and Table 10.18.6). The resolution and the separation of markers
were also unaltered.
10.4.6 Stability studies
Stability of the sample solutions was tested after 24, 48 and 72 hours after
preparation and storage at 4.0 °C and 25.0 °C separately. Stability was
assessed by comparing the chromatographic parameters of the solutions after
storage with the same characteristics of freshly prepared solutions of all the
three markers.
Methanol solution containing the mixture of all three markers showed 1.81 %
of degradation after 72 hrs at 4 °C and 2.59 % of degradation after 72 hrs at
25 °C of kaempferol. The rutin marker showed maximum degradation after 72
hrs of 1.41% at 4 °C and of 1.17% at 25°C. The percent of degradation of
quercetin after 72 hrs was 2.19 % and 2.91% at 4 °C and 25 °C respectively
(Table 10.19).
Table 10.18.1: Robustness (Mobile phase variation) studies of
Kaempferol
Sr.
No
Mobile phase composition (v/v)
Rf AUC
Chloroform Methanol Formic acid
1. 8.2 1.5 1 0.69 5531.03±0.45
2. 8.2 1.625 1 0.69 5531.51±0.23
3. 8.2 1.375 1 0.69 5531.86±0.42
S.D - - - 0.0 0.42
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 297
Table 10.18.2: Robustness (Chamber saturation time) studies of
Kaempferol
Sr. No Chamber saturation time (min) Rf AUC
1. 15 0.69 5531.03±0.45
2. 20 0.69 5530.99±0.31
3. 25 0.69 5531.54±0.28
4. 30 0.69 5531.05±0.14
S.D. - 0.0 0.26
Table 10.18.3: Robustness (Mobile phase variation) studies of Rutin
Sr.
No.
Mobile phase composition (v/v) Rf AUC
Chloroform Methanol Formic acid
1. 8.2 1.5 1 0.12 2580.41±0.15
2. 8.2 1.625 1 0.12 2580.79±0.28
3. 8.2 1.375 1 0.12 2581.07±0.51
S.D - - - 0.0 0.33
Table 10.18.4: Robustness (Chamber saturation time) studies of Rutin
Sr. No. Chamber saturation time (min) Rf AUC
1. 15 0.12 2580.41±0.15
2. 20 0.12 2580.97±0.80
3. 25 0.12 2581.06±0.24
4. 30 0.12 2581.25±0.57
S.D. - 0.0 0.36
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 298
Table 10.18.5: Robustness (Mobile phase variation) studies of Quercetin
Sr.
No.
Mobile phase composition (v/v)
Rf AUC
Chloroform Methanol Formic acid
1. 8.2 1.5 1 0.53 15787.08±0.31
2. 8.2 1.625 1 0.53 15787.48±0.24
3. 8.2 1.375 1 0.53 15788.05±0.44
S.D - - - 0.0 0.49
Table 10.18.6: Robustness (Chamber saturation time) studies of
Quercetin
Sr. No. Chamber saturation time (min) Rf AUC
1. 15 0.53 15787.08±0.31
2. 20 0.53 15787.15±0.24
3. 25 0.53 15788.06±0.14
4. 30 0.53 15787.54±0.11
S.D. - 0.0 0.45
Table 10.19: Stability Studies of solution containing three markers
Marker
compounds
Temperature
4 °C 25 °C
24 h 48 h 72 h 24 h 48 h 72 h
Kaempferol 99.87 98.10 98.19 98.93 97.39 97.41
Rutin 99.76 98.91 98.59 99.63 98.56 98.23
Quercetin 99.83 98.87 97.81 99.60 98.25 97.09
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 299
10.5 Quantitative analysis for simultaneous estimation of Kaempferol,
Rutin and Quercetin content by HPTLC
10.5.1. Procurement of plant based formulations
The following formulations were procured from the local market.
Sr. No Formulations containing
Bhuiamla Amla
1. Bhuiamla Vati Amla Vati
2. Bhuiamla Capsules Amla Tincture
10.5.2. Sample solutions
10.5.2.1 For Phyllanthus niruri Linn. extract and Emblica officinalis Gaertn
(Amla) extract
2 g of commercial methanol extracts of Phyllanthus niruri Linn. and Emblica
officinalis Gaertn each was transferred to 100 ml volumetric flask containing
50 ml of methanol and was macerated on a shaker for 24 hrs at room
temperature and volume was made upto 100ml. Then 1.0 ml of each extract
was diluted to 10 ml with methanol.
10.5.2.2 For Bhuiamla, Amla vati
2 g of powdered vati of Bhuiamla and Amla was transferred to 100 ml
volumetric flask containing 50 ml of methanol separately was macerated on a
shaker for 24 hrs at room temperature and volume was made upto 100 ml.
Then 1.0 ml of each extract was diluted to 10 ml with methanol separately.
10.5.2.3 For Bhuiamla Capsule
Sample solutions of capsule formulation were prepared same as that of vati by
transferring 2 g of capsule contents of both the plants.
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 300
10.5.2.4 For Amla tincture
10 ml of of Amla tincture (homeopathic preparation) was evaporated to
dryness. 10 mg of residue was dissolved in 10 ml methanol in a volumetric
flask.
