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LC-MS studies and antioxidant potential of the
root extracts of Achyranthes aspera Linn
Nafisehsadat Omidiani1, Dr. Rajkumar. B. Barmukh2
1,2Post Graduate Research Centre, Department of Botany, Modern College of Arts, Science and Commerce, Pune-
411005, Savitribai Phule Pune University, Pune-411007, Pune, India.
Abstract
Achyranthes aspera L. (Amaranthaceae) is an erect, annual herb. It is an herbaceous roadside weed that grows in various parts of
India and possesses several types of pharmaceutical properties. This investigation was conducted to identify the bioactive compounds
that confer antioxidant properties to the extracts from its biomass. According to the results, the root-ethyl acetate (R-EA) extracts of
A. aspera showed the highest antioxidant activity and exhibited 66.78±1.65 scavenging of DPPH radicals by 500 μg extract. This
extract was subjected to thin layer chromatography (TLC). According to the results, root-ethyl acetate revealed fourteen bands.
Among all solutions of root biomass, the solution obtained from the second band (Rf=0.16±0.02) exhibited the maximum percentages
of antioxidant activity (25.11±4.05 %). LC-MS analysis of this solution showed the presence of a few compounds have been reported
to possess antioxidant properties in different plants.
Index Terms: Achyranthes aspera L.; antioxidant activity; DPPH assay; TLC; LC-MS.
1.Introduction
Antioxidants protect cells against the damaging effects of reactive oxygen species (ROS). ROS such as superoxide anion (O2−),
hydrogen peroxide (H2O2), and hydroxyl radical (HO•) are generated due to the partial reduction of oxygen as a part of aerobic
metabolism in the body. In small doses, ROS are beneficial, play physiological roles, and are involved in signaling processes [13].
However, ROS overproduction can induce an inflammatory response, and inflammatory mediators can induce oxidative stress [4].
An imbalance between antioxidants and ROS can lead to ROS pathological quantities that can trigger coronary heart disease,
Alzheimer's disease, and cancer. Therefore, using plants that contain antioxidants decreases the risk of such diseases in the human
body. Plants are rich in secondary metabolites that possess antioxidant activities such as phenolics, flavonoids and anthocyanins.
Natural phenolic antioxidants can control various pathologies induced by oxidative stress [11] and several antioxidant-rich natural
products have protective effects against inflammation [26]. Moreover, antioxidants act as anti-aging, anticancer and anti-diabetic
agents.
Achyranthes aspera L. (Amaranthaceae) is a procumbent or erect, annual herb. It is a herbaceous roadside weed that grows in various
parts of India [20]. Since ancient times, nature has been the ultimate reservoir of medicinal agents, and many modern drugs have
been isolated from natural sources [7]. Medicinal plants contain numerous phytochemicals that confer beneficial and medicinal
properties to maintain health and cure or reduce the symptoms of diseases. Approximately 25% of modern pharmacopeia drugs are
derived from plants, and many others are synthetic analogs built on natural compounds isolated from plants [22].
Medicinal plants are a rich source of secondary metabolites. Phytochemicals play diverse roles in plant life and protect plants through
disease resistance, protection against various stresses, and defense mechanisms [12]. These bioactive components include alkaloids,
amines, steroids, glycosides, phytoestrogens, carotenoids, phytosterols, glucosinolates, terpenoids, and flavonoids [14]. Most of these
secondary metabolites have shown bioactivities, including anti-diabetic and antioxidant activities [10].
In the DPPH radical scavenging assay, the ethyl acetate and aqueous extracts of whole plants of A. aspera were shown to have the
highest antioxidant potential with IC50 values of 0.96 and 0.76 μg per ml, [2]. The leaf, stem, and inflorescence of A. aspera extracted
in different solvents have different antioxidant and antibacterial activities depending on the concentration and assay duration [1].
