Estimation of Quercetin in Mangifera
indica Leave Extract
by
Planer Chromatographic Method
Jahanvi Soni, Chintan Patel, Avni Solanki, Jinal
Tandel*, Heta Kachhiya
Department of Pharmaceutical Chemistry and Analysis,
Indukaka Ipcowala College of Pharmacy, New V.V. Nagar,
Anand.
*Corresponding Author E-mail:
jinal.tandel@cvmu.edu.in
ABSTRACT:
Mangifera indica L. belongs to family Anacardiaceae is an important
herb in ayurvedic and indigenous medical systems for over 4000 to treat numbers
of diseases like Diabetes, Dissolve gall and kidney stones, cures respiratory
problems, treat throat problems, treat restlessness, treat hiccups, helps to
lose weight and also heals burns. A sensitive, simple, and accurate High
Performance Thin Layer Chromatographic method has been used for quantitation of
Quercetin from of Mango Leaf powder. Phytochemical Screening was performed to
identify various chemical constituents present in the plant. The
chromatographic development was done on HPTLC plates recoated with Silica Gel
60 F254 utilizing a mobile phase of Toluene: Ethyl Acetate: Formic
acid (4: 5: 1 %v/v/v) as flexible stage. The eluted analytes detected at 374
nm. Phytochemical screening gives promising data for presence of various
chemical constituents like, Alkaloids, Flavonoids, Glycosides, Steroids,
Glycosides, Proteins, Tannins and Phenolic Compounds, Volatile oil etc. In
developed HPTLC method quercetin was found at 0.84 Rf as compared
with standard Quercetin. Present study confirms the presence of various
chemical constituents in the leaf of Mangifera indica. Also, it confirms
the presence of Quercetin in different extract of Mangifera indica
leaves. Highest concentration of Quercetin was found in ethyl acetate extract
where lowest amount was found in ether extract.
KEYWORDS: Mangifera indica L., HPTLC, Phytochemical tests.
INTRODUCTION:
Mango (Mangifera indica L.) belongs
to family Anacardiaceae is an important herb in ayurvedic and indigenous
medical systems for over 4000 years1. The mango tree is
an evergreen that is well-known for its mouthwatering fruits as well as its
many traditional medical applications. The cultivation of mangoes produces a
lot of agricultural residue, including leaves, flowers, stems, and bark while
trimming, in addition to its commercially valued fruit, which presents disposal
issues for farmers Mango leaves are a rich source of essential
minerals, including nitrogen, potassium, phosphorus, iron, sodium, calcium, and
magnesium, as well as vitamins A, B, E, and C. The primary bio-macromolecule in
mango leaves is protein, making them a viable alternative feed source for
livestock in developing countries, thereby helping to mitigate food shortages.
Mangiferin is the main biologically active component of mango leaves, with
phenolic acids, benzophenones, and other antioxidants such flavonoids,
carotenoids, isoquercetin, quercetin, ascorbic acid, and tocopherols. Mango
leaves are known for their various biological, pharmacological, and
phytochemical characteristics. They have been shown to have immunomodulatory,
antidiabetic, antioxidant, and antibacterial activities1-4.
Among the most notable is quercetin, a plant pigment
and powerful flavonoid antioxidant. Quercetin is a flavonol that has antiviral,
antioxidative, anti-inflammatory, antiproliferative, anticarcinogenic, and
antidiabetic qualities. This lipophilic chemical offers defense against
neurodegenerative illnesses by effortlessly passing through the blood-brain
barrier (BBB). Quercetin has been shown to be a useful therapeutic drug for
neurodegeneration, diabetes, cancer, and inflammation in a number of in vivo
and in vitro investigations. Studies have indicated that quercetin plays a
major role in the prevention of illnesses associated with aging5,6.
Presence of quercetin in mango leaves gives clarity
about the use of mango leaves in various disease. Further isolation can be
carried out to identify pharmacological role of quercetin in Mangifera
indica The present study focuses on the phytochemical screening along with
the qualitative analysis of Quercetin in the Mangifera indica L. HPTLC
method has not been reported for optimization of Quercetin in Mango leaves in
any reputed journal. Structure of Quercetin is shown in figure 1.
Figure. 1: Chemical Structure of Quercetin
MATERIALS AND METHODS:
Materials:
Preparation of mango leaves powder:
Mango leaves were collected from the mango tress of
botanical garden, New Vallabh Vidyanagar, GIDC, Anand, Gujarat 388121. The
leaves were dried in shadow. They were powdered using a mechanical mixture and
was stored in container at room temperature.’
Identification of mango leave:
The Mango species used in this study was identified
and approved by botanist, New V.V. Nagar, Anand, Gujarat.
Preparation of extract:
Dried powder was weighed accurately and prepared six
different extracts first treated with hexane to remove chlorophyll and then
different extract were prepared using six solvents (diethyl ether, ethyl
acetate, methanol, chloroform, ether and hydro-alcoholic (70:30)) extraction
was done by two different methods i.e.; maceration and decoction. For
qualitative analysis, 10mg of Quercetin powder was transferred in volumetric
flask and make up the volume upto 10ml with methanol.
