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Kanagavalli et al Journal of Drug Delivery & Therapeutics. 2018; 8(5-s):332-337
ISSN: 2250-1177 [332] CODEN (USA): JDDTAO
Available online on 15.10.2018 at http://jddtonline.info
Journal of Drug Delivery and Therapeutics
Open Access to Pharmaceutical and Medical Research
© 2011-18, publisher and licensee JDDT, This is an Open Access article which permits unrestricted
non-commercial use, provided the original work is properly cited
Open Access Research Article
Isolation and characterization of bioactive compound anthraquinone
from methanolic extract of Boerhavia diffusa linn.
Mrs. U. Kanagavalli1*, Dr. A. Mohamed Sadiq2
1*Assistant Professor, Department of Biochemistry, Adhiparasakthi College of Arts and Science, G.B. Nagar, Kalavai, Vellore
District, Tamilnadu, India
2Principal, Adhiparasakthi College of Arts and Science, G.B. Nagar, Kalavai, Vellore District, Tamilnadu, India
ABSTRACT
Boerhavia diffusa Linn. (Nyctaginaceae), commonly known as ‘Punarnava’ is a perennial creeping herb widely studied and has a
long history of uses by the tribal people and in Ayurvedic and Unani medicines. Our previous study showed that in different extracts
of Boerhavia diffusa (ethanol, methanol, petroleum ether, aqueous and hexane), methanolic extract had significant anti-
oxidant activity, but the active components present in that extracts are still unclear. In this study, Boerhavia diffusa Linn was
investigated for the bioactive compounds present in its methanolic extract. Separation and purification of the compounds in the most
active methanol extract was done using a combination of column chromatography and thin layer chromatography. The compound
was identified using gas chromatography and mass spectrophotometry. The resulted compound anthraquinone which is also called as
9, 10 anthracenedione. In addition to their known use as natural dyes, it have several biological activities like antitumor,
antiinflammatory, diuretic , antiarthritic, antifungal, antibacterial, antimalarial and antioxidant activities . This justifies the use of
this plant in traditional medicine and indicates a promising potential for the development of medicinal agents from Boerhavia
diffusa.
Keywords: Antioxidant, Boerhavia diffusa Linn, Anthraquinone, Column chromatography, Thin layer Chromatograhy, GC-MS.
Article Info: Received 17 Sep, 2018; Review Completed 08 Oct 2018; Accepted 09 Oct 2018; Available online 15 Oct 2018
Cite this article as:
Kanagavalli U, Sadiq AM, Isolation and characterization of bioactive compound anthraquinone from methanolic
extract of Boerhavia diffusa linn., Journal of Drug Delivery and Therapeutics. 2018; 8(5-s):232-237
DOI: http://dx.doi.org/10.22270/jddt.v8i5-s.1987
*Address for Correspondence:
Mrs. U. Kanagavalli, Assistant Professor, Department of Biochemistry, Adhiparasakthi College of Arts and Science, G.B. Nagar,
Kalavai- 632506. Vellore District, Tamilnadu, India
INTRODUCTION
The use of natural products with therapeutic properties
is an ancient as human civilization and for a long time.
Recently, herbal medicines have increasingly been used
to treat many human diseases. Numerous studies were
carried out on plants with antioxidant properties.
However, there is still great interest in finding new
antioxidants from natural sources1. Plant materials are a
rich source of biologically active metabolites. The active
secondary metabolites produced by some of these plants
have potential bioactive compounds of interest in the
pharmaceutical industry. Plant-derived substances have
recently become of great interest due to their
applications as drugs, as model compounds for drug
synthesis or as intermediates for synthetic drugs2.
Boerhaavia diffusa Linn.(F: Nyctaginaceae) a
medicinal plant as a whole was commonly known
to the world as “Spreading hogweed” and in
Sanskrit as “Punarnava”, is widely distributed over
the tropical, subtropical and temperate regions of
the world. It is traditionally used mostly in treating
different ailments like asthma, urinary disorders,
leucorrhea, rheumatism, and encephalitis3.
Chromatographic techniques have significant role in
natural products chemistry as well as contribute
dramatically in the discovery of novel and innovative
compounds of pharmaceutical and biomedical
importance. This study focused on step-by-step visual
demonstration of fractionation and isolation of
biologically active plant secondary metabolites using
Kanagavalli et al Journal of Drug Delivery & Therapeutics. 2018; 8(5-s):332-337
ISSN: 2250-1177 [333] CODEN (USA): JDDTAO
column-chromatographic techniques. Isolation of
bioactive compounds using column-chromatographic
involves preparation of sample, packing of column,
pouring of sample into the column, elution of fractions;
and analysis of each fractions using thin layer
chromatography. However, depending on nature of
research, compounds can be further identified using
GCMS (gas chromatography Mass spectrophotometry.
This paper focus about the isolation, purification and
characterization of bioactive compound anthraquinone, a
cardiac glycoside which is used for cardioprotective
activity and anticancer activity.