The roots and inflorescence of A. aspera contain tannins (up to 1262.50 mg/50 g d.w.), flavonoids (up to 1023.81 mg/ 50 g d.w.),
and proanthocyanidins (up to 600 mg /50 g d.w.), which are important antioxidants of plant origin [18]. Hence, the present
investigation was undertaken to determine the potential antioxidant activity of A. aspera root extracts using the DPPH assay.
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2.Methodology
Collection of plant material
Fresh Achyranthes aspera L. plants were collected in August from the campuses of Savitribai Phule Pune University and Fergusson
College, Pune. The collected plant specimens were identified using the Cooks’ Flora of Presidency of Mumbai and the identification
was authenticated from the Botanical Survey of India, Western Circle, Pune. Roots of the plants were separated and washed in
running tap water and cut into small pieces. The fresh biomass was layered on sheets of filter paper and dried in the shade at room
temperature for one week. The dried biomass was powdered using a mechanical grinder and the powders were stored at -20 °C in a
polythene zipper bag.
Extraction of phytochemicals
Phytochemicals from the powdered root (R) biomass were extracted in a Soxhlet extractor separately in ethanol (Et), ethyl acetate
(EA), acetone (Ac), distilled water (DW), and methanol (Me). Twenty grams of biomass was wrapped in Whatman No. 1 filter paper
and extracted for five h in 190 mL of solvent. The extract was suction filtered using a Whatman No. 1 filter paper disc placed in a
Buchner funnel. The extract was evaporated to dryness in a 300 mL beaker situated in a hot water bath. The dried residue was
measured using an electronic balance (CAS-250, Contech Instruments Ltd., India). The residue was suspended in 5.0 mL of solvent
for nine h, after which, it was centrifuged at 5000 rpm. The clear supernatant was collected in glass vials and stored at -20 °C for
further use. Thus, five types of root extracts R-EA, R-Ac, R-Et, R-Me, R-DW were prepared. Each of these extracts was diluted with
the solvent used for extraction to yield a working solution with concentration of 500 μg/mL.
In vitro antioxidant assay
DPPH based antioxidant assay
DPPH (1,1-diphenyl-2-picrylhydrazyl) is an oxidant having an odd electron in its structure. Its purple color is changed to yellow-
colored diphenylpicrylhydrazine when it is in contact with an antioxidant that can release a hydrogen atom or electron.
The DPPH radical scavenging assay [5] was used to evaluate the antioxidant potential of the extracts. The 3.0 mL test reaction
mixture consisted of one mL extract containing 500 μg dissolved residue and 1.0 mL 1 mM DPPH prepared in methanol. The solvent
used for extraction was used to make the or a 3.0 mL volume. The control was set with 1.0 mL of 1 mM DPPH mixed with 2.0 mL
methanol. The spectrophotometer (BioEra, India) was standardized using a 3.0 mL mixture of extract containing 500 μg dissolved
residue mixed with 1.0 mL methanol and the extraction solvent to make a final volume of 3.0 mL.
The reaction mixtures were incubated in the dark at room temperature for 20 min, and the absorbance was measured at 517 nm. Each
experiment was performed in triplicates.
The percentage scavenging of DPPH was calculated according to the following formula:
% DPPH radical scavenging= [(Ac-At)/Ac] × 100
Where Ac is the absorbance of the control mixture and at is the absorbance of the test mixture
A series of 100 to 500 μg/mL of each extract was prepared separately and treated to assess DPPH scavenging potential as described
earlier. The IC50 value of each extract was calculated from the dose-response curve. IC50 values were used to express the ability of
the extracts to scavenge 50% of DPPH. The term “IC50,” which suggests the extract's concentration needed to scavenge 50% of
DPPH radical, was calculated by plotting the dose-response curve using Microsoft Excel software.
DPPH scavenging activity of ascorbic acid
Ascorbic acid is a strong antioxidant that is used as a standard antioxidant substance in most antioxidant assays. A series of ascorbic
acid concentrations were prepared in the range of 0 to 10 μg per mL and tested for its antioxidant activity as described in 6.2.2. A
dose-response curve plotted in the MS Excel program was used to calculate the IC50 of ascorbic acid, i.e., the concentration of
ascorbic acid required to scavenge 50% of DPPH.