Preliminary phytochemical evaluation:7-9
Coarse powder of the dried leaves was prepared using a
mechanical grinder. For maceration 10g mango leaf powder was weighed and
treated with 100ml hexane solution. After 24 hours all flask were filtered and
residue were treated with different solvent (diethyl
ether, ethyl acetate, methanol, chloroform, ether and hydro-alcoholic (70:30
%v/v)) for further extraction process. All flask is kept for 72 hours in dark
place at room temperature with occasional shaking. After 72 hours’ flask was
filtered and kept the filtrate for evaporation process to collect mass of
extract4. Same process was followed for decoction method but it was
heat for 30 min. before filtration. The extract was used for the preliminary
phytochemical screening. Tests for the various phytoconstituents such as
alkaloids, steroids, carbohydrate, glycosides, flavonoids etc were carried out
by using standard pharmacopeial procedures.
HPTLC method development:10-11
Chemical and Reagents:
Analytically pure Quercetin was obtained from Euca
enterprise, India. Toluene HPLC Grade from S D Fine - Chem Ltd., Mumbai India,
Ethyl acetate from Suvidhinath Laboratories, Gujarat India, Formic acid from
Suvidhinath Laboratories, Gujarat India, Methanol HPLC Grade from– SRL Pvt.
Ltd., Mumbai India.
Instrument:
Sample application has been carried out with help of
Hamilton syringe 100μl syringe which give 6mm bands width using Camag
Linomat 5 (Switzerland) sample applicator on pre-coated silica gel aluminium
plate 60 F254, (10 cm x 10 cm with 0.2 mm thickness, E. Merck,
Germany). Camag TLC scanner is used for the densitometry scanning of the
developed chromatogram. To ensure accuracy, all drugs and chemicals were
weighed on a Mettler Toledo electronic balance (ME 204, Mettler Toledo group,
Mumbai, India).
Chromatographic Condition:
Sample application- Standard Quercetin and different extract of M.Indica
was applied on the HPTLC plates in the form of narrow bands of 6 mm length,
10 mm from the bottom and left edge, and with 9 mm space between two bands.
Samples application has been carried out under a continuous drying stream of
nitrogen gas.
Mobile phase development:
After so many trials on single
as well as in different composition of various solvent, i.e. benzene, methanol,
water, toluene, ethyl acetate, chloroform, butanol Toluene: Ethyl Acetate: Formic acid (4: 5: 1 %v/v/v)
were selected as mobile phase for chromatographic development. Plates were developed using a mobile phase
consisting of Toluene: Ethyl
Acetate: Formic acid (4: 5: 1 %v/v) Linear ascending development was carried out in a twin-trough glass
chamber equilibrated through chamber saturation with the mobile phase vapours
for 40 min at 25 ºC ± 2 ºC. 10 ml of the mobile
phase (5 ml in the trough containing the plate and 5 ml in the other trough)
was used for each development and was allowed to migrate a distance of 80 mm.
Once development was over, the HPTLC plates were dried completely.
Densitometry analysis:
Densitometric scanning was performed in
the absorbance mode under control by winCATS planar chromatography software.
The source of radiation was the deuterium lamp, and bands were scanned at
λmax 374 nm. The slit dimensions were 5 mm length and 0.45 mm width, with
a scanning rate of 20 mm/s. Concentrations of the compound were determined from
densitometric the intensity of diffusely reflected light and evaluated as peak
areas against concentrations by using a linear regression equation.
RESULT:
Phytochemical screening:
Different extract of M.Indica were used to
identify various phytoconstituent using phytochemical screening method as shown
in Table 1.
High performance thin layer chromatography:
HPTLC method development was achieved with mobile
phase Toluene: Ethyl Acetate: Formic acid (4:5:1% v/v) which gives optimum
result of quercetin with 0.84 Rf value as shown in figure 2.
Figure. 2: Photograph of TLC plate with Toluene: Ethyl
Acetate: Formic acid (4: 5: 1%v/v/v)
Selection of detection wavelength:
The solution was applied in the form of band in
concentration 400 – 2000ng/band for quercetin was prepared in methanol. The
plate was developed using Toluene: Ethyl Acetate: Formic acid (4:5:1%v/v) and
dried in air. After development of TLC plate was scanned in the UV region 200 –
400nm and overlay spectrum was recorded using Camag TLC Scanner 4. The spectra
showed the maximum drug absorbance at 374nm. The spectra of Quercetin show
promising absorbance for Quercetin at 374nm. (figure 3)
Table 1: Phytochemical Analysis of Mangifera indica
leaves by Maceration(M) and Decoction(D)
|
Sr.
