SSRN Id4233873

Title page
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Title: Mycochemistry of partial purified F12 of Astraeus asiaticus ethyl
acetate extract and its anticancerous activity on human breast, lung, and
cervical cancer cell lines
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Authors: Koushik Pandeya, *Swapan Kumar Ghosha , Pradip Kumar Surb
Affiliation: aMolecular Mycopathology Lab., Biocontrol and Cancer Research Unit, PG Department of Botany,
Ramakrishna Mission Vivekananda Centenary College (Autonomous), Rahara, Kolkata 700118, WB, India
bEx Associate Professor of Zoology department of Kanchrapara college, Kanchrapara, North 24
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pargans,743145, WB, India
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* Corresponding address: Email: gswapan582@gmail.com
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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4233873
ABSTRACT
The current research focused on anticancer/antitumor drug derived from the partial
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purification of mushroom extracts from Astraeus asiaticus to treat cervical, lung, and breast
cancer. The fruit body was extracted in ethyl acetate solvent and quantitative analysis
revealed that it contained a significant amount of total phenols, flavonoids, and ascorbic
acids. The FTIR studies revealed that different functional groups in the extracts had different
characteristic peak values. The GC-MS profile of AAEA extract exhibited several anti-
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cancerous compounds. The column chromatography of AAEA extract was performed. The
F12 fraction demonstrated the greatest radical scavenging activity. F12 had an IC50 of 21.65 ±
0.81 µg/mL. A TLC analysis of the F12 fraction revealed eight distinct spots that matched
perfectly with the standards of quinine, lupeol, quercetin, p-Coumaric acid. The mass
spectrum analysis of F12 showed different anticancer and antioxidant compounds like 3,4,5,6
Tetramethyloctane, Hexadecanoic acid, 9,12-Octadecadienoic acid, 9,12-Octadecadienoic
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acid, methyl ester. After 24 h of treatment, the percentages of cell growth inhibition of HeLa,
MCF7, and A549 cell lines by F12 were 92.03 ± 1.65, 90.38 ± 0.53, and 87.51 ± 1.36%,
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respectively and the IC50 values were 701 ± 1.52, 728.71 ±1.52, and 906.88 ± 3.51 µg/mL
respectively. The F12 fraction showed apoptosis, LDH leakage, and up regulation of protein
expression levels of Caspase 3, Caspase 9, P53, and down regulation of Bcl2 gene of all cell
lines. As a result, the use of F12 of AAEA extract in cancer treatment will be a novel study
for future drug development. er
Key words: Mycochemistry, partial purification, Chromatogram, Chemoprofiling,
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Cytotoxicity, apoptosis, gene expression
1. Introduction
Cancer is the leading cause of death in the world. In 2020, the most common cancers
were breast (2.26 million cases), lung (2.21 million cases), colon and rectum (1.93 million
cases), prostate (1.41 million cases), skin (non-melanoma) (1.20 million cases), and stomach
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(1.09 million cases). In 2020, the most common cancer deaths were lung (1.80 million), colon
and rectum (916 000), liver (830 000), stomach (769 000), and breast (685 000 deaths). Every
year, approximately 400 000 children are diagnosed with cancer. The most common cancers
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differ by country. Cervical cancer is the most common cancer in 23 different countries
(WHO,2020). Globally, breast cancer is the most aggressive cancer in woman with an
estimated 1.38 million new cases per year (Eccles et al., 2013). Combinations of some
treatments such as chemotherapy, operative, radiotherapy, immunotherapy and
photodynamics therapy are commonly used to treat cancer (Adamson and Longo, 2005). The
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current anti-cancer drugs on the market are not target specific and cause a variety of side
effects and complications in the clinical management of various types of cancer, highlighting
the critical need for novel effective and less-toxic therapeutic approaches (Breene,1990)
Mushrooms have been recognised as an important source of biologically active compounds of
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important medicinal value, in addition to being used as food materials with their distinct
flavour and texture (Sun, and Liu 2009). Secondary metabolites found in mushrooms include
phenolic compounds, polypeptides, terpenes, and steroids. Mushrooms also contain lectins,
polysaccharides, polysaccharide-peptide complexes, and polysaccharide-protein complexes,
all of which have immunomodulatory and anticancer properties (Patel, and Goyal.,2012;
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Finimundy et al., 2013; Lixia et al., 2011). In this context, the edible medicinal mushroom
Astraeus asiaticus is of particular interest, as its extract demonstrated anticancer activity in
this study. The chemical nature of medicinal mushroom compounds will provide some
information about the functional groups responsible for their medicinal properties. Das et
al.(2020) used FTIR to identify functional groups from various species of cultivated oyster
mushrooms in order to screen the bioactive group of oyster mushrooms. Plants and fungi are
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rich in bioactive compounds that can be used for the development of new novel drugs and as
a result of minimal side effects and low cost, (Jagtap et al., 2009). There has been little/no
research on the partial purification of Astraeus asiaticus that are responsible for its anticancer
activity. To determine the anticancer compounds present in the active fraction (F12) of
AAEA that mass spectrum analysis is required. A few studies using an ethyl acetate extract of
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a mushroom have shown anticancer activity. In HeLa, SiHa, and CaSki cells, extracts of
Hexagonia glebra in ethyl acetate, demonstrated anticancer activity (Ghosh et al.,2020)
Ethyl acetate fraction from Shitake mushroom occurred anticancer activity and apoptosis
(Fang et al., 2006) The ethyl acetate extract of Auricularia auricula showed cytotoxic activity
and apoptosis on cervical cancer cell line (Ekowati et al., 2020).The ethanolic extract of
Astraeus hygrometricus mushroom has a in vitro free-radical scavenging activity as well as
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anti-inflammatory activity in vivo.[ Biswas et al. 2010], hepatoprotective (Sarkar and
Acharya, 2011), and cardioprotective (Biswas and Acharya, 2013) properties.
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Astraeus asiaticus being a potential mushroom with edibility and therapeutic value and anti-
cancerous activity has not been studied so much. So, the primary goal of this study was to
introduce a new Astraeus species discovered in West Bengal, India, to determine its cytotoxic
efficacy and investigate its mycochemistry in order to promote further mushroom research
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and development worldwide.
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2. Materials and Methods
2.1 Solvent Extraction
Collected fruit bodies of A, asiaticus were oven dried at 450C for 48 h, were ground
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up, and 2kg of the powder was combined in a 1:10 solute to solvent system ratio with ethyl
acetate. It was kept in an incubator for 72 h under stirring. Whatman No. 4 filter paper and
Whatman No. 1 filter paper were used to filter the extract. The extract was dried in a rotary
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evaporator (Superfit, Model: PBV-7D) at 500 C, lyophilized (Biobase, Model: BK-FD10P)