Estimation of phenolics
Total phenolics were estimated by Swain and Hillis's (1959) method, and gallic acid (100 μg/mL) was used as a standard phenol to
estimate the phenolic content in the extracts. A series of concentrations from 0 to 100 μg gallic acid was made, and to each test tube,
7.0 mL DW, 0.5 mL Folin Ciocalteu’s phenol reagent, and 1.5 mL 20% Na2CO3 were added. The contents were thoroughly mixed,
and the absorbance was recorded at 760 nm on a spectrophotometer. The blank was set with DW. The amount of extract containing
100 μg dissolved residue was used to estimate phenolic content and the extracts were treated in the same way as the standard gallic
acid solution.
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Estimation of flavonoids
Total flavonoids were estimated using the method of Balbaa et al. (1974), and rutin (100 μg/mL) was used as a standard flavonoid.
A series of concentrations from 0 to 100 μg rutin was made, and to each test tube, 1.5 mL 95% methanol, 10% methanolic aluminum
chloride, and 2.9 mL DW were added. The contents were mixed thoroughly, and the tubes were incubated at 30 ºC in the dark. After
the incubation period, the absorbance was recorded at 420 nm on a spectrophotometer. The blank was set with DW. Finally, the
absorbance was plotted against the amount of rutin to draw a standard curve.
The volume of extract containing 100 μg dissolved residue was used to estimate flavonoid content and the extract was treated in the
same way as the standard rutin solution.
Thin layer chromatography (TLC) separation of extracts
Five types of extracts prepared from root biomass were tested for antioxidant activity. The extract from the root biomass that showed
the highest antioxidant activity was separated on a preparatory TLC plate for the separation of phytochemicals
The pre-coated silica plates (TLC Silica Gel 60 F254, Merk, India) were utilized for the separation. A preparative TLC plate of 200
mm × 200 mm was used to separate 350 μg dissolved residue in the best solvent system toluene: ethyl acetate: acetic acid (18:2:0.5)
identified from the separation on analytical plates. Two such plates were simultaneously developed in one chromatography chamber.
At the end of separation, the solvent front was marked, and the Rf value of each visible band was calculated.
Elution of separated bands
The TLC plates were air-dried, and each band was scraped off from the plate by using a scalpel, and the powder of two identical
bands from two plates was pooled and suspended in 500 μL of ethyl acetate for eight h. The suspension was then centrifuged to
eliminate the silica powder, and the clear supernatant was stored in glass vials. The solution thus prepared from each band was
subjected to antioxidant testing using DPPH assay. The solution with the highest percent inhibition was subjected to LC-MS analysis.
Among the five extracts, the R-EA extract showed the highest DPPH radical scavenging activity. When subjected to TLC separation
as mentioned earlier, R-EA, produced 14 distinct bands on a preparatory TLC plate. The Rf values of these bands ranged between
0.12±0.01 to 0.71±0.02. The antioxidant activity shown by the extracts prepared from these 14 bands is presented in (Table 2).
According to the result, among 14 bands of R-EA extract, the R-EA-B2 and S-EA-B1 showed the highest (25.11±4.05%) and lowest
(3.00±1.54%) percentage scavenging of DPPH radicals. Following the R-EA-B2 was subjected to the LC-MS analysis. From the
data on LC-MS analysis, phytochemicals with the known antioxidant activity were searched in the literature.