No.
|
Chemical Constitute
|
H2O
|
Methanol
|
Ether
|
Chloroform
|
Ethyl Acetate
|
Hydro-Alcoholic (70:30)
|
|
M
|
D
|
M
|
D
|
M
|
D
|
M
|
D
|
M
|
D
|
M
|
D
|
|
1
|
Alkaloids
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
|
2
|
Carbohydrates
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
3
|
Calcium
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
4
|
Chloride
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
5
|
Carbonate
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
6
|
Fats and Oils (Fixed Oil)
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
7
|
Flavonoids
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
|
8
|
Glycosides
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
|
9
|
Iron
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
10
|
Magnesium
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
11
|
Nitrates
|
+
|
+
|
+
|
+
|
+
|
+
|
-
|
-
|
+
|
+
|
+
|
+
|
|
12
|
Phosphates
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
13
|
Potassium
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
14
|
Proteins
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
|
15
|
Steroids
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
|
16
|
Sulphate
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
17
|
Tannins and Phenolic Compound
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
|
18
|
Volatile Oils
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
Figure. 3: UV Spectra of Quercetin
Quantification of quercetin in various extract:
Calibration curve of Quercetin was prepared in the
concentration range 400 – 2000ng/band. Densitogram and 3D Chromatogram were
shown in figure 4. calibration curve was plotted which shows linear response of
the quercetin (figure 5). Data was shown in table 2. With help of regression
equation of quercetin from the calibration data, concentration of quercetin in
different extract of Leave of M.indica were calculated based on the
absorbance value. % of amount of quercetin were shown in table 3.
Figure. 4: HPTLC Chromatogram of Calibration Curve of
Quercetin
From the regression equation concentration of
quercetin was found in different extract which was indicate that highest
concentration was found in ethyl acetate extract were lowest concentration was
found in ether extract. (figure 6) (table 3)
Figure. 5: Calibration curve of Quercetin
Table 2: Calibration curve of Quercetin
|
Sr. No.
|
Concentration (ng/band)
|
Peak Area (AU) (n=5)
|
±SD
|
%RSD
|
|
1
|
400
|
4331.8
|
50.11347
|
1.156874
|
|
2
|
800
|
6360.06
|
70.70088
|
1.111639
|
|
3
|
1200
|
8223.32
|
84.74013
|
1.030486
|
|
4
|
1600
|
9719.86
|
97.34721
|
1.001529
|
|
5
|
2000
|
11476.28
|
117.8446
|
1.026854
|
Figure. 6: HPTLC Chromatogram of different extract with
Quercetin
Table 3: Quantitative Analysis of Quercetin
|
Extract
Concentration
(2000 ng/band)
|
Peak Area (AU)
|
Concentration
found (ng/band)
|
% Quercetin
|
|
Methanol
|
1501.2
|
856.26
|
42.81
|
|
Ethyl acetate
|
2146.5
|
1501.56
|
75.08
|
|
Ether
|
671.7
|
26.76
|
1.34
|
|
Diethyl ether
|
2135.7
|
1490.76
|
74.54
|
|
Chloroform
|
1206.2
|
561.26
|
28.06
|
|
Hydro: Alcoholic
(70:30)
|
795.8
|
150.86
|
7.54
|
|
Quercetin
|
2600.2
|
1955.26
|
97.76
|
DISCUSSION:
The present study involved testing of phytochemical
compounds and development of HPTLC method for Mangifera indica leaves.
Phytochemical Testing:
The phytochemical tests should that after maceration
and decoction the test for Alkaloids, Flavonoids, Glycosides, Steroids,
Glycosides, Proteins, Tannins and Phenolic Compounds, Volatile oils were
present also Carbohydrates, Fats and Oil (Fixed Oil) were absent in the
extracts.
HPTLC Method:
The HPTLC Method was developed using mobile phase
Toluene: Ethyl acetate: Formic acid (4: 5: 1%v/v/v) and stationary phase
pre-coated silica gel G60-F254 aluminum sheet at 374nm. The Rf value
was found to 0.84. The quercetin was found to be 42.81%, 75.08%, 1.34%, 74.54%,
28.06%, 7.54% and 97.76% in Methanol, Ethyl acetate, Ether, Diethyl ether,
Chloroform, Hydro: Alcoholic (70:30), Quercetin respectively.
The highest percentage was found in Ethyl acetate
i.e.; 75.08% and the lowest was found in Ether i.e.; 1.34%
CONCLUSION:
Mangifera indica contain various chemical marker which are responsible
for their action in various disease. Study found that quercetin is present in
all different solvent extract which give Rf value 0.84 in HPTLC.
During the estimation it was found that ethyl acetate extract contain quercetin
in higher concentration and ether extract in lower concentration. Further study
can be carried out to importance of quercetin in Mangifera indica leaf
used in various diseases.
CONFLICT OF INTEREST:
The authors have no conflicts of interest
regarding this investigation.
ACKNOWLEDGMENTS:
The authors would like to thank Indukaka Ipcowala
College of Pharmacy for providing necessary requirement to carry out this
research study.
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