and stored in the 40C refrigerator.
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.
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2.2 Mycochemical Screening of AAEA extract
2.2.1. Test for Flavonoid

0.2 g of AAEA extract was dissolved in 4 ml of distilled water in a test tube, then
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diluted sodium hydroxide (1M solution) was added, resulting in a yellow solution, that
turned colourless when a few drops of dilute acid (10 % hydrochloric acid) were added,
indicating the presence of flavonoid ( Hossain et al., 2013 ).
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2.2.2. Test for Terpenoids
0.2 g of AAEA extract was confiscated and dissolved in 2 mL of chloroform and 3
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mL of concentrated sulphuric acid (90%), resulting in a reddish brown colour indicating the
presence of terpenoid (Talukdar et al., 2017).
2.2.3. Test for cardiac glycosides
0.2g of AAEA extract was mixed with 2 mL of glacial acetic acid and 1 drop of
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FeCl3 solution. 1 mL of concentrated H2SO4 was added to this mixture. The upper layer and
the interface between the two layers were found to be bluish-green and reddish-brown in
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colour, indicating the presence of cardiac glycosides (Auwal et al., 2014).
2.2.4. Test for steroid
A few drops of acetic acid anhydride were added to 0.2 g of AAEA extract. Drop by
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drop, concentrated H2SO4 was carefully added to the test tube through the inner wall. The
presence of steroids is indicated by a brown ring around the surface. (Gul et al., 2017).
2.2.5. Alkaloid test

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In a separate test tube, 0.2 g of AAEA extract was weighed and warmed for 2 min
with 2 % Sulphuric acid. It was then dribbled into a separate test tube with a few drops of
Dragendroffs reagent (Mix the two solutions: 0.5 gm of bismuth nitrate with 10 ml of HCl,
and 4 gm of KI with 5 ml of distilled water.), the presence of orange red precipitate for
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alkaloid emanation was observed (Auwal et al., 2014).
2.2.6. Tests for carbohydrate

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0.2 g AAEA was dissolved in 2 mL of water. 2-3 drops of Molisch's reagent (3.75
gm of -naphthol in 25 ml Ethanol 99 %) were added to this solution to form a layer without
shaking the test tube, followed by a drop of concentrated sulphuric acid (99 percent). The
presence of a purple colour, which indicated the presence of carbohydrates, was
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investigated at the interface. (Auwal et al., 2014).

