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Lupeol validation and quantification in Heteropogon Contortus (L.) beauv. (spear grass) through high-performance thin-layer chromatography

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Abstract

Abstract Objective: Chromatographic evaluation of triterpenoid i.e. lupeol from methanolic extracts of leaves, stem and inflorescence of Heteropogon contortus. Methods: The High-Performance Thin Layer Chromatography densitometry determination of lupeol was performed by using optimized mobile phase toluene: methanol: formic acid (7:3:0.3 v/v) with a derivatization of freshly prepared anisaldehyde-sulphuric acid. For densitometry measurements, the plates were scanned at 530 nm absorbance/reflectance wavelength. Quantification of lupeol marker compound in H. contortus leaves, stem and inflorescence are estimated using 2-12 μg/spot. Results: The appearance of light purple color bands on the chromatograms confirmed the lupeol component in plant samples. Further, the confirmation of the compound is done from the densitometric scanning by comparing λmax values. From this, it is reported that lupeol is present in leaf samples i.e. 10 mg/g of dry wt., while in rest of the two samples it is found absent. Conclusion: The leaves of H. contortus (spear grass) are good source of lupeol and can be used as an alternate natural source to synthesize herbal drugs to control cancer and other anti-inflammatory agents. The presently selected HPTLC is validated and most accurate for the quantification and identification of lupeol present in the selected plant. The leaves of the species which are rich in lupeol can be used in pharmaceutical industry.
Vol 10, Issue 12, 2017
Online - 2455-3891
Print - 0974-2441
LUPEOL VALIDATION AND QUANTIFICATION IN HETEROPOGON CONTORTUS



Received: 18 July 2017, Revised and Accepted: 05 October 2017

 This is aimed to study the chromatographic evaluation of triterpenoid, i.e., lupeol from methanolic extracts of leaves, stem, and inflorescence
of Heteropogon contortus.
 The high-performance thin-layer chromatography (HPTLC) densitometry determination of lupeol was performed using optimized
mobile phase toluene:methanol:formic acid (7:3:0.3 v/v) with a derivatization of freshly prepared anisaldehyde-sulfuric acid. For densitometry
measurements, the plates were scanned at 530 nm absorbance/reflectance wavelength. Quantification of lupeol marker compound in H. contortus
leaves, stem, and inflorescence is estimated using 2-12 µg/spot.
 The appearance of light purple bands on the chromatograms confirmed the lupeol component in plant samples. Further, the confirmation of
the compound is done from the densitometric scanning by comparing λmax values. From this, it is reported that lupeol is present in leaf samples, i.e.,
10 mg/g of dry wt., while in rest of the two samples, it is found absent.
 The leaves of H. contortus (spear grass) are a good source of lupeol and can be used as an alternate natural source to synthesize herbal
drugs to control cancer and other anti-inflammatory agents. The presently selected HPTLC is validated and most accurate for the quantification and
identification of lupeol present in the selected plant. The leaves of the species which are rich in lupeol can be used in pharmaceutical industry.
 Lupeol, High-performance thin-layer chromatographic, Triterpenoid, Heteropogon contortus.