MATERIALS AND METHODS
1. Collection of Plant material:
Healthy, ailment free whole Boerhaavia diffusa L. plant
were collected from Samanthipuram village, Arcot,
Tamil Nadu. The taxonomical identification was done
by Dr. P. JAYARAMAN, Director, Plant anatomy
research center, Chennai and voucher specimen was
kept for further reference with register number
PARC/2014/2078.
2. Preparation of extract:
The plant was shade dried and ground to make fine
powder that was then extracted with methanol. The
extract solution was evaporated under reduced pressure
to obtain solid crude extract.
3. Column-chromatography:
A cylinder shaped glass column containing stationary
phase (silica gel) is encountered slowly from the top
with a liquid solvent (mobile phase) that flows down
the column with the help gravity or external pressure
applied. This technique is used for the purification of
compounds from a mixture. Once the column is ready,
the sample is loaded inside the top of the column. The
mobile solvent is then allowed to flow down through the
column. The compounds in mixture have different
interactions ability with stationary phase (silica gel), and
mobile phase, thereby will flow along the mobile phase
at different time intervals or degrees. In this way, the
separation of compounds from the mixture is
achieved. The individual compounds are collected as
fractions and analyzed further for structure elucidation.
Isolation and purification of bioactive
compounds
from plant samples
A suitable size long cylindrical glass column (based on
the amount of the sample) should be stand firm on a
column-chromatography stand. Completely dried plant
extract sample should be mixed with silica gel to make
a fine powdered form for easy distribution of sample in
already packed silica gel column. Sample powdered
mass should be placed on the top of the pre-packed
silica column and sample should be covered with a
layer of cotton. Then solvents of different polarities
were passed through column at uniform rate under
gravity to fractionate the sample extract. Each fraction
was collected separately in a test tube and numbered
consecutively for further analysis on thin layer
chromatography.
Thin layer chromatography (TLC) provides partial
separation of both organic and inorganic materials using
thin-layered chromatographic plates especially useful for
checking the purity of fractions. Each fraction is applied
on activated TLC plates with the help of capillary tube
at a 1/2 inch apart from the lower edge of TLC plate,
and plate is kept in a developing chamber containing
suitable solvent system for specific time until the
developing solvent reaches top of the upper edge of
TLC plate. Plate is taken out from developing chamber,
dried and solvent front is marked by lead pencil.
Compound bands/spots visualized on TLC
chromatoplate can be detected by visual detection,
under UV light (254 nm), in iodine chamber and by
using spray reagent (vanillin-sulfuric acid) for the
presence of specific compounds. The visualized spots of
the components in the chromatoplate are marked and the
Rf
value of each spot is calculated by the formula:
Rf =
distance travelled by the sample (cm)/distance
travelled by the solvent (cm). TLC plate showing
number of bands (compounds) for each fraction can be
further compounds are identified using Gas
chromatography Mass spectrophotometry.(GCMS )5.
4. Gas chromatography mass spectrophotometry:
GC-MS analysis of the methanolic extract of B. diffusa
was performed using a Perkin–Elmer GC Clarus 500
system comprising an AOC-20i auto-sampler and a Gas
Chromatograph interfaced to a Mass Spectrometer (GC-
MS) equipped with a Elite-5MS (5% diphenyl/95%
dimethyl poly siloxane) fused a capillary column (30 ×
0.25 μm ID × 0.25 μm df). For GC-MS detection, an
electron ionization system was operated in electron
impact mode with ionization energy of 70 eV. Helium
gas (99.999%) was used as a carrier gas at a constant
flow rate of 1 ml/min, and an injection volume of 2 μl
was employed (a split ratio of 10:1). The injector
temperature was maintained at 250 °C, the ion-source
temperature was 200 °C, the oven temperature was
programmed from 110 °C (isothermal for 2 min), with
an increase of 10 °C/min to 200°C, then 5 °C/min to
280°C, ending with a 9 min isothermal at 280 °C. Mass
spectra were taken at 70 eV; a scan interval of 0.5 s and
fragments from 45 to 450 Da. The solvent delay was 0
to 2 min, and the total GC/MS running time was 36 min.
The relative percentage amount of each component was
calculated by comparing its average peak area to the
total areas. The mass-detector used in this analysis was
Turbo-Mass Gold-Perkin-Elmer, and the software
adopted to handle mass spectra and chromatograms was
a Turbo-Mass ver-5.2.6
RESULT AND DISCUSSION
Purification and isolation of bioactive compounds from
plants is a technique that
has
undergone new
development in recent years 7,8. This modern technique
offers the ability to parallel the development and
availability
of many advanced bioassays on the one
hand, and provided precise techniques of isolation,
separation, and purification on the other. The goal when
searching for bioactive compounds is to find an
appropriate method that can screen
the
source
material
for bioactivity
such
as antioxidant, antibacterial, or
Kanagavalli et al Journal of Drug Delivery & Therapeutics. 2018; 8(5-s):332-337
ISSN: 2250-1177 [334] CODEN (USA): JDDTAO
cytotoxicity, combined with simplicity, specificity, and
speed
9
.