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2.2.7. Test for phlobatannins
0.2 g AAEA was dissolved into 2 mL of water and it was added into 3mL of dilute
HCl (10%). The presence of phlobatannins was determined by the presence of a red
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precipitate (Auwal et al., 2014).
2.2.8.Test for Phenol
0.2 g of AAEA extract was dissolved in 2 ml of distilled water and after that the
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solution was treated with two drops of 5% FeCl3, producing a deep blue colour which
indicated the presence of phenol (Talukdar et al., 2017).
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2.2.9. Test for Tannin
0.2 g AAEA extract was dissolved in 4 ml of distilled water and heated in a water
bath (50ºC). The mixture was bled, and two drop of 1% FeCl3 was added to the filtrate,
revealing a dark green solution indicative of tannin presence (Usman et al., 2009; Fransina
et al., 2019).
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2.3. Divergent biochemical assay of AAEA extract
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2.3.1. Comprehensive phenol content assessment
Folin-reagent Ciocalteu's method was used to determine the total phenol content of
extract (McDonald et al., 2001). A mixture of 0.1 mL Folin-reagent Ciocalteu's (0.5 N)
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reagent and 0.5 mL (1g in 1 mL ethyl acetate) extract solution was mixed and incubated at
room temperature (35 0C) for 15 min. The mixture was then incubated at room temperature
(35 0C) for 30 min with 2.5 mL of saturated sodium carbonate solution. The absorbance was
measured at 760 nm wave length. The result was expressed as mg of Gallic acid equivalent
(GAEs) per gm of mushroom dry weight and presented as the mean standard deviation (SD)
of three replicates.
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2.3.2 Totality of flavonoid (TFC) content assay
TFC was calculated for the extracts using a standard method proposed by Meda et al
(2005). Briefly in a glass tube, equal parts of 2% AlCl3 (prepared in absolute methanol) and
extract (1 mg/mL) were incubated at room temperature for 10 min. After incubating, the
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absorbance at 415nm was measured. Finally, the result was expressed as mg of Quercetin
equivalent (QEs) per gm of dry mushroom weight. and was shown as the mean standard
deviation of three replicates.
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2.3.3. Ascorbic acid content assay
With minor modifications, the total ascorbic acid content was determined using the
Folin ciocalteu reagent method proposed by Jagota and Dani (1982).
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0.5 mL extract solution (1 gm AAEAE in 1 ml ethyl acetate) was quickly mixed with 0.8 mL
of 10% trichloroacetic acid and refrigerated for 5 min before centrifugation at 3000 rpm for 5
min. 0.2mL of taken from the mixture and it was diluted to 2 mL by adding 10 ml distilled
water. To the diluted extract mixture, 0.2 mL of diluted Folin-Ciocalteu ( 1:9 ) was added.
After a 10 min incubation period at room temperature (37 0C), the absorbance at 760 nm was
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measured. Finally, the results were expressed as mg of ascorbic acid equivalents (AAEs) per
gm of mushroom extract, with the mean standard deviation (SD) of three replicates.
2.4. Fourier Transform Infrared Spectrophotometer (FTIR) analysis of AAEA extract
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The Fourier Transform Infrared Spectrophotometer (FTIR) is the most powerful tool for
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determining the types of chemical bonds (functional groups) present in compounds. As
shown in the annotated spectrum, the wavelength of light absorbed is characteristic of the
chemical bond. The chemical bonds in a molecule can be determined by interpreting the
infrared absorption spectrum.
10 mg of crude powder of ethyl acetate was encapsulated in 100 mg of KBr pellet. The
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sample powdered specimen was stuffed into an FTIR spectroscope with a scan range of 400
to 4000 cm-1 and a resolution of cm-1(Ashokkumar and Ramaswamy., 2014).
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2.5 GCMS analysis of AAEA extract
2.5.1. Preparation of the AAEA

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The AAEA extract was analyzed using– Agilent Technologies, GC-6860N Network GC
System with 5973 inert Mass Selective Detector. Mass Spectrometry (GC-MS) for the
identification of myco-compound present. A solvent blank analysis was first conducted using
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1 μL of ethyl acetate. Then 1μL of the reconstituted ethyl acetate extract solution was
employed for GC-MS analysis using direct liquid injection.
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2.5.2. Analysis
GC-MS analysis was carried out on a GC system comprising a Gas Chromatograph

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interfaced to a Mass Spectrometer (GC-MS) instrument; Agilent Technologies, GC-6860N

Network GC System with 5973 inert Mass Selective Detector: fused silica capillary column
(HP- 5MS (19091S-602), operating in electron impact mode of 70 eV; helium (99.999%) as
carrier gas at a constant flow of 1mL/min and a sample injection volume of 1 μL which was
employed (split ratio of 10:1) injector temperature 280⁰C, ion source temperature 280⁰C. The
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oven temperature was programmed from 50⁰C (isothermal for 2 min), with an increase of
10⁰C/minute to 280⁰C, then. 5⁰C/min to 300⁰C. Mass spectra were taken at 70eV and scan
interval of 0.2 seconds and fragments from 40 to 550 Da. Total run time was 30 min.
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2.5.3. Identification of compound
The compounds were then identified from the GC-MS peaks, using library data of the
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corresponding compounds. GC-MS was analyzed using electron impact ionization at 70eV
and data was evaluated using a total ion count (TIC) for compound identification and
quantification. The spectrums of the components were compared with the database of
spectrum of known components stored in the GC-MS library using National Institute of
Standards and Technology (NIST) Search. The relative % amount of each component was
calculated by comparing its average peak area to the total areas. Measurement of peak areas
and data processing were carried out by GC-MS Post Run Solution Software.s (Sanyal and
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Ghosh., 2019).
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2.6. Fractionation of AAEA extract by Column Chromatography
Purification of AAEA extract was carried out by column chromatography (Bajpai et

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al.,2016).
2.6.1. Charging of the Column

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A glass column was rinsed with hexane, a cotton layer was placed at the bottom, and it
was charged with solvents; In order to create the gel bed for complete separation, 1:7 silica
gel (60-120 mesh) was used in the extract. The solvent was eluted up to the column bed level,
and the dried extract was charged into the column. Another layer of cotton was placed over
the charged matter to prevent the extract bed from being disturbed while the eluting solvent
was poured from the top. The charged column was left for 4 h to ensure complete saturation
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and the removal of air bubbles, resulting in a static bed.