Triterpenoids are the secondary metabolites and have great
importance in medicinal plants [1]. These are derived from vegetable
oils, cereals, fruits, and vegetables and are consumed at an average of
250 µg/day [2]. Lupeol is an active triterpenoid. It exhibits important
biological activities such as antiprotozoal, antitumor (antiprostate
cancer, anti-melanoma, anti-head and neck squamous cell carcinoma,
and anti-pancreatic cancer) [3-5], nutraceutical or chemopreventive
agent [6-8], anti-inflammatory [9], and hepatoprotective [10].
Heteropogon contortus (L.) Beauv. (= Andropogon contortus L.),
belonging to the family Poaceae (Gramineae), is commonly known as
spear grass, black spear grass, bellary grass, kher (Hindi), gantegawata
(Marathi). It is distributed in Southern Asia, Southern Africa, and
Northern Australia. According to Blake and Richards [11], the grass is
reported to have myo-inositol, galactinol, and raffinose. Polysaccharides
were also observed in the plant species. It is medicinally important
grass and is found useful in toothache, fever, atrophy, emaciation
or cahexy, muscular pain, hematological disorders, dysentery, and
scorpion sting [12,13]. Roots have diuretic and stimulant properties.
The whole plant is used for asthma in the form of extracts or steam
distillation product [14]. Oil distilled from the awns has been found to
be useful in asthma.
Previously, Ghante et al. [15] obtained the results from the methanolic
extract of H. contortus that it is used for the treatment of pathologies
caused by mast cell destabilization, membrane destabilization, and
free radical generation, which mainly include acute and chronic
inflammatory response such as asthma, arthritis, cardiovascular, and
neural diseases. Further, it was reported that H. contortus extract inhibits
bronchoconstriction induced by histamine or acetylcholine [16].
Grasses are very important both economically as well as medicinally
from the ancient time of herbal medicine use [17]. Compared the
antimicrobial activities of ethanolic and hydroalcoholic extract of
Vetiveria zizanioides root (a grass) and analyze the major bioactive
compounds present in those extracts through high-performance
thin-layer chromatography (HPTLC). It also inhibits inflammation
induced by carrageenan and egg albumin. Some medicinally important
grasses also show cytomorphological variations such as meiotic
abnormalities and reduced pollen fertility [18]. Extraction-based
studies were made on Ormocarpum cochinchinense and resulted that
phenols in acetone and terpenoids in methanol extracts give better
results for phytochemical analysis [19]. Thus, during the present study,
methanol is used as a solvent for the quantification of lupeol from
leaves, stem, and inflorescence of H. contortus.


Reference standard, i.e., lupeol was purchased from Himedia. Chemicals
such as toulene, formic acid, and methanol were obtained from S.D.
Fine Chemicals, Mumbai, India. Pre-coated silica gel 60 F254 HPTLC
aluminum plates (20×20 cm2 layer thickness-0.2 mm, 5-6 µm particle
size; E. Merck, Darmstadt, Germany) was obtained from E. Merck Ltd.
(Mumbai, India).

The material for the present study was collected from different
localities of Udaipur district of Rajasthan, India. The plant is
authenticated from Botanical Survey of India, Arid Zone Regional
Center, Jodhpur, Rajasthan. The leaves, stem, and inflorescence (with
awns) were excised from the plant. The collected material is then
washed under running tap water, air-dried, and grinded to form a fine
powder. 5 g dried powder of each plant part was extracted with 150
ml methanol using Soxhlet extractor for 8 hrs. The obtained extracts
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.
org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2017.v10i12.21431
Research Article
393
Asian J Pharm Clin Res, Vol 10, Issue 12, 2017, 392-395
Kaur and Gupta
were then filtered and concentrated using rotary vacuum evaporator
and then lyophilized with Allied Frost Lyophilizer-FD-3. The obtained
lyophilized powder of samples was accurately weighed and then
dissolved in methanol (1 mg/1 ml). These solutions were then used as
test solutions for HPTLC analysis.

Spotting device-CAMAG Linomat V sample applicator (CAMAG,
Switzerland)
Syringe-100 µl Hamilton syringe
TLC Chamber-CAMAG twin trough chamber.
Scanner-CAMAG TLC scanner with D2 and Hg lamp, Reprostar and
win CATS planar chromatography manager and CAMAG integration
software and TLC viewing cabinet (all from CAMAG, Muttenz,
Switzerland).

Accurately weighed reference standard lupeol (5 mg) was transferred
to 10 ml volumetric flask, dissolved in 5 ml methanol (1 mg/1 ml). The
stock solution was ready to use for HPTLC.

The HPTLC analysis was performed using precoated silica gel 60 F254
aluminum plates. Linomat V autosampler was used for spotting of
standard lupeol (2, 4, 6, 8, 10, and 12 µl each) and sample solutions
(10 µl each) operated with 6 mm band length, distance between the
tracks 10 mm, distance from the bottom of the plate, 8.0 mm. The
linear ascending developments were carried out up to a distance of
75 mm in a CAMAG twin trough chamber presaturated with mobile
phase toluene:methanol:formic acid (7:3:0.3 v/v) for 25 minutes, and
anisaldehyde is used as derivatizing reagent. The plates were then
scanned in the CAMAG TLC scanner V, operated by win CATS software
in the absorbance mode at 530 nm. The images of the plates were then
taken in the visible mode.