A constant application volume of 10.0 µl/spot was employed for all the
sample solutions.
Validity of the proposed method was applied to standardization for both
traditional and modern dosage forms of three plants viz. Bhuiamla (extract,
vati and capsule), Amla (vati and tincture). The shape of the peaks was not
altered by other substances present in the matrix. The developed and validated
HPTLC method gave well resolved peaks of three respective markers in all
the formulations (Fig.10.28 and Fig.10.29). The percent content of
kaempferol, rutin and quercetin in all three products each of two plants are
indicated in Table 10.20.
The rapid, simple, precise, accurate and reproducible HPTLC method was
successfully developed and validated for simultaneous analysis of kaempferol,
rutin and quercetin in formulations containing Bhuiamla and Amla. The
various plant based products analysed were vati (an ayurvedic preparation),
capsule (a modern based formulation) and Amla tincture (homeopathic
medicine). Thus, this method can be applied for a wide range for plant based
medicines ranging from traditional to modern dosage form.
This method enabled to detect and quantified the amount of all three markers
as least as 0.199% of kaempferol to as high as 0.501 % of rutin in analyzed
plant-based products.
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Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 301
Table 10.20: Percent Content of Kaempferol, Rutin and Quercetin in
Bhuiamla and Amla products
Extract &
Formulati
on
Percent Content
± S.D. (%)
Weight of
formulation
per unit
(mg)
Content per unit of
dosage form (mg)
K R Q K R Q
Methanol
extract of
Bhuiamla
0.298
±0.81
0.441
±0.46
0.407
±0.30
- - - -
Bhuiamla
Vati
0.301
±0.24
0.420
±0.12
0.367
±0.88
250 0.602 1.05 0.9175
Bhuiamla
Capsule
0.278
±0.11
0.470
±0.15
0.389
±0.75
200 0.556 0.94 0.778
Methanol
extract of
Amla
0.219
±0.17
0.439
±0.060
0.322
±0.15
Amla Vati 0.212
±0.55
0.472
±0.31
0.314
±0.10
250 0.530 1.18 0.785
Amla
Tincture
0.199
±0.42
0.501
±0.15
0.306
±0.22
1000
(10 ml)
2 5 3.1
* K=Kaempferol, R=Rutin, Q=Quercetin
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 302
Fig 10.28: HPTLC chromatogram of Bhuiamla extract showing
all three markers using optimized parameters
Fig 10.29: HPTLC chromatogram of Amla extract showing all
three markers using optimized parameters
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 303
10.6 Quantitative analysis of formulations containing Clitoria ternatea
Linn (Aparajita) by HPTLC for Kaempferol content
The Clitoria ternatea Linn known as the Aparajita is an Indian Miracle Plant
which facilitates Normal Chilbirth even when a Cesarean is predicted.
Aparajita means "The Undefeated". This plant is a trailing creeper with the
usual Indigo Blue colour flowers and the rare White ones which is more of a
pale cream with a hint of green at the edges.
Leaves contain glycosides of kaempferol. The leaves are useful in otalgia,
hepatopathy and eruptions.
10.6.1. Procurement of plant based formulations
Two formulations of Clitoria ternatea Linn (Aparajita) namely Vati and
Capsules were procured from the local market.
10.6.2 Sample solutions
10.6.2.1 For Aparajita vati
2 g of powdered vati of Aparajita was transferred to 100 ml volumetric flask
containing 50 ml of methanol separately was macerated on a shaker for 24 hrs
at room temperature. Then 1.0 ml of each extracts were diluted to a 10 ml
with methanol separately.
10.6.2.2 For Aparajita Capsule
Sample solution of capsule formulation were prepared same as that of vati by
transferring 2 g of capsule contents of both the plants.
A constant application volume of 10.0 µl/spot was employed for all the
sample solutions.
The developed and validated HPTLC method for simultaneous estimation of
kaempferol, rutin and quercetin was employed to determine the content of
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 304
kaempferol alone in Aparajita containing traditional (vati) as well as modern
(capsule) dosage form. The method was well adapted and gave well resolution
of kaempferol peak in the extracts of formulations containing Aparajita
(Fig.10.30).
The percent content of Kaempferol in formulations (vati and capsule)
containing Aparajita are calculated and shown in Table 10.21.
Table 10.21: Percent Content of Kaempferol in Aparajita formulations
Aparajita
Formulations
Percent Content ±
S.D. (%) of
Kaempferol
Weight of
formulations per
unit
Content per
unit of
dosage form
Aparajita Vati 1.032±0.34 250 mg 2.58 mg
Aparajita Capsule 2.101±0.52 200 mg 4.202 mg
Fig 10.30: HPTLC chromatogram of Aprajita extract showing
Kaempferol marker using optimized parameters
Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10
Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 305
The same method was successfully employed for estimation of single marker
i.e, Kaempferol in two formulations containing Aparajita (vati and capsule)
(Fig.10.24). The vati contained 1.032% of Kaempferol whereas 2.101% of
kaempferol was found to be present in capsule.
Thus, the method has a broad range of applicability to various plant products
and even various preparations of different system of medicines.