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2.6.2. Elution
The charged column was eluted with various mobile phases (Gradient elution
technique), with a gradual increase in polarity with hexane and hexane in ethyl acetate in the
ratios of 9:1, 8:2, 7:3, 6:4, and 5:5 and 0:10. Similarly, ethyl acetate in methanol in the
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following ratios: 9:1, 8:2, 7:3, 6:4, and 5:5 and 0:10. Each fraction (2ml) was collected
separately and concentrated using a vacuum rotary evaporator (Superfit,Model:PBV-7D), and
the preliminary antioxidant activity was determined using the DPPH method.
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2.7.Antioxidant test of each fraction
The antioxidant test of the each fraction was performed using the Bondet et al. (1997)
technique of DPPH (1,1 diphenyl-2- picrylhydrazyl) free radical scavenging .The
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concentration of the fraction sample solution was 10-100 µg/mL, and the concentration of the
DPPH solution in methanol was 0.4 mM. BHA was employed as a positive control. After two
hours of dark incubation, the absorbance at 517 nm was measured using a spectrophotometer.
The DPPH reduction was calculated using the following formula:
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Ac– As
% DPPH = ∗ 100
Ac
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Where as
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AC=The absorbance of the solvent served as a control,
AS= the absorbance of the extract served as a sample.
2.7 .TLC Profiling of F12 of AAEA extract

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After screening of 20 fractions as antioxidant activity, the best fraction 12 (F12) of
AAEA extract was used for TLC profiling. The solvent system was ethyl acetate:
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chloroform: hexane in 40:40:20 ratio (standardized by trials). Pre coated silica gel 60 F254
plates (E. Merck) of uniform thickness of 0.2 mm was used as the stationary phase. 1.5 cm
from one edge of the plates, pencil line was drawn. Using thin capillary pipettes, 20 µl of the
test solution was applied to TLC plate. The plates were then placed in a development
chamber with a solvent system height of 8 cm. The solvent front was allowed to move until it
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was about 1 cm from the top end. After removing the TLC plates, the solvent front was
marked with a soft pencil. The plate was examined under UV light (254 nm and 366 nm)
.The resulting chromatograms were photographed. Following that, we labelled the
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chromatograms spots in various exposures and calculated and recorded the retention factors
(Rf)( Bele and Khale, 2011). The Rf values of the various bands were then calculated using
the following equation:
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Retention factor (Rf) =
Where as-
a = Distance travelled by solute (spot/compound)
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b = Distance travelled by solvent

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2.8. Mass sprectrum analysis of F12
Thermo Polaris Q mass spectrometry equipment was used to analyse the mass spectrum
of F12 (Anderson et al.,1972).
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2.9.Anti- cancerous activity of F12 of AAEA extract
2.9.1.Cell Culture
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The Human cancer cell lines like Lung cancer (A549), breast cancer (MCF-7), and
cervical cancer (HeLa) at passage number 30 were cultured in DMEM supplemented with L-
glutamine, 10% (v/v) foetal bovine serum, 100 µg.mL-1 streptomycin, and 250 IU.mL-1
penicillin (all purchased from Hi Media Laboratories Pvt. Ltd., Mumbai, India) in 75-mm
tissue culture flasks at 37°C in Cultures were passaged once a week for maintenance, and the
culture medium was changed twice a week.( Freshney, 2015)
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2.9.2. Cytotoxicity/cell proliferation assay
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A methylthiazolyl tetrazolium (MTT) assay was used to assess the effect of F12
AAEA on cell proliferation in the HeLa, A549, MCF-7 cell line (reagents provided by
HiMedia Laboratories Pvt. Ltd.), In a 96-well culture plate, 10x104 cells were seeded with
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fresh DMEM medium containing 10% FBS and antibiotics overnight until the cells reached
80% confluence. The cultures were then washed with 10% PBS and treated with AAEA
dissolved in DMEM at various concentrations (250, 500, 750, 1000, and 1500 µg.mL-1), and
incubated at 37°C in a 5 % CO2 95 percent air atmosphere, with DMEM serving as a vehicle
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control. Cells were washed with PBS (Phosphate Buffered Saline) after 24 h of treatment, and
100 µL of 500 μg.mL-1 MTT solution (dissolved in phenol red-free DMEM) was added to
each well. After discarding the media, the cells were incubated for another 3 h before being
given 100 µL dimethyl sulfoxide (DMSO) to dissolve the crystals. A microplate reader (Bio-
Rad Laboratories, Inc., Hercules, CA, USA) was used to read the plate at an absorbance of
570 nm (Vorauer et al.1996). The LC50 value (i.e., the concentration that caused 50% cell
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death) was determined by plotting a dose-response graph of the cytotoxicity values obtained
using the formula:
% Cell cytotoxicity= 100 − ( ) ∗ 100
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Where as
Ac= Absorbance of the control.
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At = Absorbance of the test.