The standard stock solutions (1 mg/1 ml) of lupeol (2-10 µg spot−1) were
applied in triplicate on an HPTLC plate. These plates were developed
with the mobile phase toluene:methanol:formic acid (7:3:0.3 v/v) for
25 minutes. After development, the plates were air-dried and scanned
at 530 nm absorbance using deuterium lamp. The resolved peak areas
were recorded for all the standards separately. The calibration curve
of lupeol was plotted by taking peak area versus concentrations of
standards (Fig. 1).

The HPTLC method was validated using the ICH guidelines for
specificity, linearity, limits of detection and quantification, precision
and accuracy, robustness, and stability [20].

The specificity was revealed by analyzing the standard marker
compound and plant samples. The band of lupeol was confirmed by
comparing the Rf and ultraviolet (UV) spectra of the spots to those of
the standard.

The different concentrations (2, 4, 6, 8, 10, and 12 ng spot−1) of lupeol
were applied on the HPTLC plate in triplicates, separately. The plate
was developed as prescribed above. The calibration curve was drawn
by plotting the standard concentrations ranges from 2 to 12 µg spot−1
versus peak area. The linear calibration curve was obtained from
which linear regression equation and correlation coefficients were
obtained.

The LOD and LOQ for the present marker compound, i.e., lupeol was
calculated using the following equations:
LOD=3.3 SD/S
LOQ=10 SD/S
Where SD stands for the standard deviation of replicates under the
same conditions, and S is the slope of the calibration curve.

Robustness was studied in triplicate at 800 ng spot−1 by making small
changes to the volume of the mobile phase, the composition of the
mobile phase, and saturation time of development chamber. The effects
on the results were examined by calculation of relative SD (RSD) (%)
and Rf values.

Instrumental precision was checked by repeated scanning (n=6) of
same spot for each standard separately (10 µg spot−1) and expressed
as % RSD. The accuracy of the present method was checked using a
recovery study by spiking the sample with two levels of standards
(6 and 12 µg band−1). The % recovery was calculated using the formula
given by the [21].

The samples prepared from leaves, stem, and inflorescence of
H. contortus were analyzed by using the validated HPTLC method, and
the amount of the referred standard was calculated from the standard
calibration curve of lupeol.

During the present investigation, chromatographic conditions were
optimized for the detection of lupeol using modified mobile phase
toluene:methanol:formic acid (7:3:0.3 v/v) which gave the better


Heteropogon
contortus
394
Asian J Pharm Clin Res, Vol 10, Issue 12, 2017, 392-395
Kaur and Gupta
results, i.e., sharp and well-resolved bands of lupeol at Rf value of 0.21
(Fig. 2). Besides, the plates were derivatized with freshly prepared
anisaldehyde-sulfuric acid and were heated at 110-120°C and
scanned for densitometry measurements at an absorbing wavelength
of 530 nm (Fig. 3). The identity and purity of the bands of selected
sterols resolved in the plant samples (leaves, stem, and inflorescence)
were confirmed by overlaying their UV-visible absorption spectra
with those of the standard compound using TLC scanner V (Fig. 4).
Line-to-line overlay spectra of leaf sample were obtained which
shows that the identity and purity of the bands matches with those of
the standard compound while stem and inflorescence show little bit
variation in λmax. It shows that lupeol is absent in leaf and inflorescence
of H. contortus.
The method is validated as per the ICH guidelines in terms of
precision, repeatability, and accuracy (Table 1). The linearity range
for lupeol was found to be 2-12 µg spot−1 with correlation coefficient,
i.e., 0.997. A linear calibration curve was obtained for the standard
compound as described above. LOD value for a standard compound
is 0.84 ng spot−1, whereas LOQ value is 0.25 ng spot−1. The average
% recovery at 3 different levels of the referred marker compound
was found to be 99.45 (Table 1). The accuracy of the present method
was determined by spiking the sample along with known amounts of
standard (100 µg band−1). The present method showed a recovery of
99.05% for the addition of 100 µg band−1 of standard correspondingly
(Table 2).
The results of quantification of lupeol are given in Table 3. During the
present investigation, lupeol is only detected in leaf samples, whereas
in rest of the two samples, i.e., stem and inflorescence, it is found to be
absent. The amount of lupeol present in the leaf sample is 10±0.13 mg/g
of dry wt.