3.Results
Phenolic and flavonoid content in the extracts
Out of ten different extracts of root biomass, the R-EA extract showed the highest contents of phenolics and flavonoids. Hundred µg
R-EA extract was equivalent to 4.83±0.97 µg gallic acid (phenolics) and 2.69±0.59 µg rutin (flavonoids). The R-EA extract was thus
superior over all other extracts for the phenolics and flavonoid contents. Among the root extracts, the phenolic content in the R-EA
extract was about eight times higher than the lowest content observed in the R-DW extract. As for flavonoid content, the R-EA
extract had almost 18 times higher flavonoid content than the R-Ac extract that showed the lowest flavonoid (0.15±0.02 µg) content
in terms of rutin equivalence with 100 µg extract.
To summarize, the R-EA extract was superior over all other extracts in having the highest contents of phenolics and flavonoids in
terms of gallic acid and rutin equivalence, respectively, per 100 µg extract.
On the other hand, phenolics and flavonoids are known to be potent antioxidant substances. In all five root extracts prepared from
A. aspera, phenolics and flavonoids were present at varying concentrations (Table 1). In general, the levels of phenolics and
flavonoids did not follow any pattern concerning the polarity of the solvents used for extraction.
.
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Table 1 Phenolics and flavonoids in various root extracts of Achyranthes aspera
Extract type
Gallic acid
equivalence of
100 μg extract
Rutin equivalence of 100
μg extract
% scavenging of
DPPH radicals by
500 μg extract
IC50 for % DPPH
scavenging
R-EA
4.83±0.97
2.69±0.59
66.78±1.65
466.85±11.79
R-Ac
0.81±0.26
0.15±0.02
44.22±1.42
449.23±13.75
R-Et
1.73±0.08
0.91±0.08
51.91±2.35
450.67±20.21
R-Me
3.84±0.86
1.59±0.04
50.21±1.04
454.33±09.50
R-DW
0.59±0.04
0.21±0.06
13.93±0.45
1712.35±18.45
(Values represent mean±SD. Values followed by similar letters in a column do not differ significantly at p =0.05 as per DMRT
performed separately for a group of extracts from root biomass).
Antioxidant status of A. aspera extracts
Each of the ten extracts prepared from roots of A. aspera showed the potential to scavenge free radicals of DPPH
(Table 1). The percent scavenging of DPPH radicals by 500 μg/mL extract ranged from a minimum of 13.93±0.45 %
(R-DW extract) to a maximum of 66.78±1.65% (R-EA extract).
According to the results, the R-Et and R-Me extracts showed almost the same levels of antioxidant activities (50-
51%). The R-DW extract showed the lowest DPPH scavenging potential, and it was about 80% lower (13.93±0.45%)
than the R-EA extract.
The inverse relationship between the antioxidant potential of extract and its IC50 value is evident in Table 1. Thus,
the extracts that showed the highest antioxidant potential had the lowest IC50 value. Among the 5 extracts, R-EA
extract showed the highest antioxidant potentials, and consequently, showed the lowest IC50 values of 466.85±11.79
μg/mL. Therefore, the R-EA extract of A. aspera biomass were separated by TLC.Next, the separated bands on the
TLC plates were eluted in the solvent used for extraction as described above. and the extracts thus obtained were
subjected to the DPPH free radical scavenging assay as described earlier.
Table 2 Antioxidant potential of TLC bands of R-EA extract of A. aspera
Band No.
Root/EA
Rf
% Antioxidant
activity
Band No.
Root/EA
Rf
% Antioxidant
activity
B1
0.12±0.01
7.40±2.60
B8
0.44±0.04
9.27±0.67
B2
0.16±0.02
25.11±4.05
B9
0.48±0.03
3.83±0.61
B3
0.21±0.03
11.02±0.49
B10
0.50±0.03
9.33±1.21
B4
0.24±0.04
8.12±2.93
B11
0.58±0.03
6.43±1.29
B5
0.30±0.04
11.44±1.94
B12
0.65±0.04
5.98±1.59
B6
0.37±0.05
15.29±1.84
B13
0.68±0.02
6.44±1.94
B7
0.40±0.05
8.12±1.25
B14
0.71±0.02
3.35±0.62
(Values represent mean ± SD. Values followed by similar letters in a column do not differ significantly at p =0.05 as
per DMRT).