Each experiment's mean and standard deviation are represented by data points, and the
experiments were carried out at least three times.
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2.9.3. Cell morphology examined under a phase contrast microscope
To investigate the cell morphology, 2x105 HeLa, A549 and MCF-7 cells/well were
seeded into 6-well plates, and at 80-90% confluence the cells were treated with various
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concentrations of AAEA (250,500, 750, and 1,000,1500 μg.mL-1) where DMEM used as
vehicle control.
2.9.4. Apoptosis analysis
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After being stained with DAPI, the nuclear morphology of three cell lines were
examined to determine whether or not apoptosis had occurred. Each experiment involved the
observation and counting of apoptotic cells using an inverted fluorescent microscope. At least
five optical fields containing a total of 200 cells were counted in each experiment.
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The formula used to calculate the percentage of apoptotic cells was percentage of apoptotic
cells = [number of apoptotic cells / total cells counted (usually 200)] x 100%.
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2.9.5. Lactate dehydrogenase assay
Cancer cells were seeded and grown separately in 96-well plate (4x10 cells/well) in
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medium containing 10% FBS and incubated for 24 h under 5% CO2 and at 37°C. Then the
cells were washed with PBS, 100 μL of the each concentration of F12 fraction of AAEA was
added separately to the wells, and 100 μL of medium was added to the control well and
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incubated for 24 h. The cells were harvested, and the assay was carried out using the lactate
dehydrogenase (LDH) kit (Thermo Fisher Sci. Ltd, USA). Hundred microliter of (10:1000)
sample with working reagent was mixed and incubated for 1 min at 37°C, observed at 340
nm and calculated according to the manufacture's instruction.
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2.9.6. Western blot analysis for study of expression of genes

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Hela, MCF-7, and A549 (2x105) cells were treated separately with 750 µg/mL of F12
fraction of AAEA for 24 h. Following treatment, cells were lysed in RIPA buffer (Abcam,
USA). The effect of treatment on the expression of certain genes like p53 (tumour guard) as
well as apoptotic caspase-3, and caspase-9 and anti-apoptotic gene BcL (Santacruze
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Biotechnology, USA) were studied.

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2.10.Statistical analysis
For data analysis, GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) was
used, and one-way analysis of variance (ANOVA) was used to compare concentration
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differences. The results represent means ± standard deviation/standard error (SD/SE) from
triplicate experiments done in a parallel manner, unless otherwise indicated. All of the
figures were obtained from at least three separate experiments. A statistically significant
difference was defined as P<0.05
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2.11. Research Design of the study at a glance
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The research design at a glance is presented in the figure 1.
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Fig. 1. A brief summary of the general approaches in extraction, functional group

identification, partial purification, structural elucidation and anticancerous activity of the
bioactive compound from AAEA extract
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3. Results
3.1. Mycochemistry study
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Crude Product
The extraction yield of AAEA was 45.25±1.23 gm, and the percentage of yield was
2.25 ± 0.12%.
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3.2. Mycochemical screening of Ethyl acetate extract of A.asiaticus
A variety of qualitative tests were used to screen ethyl acetate extract of A.asiaticus for
mycochemical activity. Flavonoid, Cardiac glycoside, Alkaloid, Phenol, Carbohydrate, were
detected in this extract, while phlobatannins, terpenoids, tannins, and steroids were absent.
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(Table 1).
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Table 1. Mycochemical screening of Ethyl acetate extract of A. asiaticus
Mycocompound/class AAEAE
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Flavonoid +
Terpenoid -
Cardiac glycoside +
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Steroid -
Alkaloid +
Tannins -
Carbohydrate +
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3.3. Total Phenol, flavonoid, Ascorbic acid content assay of AAEA extract
The total phenolic content of ethyl acetate extract of A. asiaticus was 8.13 ± 0.58 mg
GAE/g, as well as the total flavonoid content of ethyl acetate extract of A. asiaticus was 3.03
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± 0.39 mg QE/g and the ascorbic acid content was 1.59 ±0.33 mg AAE/g (Fig. 2
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Fig.2. Bar diagram showed total phenol content, total flavonoid content and ascorbic acid
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content (mg/g) of AAEA extract.
3.4. FTIR spectral data interpretation of AAEA extract