The presently selected HPTLC is validated and most accurate for the
quantification and identification of lupeol in medicinally important
grass H. contortus. The developed method has been found to be
sensitive, accurate, precise, specific, and robust for the screening and
quantification of sterols. Although, HPTLC has a few limitations like
limited developing distance and lower plate efficiency in comparison
to HPLC and gas chromatography. HPTLC is still an effective tool for
quality evaluation of medicinal plants due to its simplicity, low cost, and
low requirements. Thus, leaves of the species which are rich in lupeol
can be used in pharmaceutical industry.



Parameters Lupeol
Wavelength (nm) 530
Rf0.28
Selectivity Selective
Specificity Specific
Linearity range (µg/spot) 2-12
Correlation coefficient (R2) 0.997
Linear regression equation (y) 2762×
LOD (ng spot−1) 0.84
LOQ (ng spot−1) 0.25
Accuracy (average % recovery) 99.05
LOD: Limit of detection, LOQ: Limit of quantification

plant samples of Heteropogon contortus
max

H. contortus

 Amount present

 

  

100 75 175 172.62 98.64
Lupeol 100 100 200 198.46 99.23 99.05
100 125 225 223.41 99.29
HPTLC: High-performance thin-layer chromatography

H. contortus
Plant parts  

Leaves HCLM 10±0.13
Stem HCSM ND
Inflorescence HCIM ND
ND means not detected. H. contortus: Heteropogon contortus
of this study. The authors are also
thankful to Head, Department of Botany, Punjabi University, Patiala.
The authors would like to express their profound gratitude and sincere
appreciation to UGC-BSR Single Girl Child Fellowship (Award
letter no. and dated. F7-152/2007 BSR; 16-12-2013) and
DBT-IPLS project (Project no. BT/PR-4548/INF/22/146/2012)
sanctioned to Navjot Kaur, Punjabi University, Patiala, for using
the facilities and financial support
395
Asian J Pharm Clin Res, Vol 10, Issue 12, 2017, 392-395
Kaur and Gupta

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... Roots of the plant have diuretic and stimulant action and the plant has been also used in the treatment of asthma, bronchial diseases, jaundice, toothache, fever, dysentery, muscular pain and scorpion sting (Ghante et al., 2013;Jayaweera, 1981) [10,11] . The broncho relaxant activity and anti-inflammatory activity of the plant and the quantification of lupeol from leaves, stem, and inflorescence of plant have been well-established (Ghante et al., 2013;Kaur and Gupta, 2017) [10,12] . In present investigation, an attempt has been taken to investigate the antimicrobial activity of the aqueous extracts of whole plant, leaves, flowers, stem and roots of the Sri Lankan grass H. contortus against clinically important two pathogenic bacteria Staphylococcus aureus and Escherichia coli and to evaluate the phytochemical composition of the plant. ...
... Roots of the plant have diuretic and stimulant action and the plant has been also used in the treatment of asthma, bronchial diseases, jaundice, toothache, fever, dysentery, muscular pain and scorpion sting (Ghante et al., 2013;Jayaweera, 1981) [10,11] . The broncho relaxant activity and anti-inflammatory activity of the plant and the quantification of lupeol from leaves, stem, and inflorescence of plant have been well-established (Ghante et al., 2013;Kaur and Gupta, 2017) [10,12] . In present investigation, an attempt has been taken to investigate the antimicrobial activity of the aqueous extracts of whole plant, leaves, flowers, stem and roots of the Sri Lankan grass H. contortus against clinically important two pathogenic bacteria Staphylococcus aureus and Escherichia coli and to evaluate the phytochemical composition of the plant. ...
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