Statistical analysis
All experiments were performed in three replicates, and the values were expressed as mean ±SD or mean ±standard error (SE) of
means.
Antioxidant phytochemicals from the eluted TLC band
LC-MS analysis
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LC-MS profiling of TLC R-EA-B2 that showed the highest antioxidant activity were performed on Agilent G6540B Quadrupole
Time of Flight (QTOF) binary LC system equipped with Dual Agilent Jet Stream Electrospray Ionization (AJS ESI) and separated
on Agilent Zorbax column (50 × 2.1 mm, 1.8 μm) using aqueous 0.1% formic acid (Solvent A) and acetonitrile (Solvent B). A
stepwise increasing polarity gradient of solvent A and solvent B was applied at the flow rate of 0.3 mL/min for 30 minutes. The
capillary voltage, cone voltage, and fragmentor voltage were 3.5 kV, 45 V, and 150 V, respectively. The gas temperature was set to
325 °C. Electroscopy mass spectra data were recorded in a positive ionization mode. The data was acquired at a scan rate of 2
spectra/sec in the mass range of 60-1700 m/z and analyzed with Mass Hunter Qualitative Software and METLIN database. The class
of the putatively identified compounds was deduced from the websites of the metabolomic workbench
(www.metabolomicsworkbench.org) and the Lipidomics Gateway (www.lipidmaps.org).
Out of 36 compounds identified in the R-EA-B2 extract respectively ( Table 3), phytol and dihydrosphingosine (Table 3) have been
reported possessing antioxidant activity in different investigations. Moreover, the structures and spectra of these antioxidant
phytochemicals have been brought in Table 4.
Phytol (C20H40O, RT= 13.50) is a diterpene and a member of the group of branched-chain unsaturated alcohols. A strong antioxidant
property of this compound has been reported. It can remove hydroxyl radicals and nitric oxide and prevent the formation of
thiobarbituric acid reactive substances (TBARS) in in vitro assay. In addition, it has shown antioxidant properties in terms of
scavenging hydroxyl radicals. Mere 0.9 ng/mL of phytol led to an increase in the removal of hydroxyl radicals by 9.66% and 8.62%
compared to the 5.4 and 7.2 ng/mL concentrations, respectively. Phytol had also exhibited antioxidant properties in terms of
scavenging nitrites and inhibited nitrite production at all the concentrations used [17].
The antioxidant property of phytol through non- and pre-clinical models has also been reported in vitro and in vivo methods where
DPPH and ABTS•+ radical scavenging tests were used as in vitro methods, and Saccharomyces cerevisiae test was used as in vivo
method. At 7.2 μg/ml, phytol was shown to reduce 59.89% and 62.79 % scavenging capacity of DPPH• and ABTS•+, respectively.
In the Swiss mouse hippocampus, phytol decreased the NO2 and LP - contents while increasing the GSH, SOD, and CAT activities
[6].
Dihydrosphingosine (C18H39NO2, RT= 12.15) had shown the antioxidant property by triacylglycerol (TAG) oxidation with and
without α-tocopherol. Three types of TAG from linseed, fish, and soybean oil were oxidized at 50 °C to determine the effect of
dihydrosphingosine (d18:0) with or without α tocopherol by using triacylglycerol (TAG) oxidation. Based on the oxygen
consumption and total volatile formation, dihydrosphingosine was shown to have antioxidant properties on TAG oxidation in the
absence of α-tocopherol. Further, the combination of dihydrosphingosine with α-tocopherol exhibited strong antioxidant properties
[24]. Shinde et al. have also concluded dihydrosphingosine to be the most probable antihaemolytic and antioxidant compound [19].