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AAEA extract showed the different absorption bands (functional groups) like 3426.8cm-
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(for O-H stretching), 2926.2 cm-1 (C-H stretching), 2854.7 cm-1 (C-H stretching),1718.4 cm-
1
(C=O ), 1460 cm-1 (ring C-C stretch),1380.2 cm-1 ( CH3C-H bend), 1261.6 cm-1 (O=C-O-
C), 1182.7 cm-1 (C-O stretch), 1069 cm-1 (C-F stretch), 978.6 cm-1 (=C-H bend), 802.4 cm-1
(C-Cl stretch) and 722.6 cm-1 (- (CH2)n bend ) (Fig. 3 ,Table 2).
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Fig. 3. FTIR spectrum of AAEA extract
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Table 2 FTIR Interpretation of compounds-AAEA extract
Sl.No Wave no(test Wave no (Reference Intensity Functional group Myco compounds
sample) article) assignment identified
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1 3426.8 2500-3500 s,broad O-H stretch,Hydroxy Carboxylic acid
group group
2 2926.2 2850-3000 S C-H stretch Alkanes and Alkyls

group
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3 2854.7 2850-3000 S C-H stretch Alkanes and Alkyls
group
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4 1718.4 1710-1720 S C=O stretch Ketone compound
5 1460 1600-1450 m-s ring C-C stretch Aromatic compound
6 1380.2 1390-1370
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group
7 1261.6 1310-1250 S-VS O=C-O-C Esters group
8 1182.7 1260-1180 m-s C-O stretch Alcohols group
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9 1069 1350-1000 VS C-F stretch Alkyl halides group
10 978.6 980-960 S =C-H bend Alkens group

11 802.4 850-750 S C-Cl stretch Alkyl halides group
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12 722.6 725-715 W - (CH2)n bend Alkanes and Alkyls

group
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3.5. Chemo- profiling of AAEA extract using GCMS analysis
An ethyl acetate extract was analysed by GC-MS and chemo profiling study revealed 61
compounds (Fig. 4, Table 3). These compounds were identified using the NIST mass spectral
library database. A peak area of 1% or more was found in 12 compounds (Table 4). The
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compounds were benzene 1,4 dichloro (15.35%), 2,2,5-Trimethylhexane-3,4-dione (1.25%),
2,5-Dimethyl-3-hexanone (1.64%), 3,4,5,6-Tetramethyloctane (1.17%),1-cyclododecyne
(2.16%), cis-9,10-Epoxyoctadecan-1-ol (2.52%).
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Fig. 4. GC-MS chromatogram of the AAEA extract

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Table 3 GCMS profiling of AAEA extract

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S/N of compound Apex RT Start RT End RT Area % Area Height %Height

1 6.11 5.84 6.21 739.164 0.24 46.572 0.15
2 6.26 6.21 6.52 491.807 0.16 42.421 0.14
3 8.42 8.33 8.9 1972.301 0.65 94.87 0.31
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4 10.8 10.7 11.15 46684.75 15.35 3321.27 11

5 12.75 12.66 13.08 1489.6 0.49 89.314 0.3
6 14.58 14.49 14.91 3811.279 1.25 279.346 0.92
7 16.36 16.27 16.64 4984.504 1.64 455.69 1.51
8 17.63 17.5 17.68 692.947 0.23 86.211 0.29
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9 17.73 17.68 17.84 628.905 0.21 91.086 0.3
10 18.02 17.94 18.29 3552.662 1.17 365.545 1.21
11 18.72 18.61 18.98 1997.126 0.66 173.679 0.58
12 19.27 19.2 19.35 421.991 0.14 84.032 0.28
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13 19.54 19.47 19.63 2223.761 0.73 404.428 1.34
14 19.74 19.63 19.83 1342.709 0.44 175.422 0.58
15 19.95 19.88 20.07 616.048 0.2 69.603 0.23
16 20.14 20.07 20.26 1459.767 0.48 319.911 1.06
17 20.49 20.42 20.64 1959.476 0.64 354.507 1.17
18 20.78 20.64 20.83 1121.666 0.37 320.247 1.06
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19 20.94 20.86 20.99 2335.995 0.77 608.907 2.02
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20 21.05 20.99 21.2 6557.796 2.16 924.893 3.06
21 21.3 21.2 21.44 7662.807 2.52 1180.111 3.91
22 21.61 21.56 21.65 682.568 0.22 234.84 0.78
23 21.77 21.66 22.15 72321.6 23.78 4544.883 15.05
24 22.33 22.29 22.38 264.777 0.09 91.293 0.3
25 22.67 22.45
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22.91 8596.27 2.83 870.255 2.88
26 23.03 23 23.06 0 0 68.602 0.23
27 23.15 23.06 23.22 27005.31 8.88 3913.678 12.96
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28 23.3 23.22 23.41 43304.58 14.24 4189.983 13.87
29 23.46 23.41 23.67 35280.31 11.6 4068.666 13.47
30 23.74 23.7 23.77 133.274 0.04 43.437 0.14
31 23.87 23.81 23.96 1028.427 0.34 225.772 0.75
32 24.04 24.01 24.08 122.253 0.04 40.677 0.13
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33 24.17 24.12 24.21 106.096 0.03 82.244 0.27

34 24.28 24.21 24.31 0 0 39.291 0.13
35 24.38 24.35 24.41 1.074 0 45.213 0.15
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36 24.49 24.44 24.54 285.437 0.09 68.851 0.23