In vitro methods are usually more desirable than in vivo
assays because animal experiments are expensive, take
more time, and are prone to ethical controversies. There
are some factors that make it impossible to find final
procedures or protocols to isolate and characterize
certain bioactive molecules. This could be due to
different parts (tissues) in a plant, many of which will
produce quite different compounds, in addition to the
diverse chemical structures and physicochemical
properties of the bioactive phytochemicals10.
Figure 1: Isolation of compound through column chromatography
Purification of the Bioactive Molecule.
Many bioactive molecules have been isolated and
purified by using paper thin-layer and column
chromatographic methods. Column chromatography and
thin-layer chromatography (TLC) are still mostly used
due to their convenience, economy, and availability in
various stationary phases. Silica, alumina, cellulose, and
polyamide exhibit the most value for separating the
phytochemicals. Plant materials include high amounts of
complex phytochemicals, which make a good separation
difficult. Therefore, increasing polarity using multiple
mobile phases is useful for highly valued separations.
Thin-layer chromatography has always been used to
analyze the fractions of compounds by column
chromatography. Silica gel column chromatography and
thin-layer chromatography (TLC) have been used for
separation of bioactive molecules with some analytical
tools11.
Figure 2: Thin layer chromatogram identified spot at different fractions using chloroform:methanol(9:1) solvent system.
Figure 3: Thin layer chromatogram showing fractions in short UV.
Kanagavalli et al Journal of Drug Delivery & Therapeutics. 2018; 8(5-s):332-337
ISSN: 2250-1177 [335] CODEN (USA): JDDTAO
Figure 4: Thin layer chromatogram showing fractions in Long UV.
The visualized spots of the components in the
chromatoplate were marked and the Rf value of each
spot was calculated by the formula: Rf = distance
travelled by the sample (cm)/distance travelled by the
solvent (cm).The several fractions gave a many spot at
different Rf on silica gel 60 F254 pre-coated aluminum
plate, of 0.2 mm thickness using Chloroform: Methanol
(9:1) as the developing solvent system. Visualization
was carried out by dipping the plate in vanillin-
sulphuric acid reagent and heating at 105°C till the color
of the spot appeared (Figure:2). Thin layer
chromatogram fractions in long and shot UV is shown in
figure 3 and 4.
Structural Clarification of the Bioactive
Molecules
Determination of the structure of certain molecules
uses
data from a wide range of spectroscopic techniques such
as UV-visible, Infrared ( IR), Nuclear Magnetic
Resonance (NMR), and mass spectroscopy. The basic
principle of spectroscopy is passing electromagnetic
radiation through an organic molecule that absorbs some
of the radiation, but not all. By measuring the amount of
absorption o
f
electromagnetic radiation, a spectrum can
be produced. The spectra are specific to certain bonds
in
a molecule. Depending on
these
spectra, the structure of
the organic molecule can be
identified.
Scientists
mainly
use
spectra produced from either three or four
regions—Ultraviolet
(UV),
V
isible, Infrared (IR), radio
frequency, and electron beam
for structural
clarification12.
Figure 5: GCMS Chromatogram fractions isolated from methanolic extract of Boerhavia diffusa containing
anthraquinone.
Kanagavalli et al Journal of Drug Delivery & Therapeutics. 2018; 8(5-s):332-337
ISSN: 2250-1177 [336] CODEN (USA): JDDTAO
Table 1: GCMS Data of bioactive compounds detected with their percentage area and retention time.
Figure 6: GC-MS data analysis identified compound as anthraquinone.
The results pertaining to GC-MS analysis of the isolated
fraction from methanolic extract of Boerhavia diffusa
lead to the identification of a bioactive compound
anthraquinone. This compound is identified through
mass spectrometry attached with GC at Retention time
(34.834). The detected compound present in the entire
herb of Boerhavia diffusa by the GC-MS was shown in
[Table 1 ].
The GC-MS spectrum confirmed the presence of
bioactive components with different retention times as
illustrated in [Figure 5, 6]. The mass spectrometer
analyzes the compounds eluted at different times to
identify the nature and structure of the compounds. The
large compound fragments into small compounds giving
rise to appearance of peaks at different m/z ratios. These
mass spectra are fingerprint of that compound which can
be identified from the data library.
These results reveals that the plant has the antioxidant
compound anthracenedione (Also called as
Anthraquinone) and further investigation required to
identify the cardioprotective activity of this compound
through biochemical parameters, gene expression study
and histopathological study.
Kanagavalli et al Journal of Drug Delivery & Therapeutics. 2018; 8(5-s):332-337
ISSN: 2250-1177 [337] CODEN (USA): JDDTAO
CONCLUSION
Phytochemicals are naturally occurring and biologically
active plant compounds that have potential disease
inhibiting capabilities. Hence the medicinal values of
these plants lie in their phytochemicals, which produce
definite physiological actions on the human body. This
is the first report that studied isolation and identification
of anthraquinones from methanolic extract of Boerhavia
Diffusa Linn. This identified important compound which
may be used to develop biopharmaceuticals against
infectious diseases with antioxidants source in future.
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