Table 3 Phytochemicals from R-EA-B2 and /S/EA/B4 extracts
Name/R/EA/B2
Type of
compound
Empirical
Formula
Retention
time (min)
m/z
Name/R/EA/B2
Type of
compound
Empirica
l
Formula
Retentio
n time
(min)
m/z
Terephthalic acid
Carboxylic
acid
C8H6O4
15.143
49.0233
4,8-dimethyl-
dodecanoic acid
Fatty acyl
C14H28
O2
8.832
228.2097
Ambelline
Alkaloid
C18H21 N
O5
7.778
14.1387
1-hexadecanoyl-sn-
glycerol
Glyceroli
pid
C19H38
O4
17.827
330.278
N-Acetylsphingosine
Sphingolipid
C20H39 N
O3
15.978
324.29
Dodecyl glucoside
Carbohyd
rate
C18H36
O6
10.554
348.2523
2-Hydroxymyristic
acid
Fatty acyl
C14H28 O3
7.854
262.2381
6-deoxyerythronolide
B
Polyketid
e
C21H38
O6
14.027
386.2679
4-hydroxy palmitic
acid
Fatty acyl
C16H32 O3
9.587
290.2694
2,2,9,9-tetramethyl-
undecan-1,10-diol
Fatty acyl
C14H30
O2
11.001
230.2256
6b,11b,16a,17a,21-
Pentahydroxypregna
-1,4-diene-3,20-
dione 16,17-
acetonide
-
C24H32 O7
11.996
415.212
3-hydroxy-eicosanoic
acid
Fatty acyl
C20H40
O3
14.066
328.2989
2E,4Z,6Z,8Z-
Decatetraenedioic
acid
-
C10H10 O4
8.528
177.0551
1alpha,25-dihydroxy-
26,27-dimethyl-24a-
homo-22-oxavitamin
D3 / 1alpha,25-
dihydroxy-26,27-
dimethyl-24
-
C29H48
O4
16.593
460.356
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*Identified phytochemicals with known antioxidant activity
Table 4 LC-MS spectra of antioxidant phytochemicals detected in the R-EA-B2 and S-EA-B4 extracts of A. aspera
Name of
biomass
Name of the
compounds
Structure
Spectrum
Root
Phytol
Root
Dihydrosphingosine
Phytosphingosine
Sphingolipid
C18H39 N
O3
11.04
318.3008
Trp Trp Tyr
Amino
acid
C31H31
N5O5
18.129
553.2341
*Phytol
Diterpenes
C20H40O
13.50
314.28
Stearaldehyde
Fatty acyl
C18H36
O
12.271
268.2777
*Dihydrosphingosi
ne
Amine
C18H39
NO2
12.15
302.3056
4-oxo-docosanoic acid
Fatty acyl
C22H42
O3
13.481
354.3147
1-Hexadecyl-2-O-
methyl-glycerol
Sugar
alcohol
C20H42 O3
13.803
330.3371
8,13-dihydroxy-9,11-
octadecadienoic acid
Fatty acid
C18H32
O4
10.54
312.2313
1,3-
Dicyclohexylurea
Organic
compound
C13H24 N2
O
11.1
225.1969
12-oxo-9-
octadecynoic acid
Fatty acyl
C18H30
O3
10.971
294.2207
C8-
Dihydroceramide
Amine
C26H53
NO3
16.726
428.4109
N-depyridomethyl-
Indinavir
-
C30H42
N4O4
19.428
522.3196
N-(3-hydroxy-7Z-
tetradecenoyl)-
homoserine
lactone
Fatty acyl
C18H31
NO4
8.099
308.2229
1R,9S-
HYDRASTINE
Isoquinoli
ne
C21H21
N O6
8.645
383.1351
4-hydroxy lauric
acid
Fatty acyl
C12 H24
O3
7.914
216.1733
Lys Cys His
Amino
acid
C15H26
N6 O4
S
11.408
386.1737
C16 Sphinganine
Sphingolipi
d
C16H35
NO2
10.863
274.2746
1-Hexadecyl-2-O-
methyl-glycerol
Sugar
alcohol
C20H42
O3
13.803
330.3138
13-hydroxy-
docosanoic acid
Fatty acyl
C22H44 O3
14.22
356.3532
calcifediol
Sterol lipid
C22H44 O2
15.389
340.3348
2-Acetyl-1-oleoyl-
sn-glycerol
Lipid
C23H42 O5
19.702
398.3031
Dihydroceramide
C2
Carbohydra
te
C20H41 N
O3
17.162
343.3095
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4.Discussion
In the present investigation roots of A. aspera extracted in solvents of different polarities showed phenolic compounds and substantial
antioxidant potential in the DPPH scavenging assay.