37 24.57 24.54 24.61 211.332 0.07 70.3 0.23
38 25.05 24.63 25.1 6603.032 2.17 320.163 1.06
39 25.15 25.1 25.3 3126.372 1.03 320.612 1.06
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40 25.36 25.3 25.77 4495.222 1.48 258.873 0.86

41 26.32 26.24 26.57 1803.605 0.59 244.829 0.81
42 26.62 26.57 26.67 74.298 0.02 41.879 0.14
43 26.8 26.7 26.85 65.159 0.02 34.046 0.11
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44 26.89 26.85 26.93 35.277 0.01 27.51 0.09

45 27.01 26.93 27.18 183.97 0.06 31.099 0.1
46 27.23 27.18 27.35 179.414 0.06 37.816 0.13
47 27.92 27.77 28.06 457.143 0.15 43.835 0.15
48 28.12 28.09 28.3 302.666 0.1 54.161 0.18
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49 28.83 28.8 28.95 232.886 0.08 42.56 0.14

50 29.21 29.11 29.33 518.337 0.17 44.063 0.15
51 29.37 29.33 29.48 307.24 0.1 51.055 0.17
52 30.22 30 30.26 567.403 0.19 73.367 0.24
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53 30.34 30.26 30.46 964.251 0.32 113.914 0.38
54 30.51 30.46 30.69 806.835 0.27 107.705 0.36
55 30.97 30.89 31.04 196.547 0.06 28.294 0.09
56 31.52 31.36 31.57 142.71 0.05 31.467 0.1
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57 31.66 31.62 31.74 100.323 0.03 37.598 0.12
58 31.85 31.78 31.91 285.941 0.09 63.268 0.21
59 31.98 31.91 32.08 411.163 0.14 60.918 0.2
60 32.2 32.17 32.25 82.113 0.03 26.874 0.09
61 32.31 32.26 32.35 79.991 0.03 22.928 0.08
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Table 4 Compounds with the highest percentage area identified in ethyl acetate extract,
with molecular weight and formula
S/N RT %Area
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Compound name Molecular Weight Molecular formula
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1 10.8 15.35 Benzene 1,4 dichloro 147 C6H4Cl2
2 14.58 1.25 2,2,5-Trimethylhexane-3,4-dione 156 C9H16O2
3 16.36 1.64 2,5-Dimethyl-3-hexanone 128 C8H16O
4 18.02 1.17 3,4,5,6-Tetramethyloctane 170 C12H26
5 21.05 2.16 1-cyclododecyne 164 C12H20
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6 21.3 2.52 cis-9,10-Epoxyoctadecan-1-ol 284 C18H36O2

7 21.77 23.78 Hexadecanoic acid 256 C16H32O2
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8 22.67 2.83 3-Ethyl-3-undecanol 200 C13H28O

9 23.15 8.88 (z)-9,17-octadecadienal 264 C18H32 O
10 23.3 14.24 9,12-Octadecadienoic acid 280 C18H32O2
11 23.46 11.6 · 9,12-Octadecadienoic acid, methyl 294 C19H34O2
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ester
12 25.05 2.17 Heptaethylene glycol 326 C14H30O8

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3.6. Column Chromatography and Antioxidant assay of Different fraction of AAEAE extract
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The column chromatography of AAEAE extract yielded 20 fractions. Each fraction was
subjected to an antioxidant test. In comparison to the values of various antioxidant
scavenging activity parameters, the yielded fraction showed a significant fluctuation (p<0.05)
(Fig.5). F12 showed very strong antioxidant scavenging activity than others, and the value
ed
was 21.65 ± 0.81 µg/mL, while IC50 of synthetic antioxidant BHA was 20.19±0.59 µg/mL.
So, this F12 fraction was tested as anticancer against human cancer cell line.
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Fig. 5. Antioxidant activity analysis of different fraction. Data are expressed as mean ± S.E.M;
n=6 Anova followed by Turkey test (P <0.5). The same letter indicates that they are statistically the
same, while a different letter indicates that they are statistically different as per Tukey Multiple range
test.
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3.7. Analytical TLC of the F 12 of AAEA extract
When the F12 of AAEA extract was run on an ethyl acetate, chloroform, hexane,
(40:40:20) solvent system, eight spots were visualised under UV 366 nm with Rf values of
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0.87, 0.74, 0.54, 0.50,0.47,0.45,0.37 and 0.26 respectively and three spots under UV 254 nm
with Rf values of 0.87, 0.50 and 0.26 respectively (Fig. 6, Table5). As a result, Fraction 12
contained eight spots or compounds. In this solvent system, four standard compounds
(quinine, lupeol, quercetin, and p-coumaric acid) were run. Quinine gave one spot with an Rf
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value of 0.37 under both UV light, matching perfectly with one spot of F12 (Rf 0.37).(Fig. 6 )
. It is noted that one of the eight compounds could be Quinine. Under UV 366 nm light,
Lupeol, Quercetin, and p-Coumaric acid gave one spot with Rf values of 0.26,0.74, and 0.87,
(Fig. 6) which matched perfectly with the spots (Rf 0.26,0.74,0.87) of AAEA extract. It
should be noted that the other compounds in the AAEA extract may be Lupeol, Quercetin,
and p-Coumaric acid implying that Fraction 12 contains an alkaloid, triterpene, flavonoid,
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and phenol group.
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Fig. 6. Thin Layer Chromatography (TLC chromatogram) of sample and standards
A. standard (Quinine) and B.F12 showing 3 spots at 254 nm UV light. C (I) standard
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quinine, C (II) lupeol, C(III) quercetin, C(IV) p-Coumaric acid and D. F12 showing 8 spots
at 366 nm UV light
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Table 5 Analytical TLC of F12 of AAEA extract, its different spots and Rf value using a
specific solvent system
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Sample/ Stationary Mobile Run time No of spots Rf value