The phenolic and flavonoid contents in A. aspera differed with the type of plant biomass and solvent used for extraction. The
maximum phenolic content was observed in the R-EA extracts of A. aspera, and these extracts also showed the highest antioxidant
activities. These results corroborate those reported by Sharma et al. [18]. They have shown the maximum phenolic content (400 mg
gallic acid equivalent per 50 g tissue) in the ethyl acetate extracts of root and inflorescence (291.67 mg gallic acid equivalent per 50
g tissue). Among the solvents used for extraction in the present investigation, those with a relatively higher non-polar nature (ethyl
acetate and acetone) seem to have better extracted the potential antioxidant phytochemicals. Similar results have been reported
earlier. The ethyl acetate extracts of leaves, stem, and inflorescence of A. aspera were shown to possess excellent antioxidant property
in DPPH assay with 20 – 100 μg/ml extract [16]. The ethanol leaf extract of A. aspera was shown to have antioxidant activity with
IC50 of 7.49 μg/ml, whereas the ascorbic acid standard had IC50 of 11.73 μg/ml in the phosphomolybdinum method [21]. The
ethanol and aqueous extracts of leaves of A. aspera have shown significant wound healing and antioxidant activities in Wistar rat
models [9]. However, better antioxidant activities were observed in extracts prepared from a more non-polar solvent like acetone
and ethyl acetate in the present investigation. The methanolic extract of A. aspera leaves at 100 μg/mL concentrations was reported
to have the maximum antioxidant activity in DPPH radical scavenging and ferric ion reduction assays [15].
Higher antioxidant potential of the methanol extracts of the plant's roots and leaves has been reported from the DPPH scavenging
assay [8].In the present investigation, despite a very high antioxidant activity in the selected TLC bands of S-EA, and R-EA extracts,
they did not show the presence of any phenolic substances or flavonoids. It seems to be a very strange result. This TLC band showed
high potential to scavenge the DPPH free radicals, yet they contained mainly lipids. These results tend to indicate that these
phytochemicals are at par with the widely accepted phytochemicals of phenolics and flavonoids classes in having a better DPPH free
radical scavenging potential. For example, the GC-MS analysis of a hydro-alcoholic leaf extract of A. aspera showed antioxidant
components like lupeol, hexadecanoic acid, and ethyl ester [25].
5.Conclusion
In conclusion, most of the earlier studies have shown that A. aspera extracts prepared in more polar solvents have better higher
antioxidant activities. Further, the extracts that showed higher phenolic and flavonoid contents also showed higher antioxidant
activities. Therefore, the results of the present investigation are in line with such earlier reports. However, when the extracts were
separated by TLC, the higher antioxidant activities were noted in the fractions containing mostly lipids. This is because lipids being
non-polar are better extracted in more non-polar solvents. Therefore, it is reasonable to conclude that the extracts of A. aspera that
contain more lipids are likely to offer better antioxidant potential.
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Acknowledgements
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. The author is
grateful to Dr. Neeta M. Patil, Head, Department of Botany, Dr. Rajendra S. Zunjarrao, Principal, Modern College of Arts, Science
and Commerce, Shivajinagar, Pune-5, for their continuous support, motivation, and for providing the necessary infrastructure to
conduct the experiments.I would like to thank Afsanehsadat Omidiani, Dr. Milad Ghiasi and Dr. Somayehsadat Omidiani for their
help in the and arrangement of tables and figures of the article.