Standard Phase Phase (Min)
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UV UV
254 366nm Rf value Rf value(366
nm (254 nm) nm)
F 12 SILICA gel- Chloroform 20 3 7 0.26 0.26
G : 0.54 0.37
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Ethyl 0.87 0.45

acetate 0.47
: 0.50
hexane 0.54
40:40:20 0.74
0.87
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Quinine SILICA gel- Chloroform 20 1 1 0.37 0.37

G :
Ethyl
acetate
:
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hexane
40:40:20
Lupeol SILICA gel- Chloroform 20 - 1 0.26
G :
Ethyl
acetate
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:
hexane
40:40:20
Quercetin SILICA gel- Chloroform 20 - 1 0.74
G :
Ethyl
acetate
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:
hexane
40:40:20
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p-Coumaric SILICA gel- Chloroform 20 - 1 0.87
acid G :
Ethyl
acetate
:
hexane
40:40:20
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3.8. Mass spectrum analysis of F 12 of AAEA extract
Mass spectrum analysis was done and the respective compounds were searched in NIST
database (National Institute of Standards and Technology). Six compounds were
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identified in the F12 fraction, three of which had anticancer activity and one of which had
antioxidant properties.The six compounds were 3,4,5,6 Tetramethyloctane, Hexadecanoic
acid, 9,12-Octadecadienoic acid, 9,12-Octadecadienoic acid methyl ester,1-
cyclododecyne ,cis-9,10-Epoxyoctadecan-1-ol, in which 3,4,5,6 Tetramethyloctane,
Hexadecanoic acid, 9,12-Octadecadienoic acid exhibited anticancer activity and 9,12-
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Octadecadienoic acid methyl ester showed antioxidant activity (Fig.7,Table 6).

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Fig. 7. Mass spectrum analysis of F12. A. 3,4,5,6 Tetramethyloctane, B. Hexadecanoic acid.
C. 9,12-Octadecadienoic acid. D.9,12-Octadecadienoic acid, methyl ester E. 1-cyclododecyne
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F. cis-9,10-Epoxyoctadecan-1-ol
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Table 6 The antioxidant or anticancer compound of F2 identified by mass spectrum

analysis
Compound name Anticancer/Antioxidant activity

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3,4,5,6-Tetramethyloctane Anticancer
9,12-Octadecadienoic acid Anticancer
Hexadecanoic acid Anticancer
9,12-Octadecadienoic acid ethyl ester Antioxidant
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3.9. Anti-cancerous activity
3.9.1. Cytotoxic effect of F12 fraction on HeLa, MCF-7 and A549 cells
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HeLa, MCF-7 and A549 cells were subjected to cytotoxicity assay with the
bioactive fraction F12 of AAEA. In case of HeLa cell line the highest cytotoxicity with 92.03
± 1.65 % of inhibition was observed at the higher concentrations of 1500 μg/mL, A dose-
dependent manner the inhibition rates were 19.00 ± 1.86%, 38.20 ± 2.34%, 65.83 ± 4.32%,
and 83.48 ± 2.17 % respectively at concentrations of 250, 500,750, and 1000 µg/mL of F12,.
After 24 h of treatment with 250, 500, 750, 1000, 1500 µg/mL of F12, the inhibition rates of
MCF-7 were 17.94 ± 3.26, 37.68 ± 3.07, 62.63 ± 3.22, 82.06 ± 2.27, 90.38 ± 0.53. In case of
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A549 cell line after 24 h treatment with 250, 500, 750, 1000, 1500 µg/mL of F12 the
inhibition rates were 9.02 ± 1.28, 23.18 ± 3.94, 42.39 ± 3.34, 62.71 ± 2.79,87.51 ± 1.36 (Fig.
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8) respectively. The relative IC50 was determined to be 701 ± 1.52, 728.71 ±1.52, and 906.88
± 3.51 µg/mL (Table 7) of Hela, MCF-7 and A549 for the F12 fraction.
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Fig. 8. Percentages of inhibition of growth of HeLa, MCF-7 and A549 by different

concentration of F12 of AAEA extract. (± SD (> 0.05) bars were inserted at the top of bars
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of data).
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Table 7: IC50 value of fraction 12 AAEAE extract on HeLa, MCF-7 and A549
Cell line IC 50 (µg/ml)

HeLa 700.00
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MCF-7 727.38
A549 906.55
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3.9.2. Cell morphology examined under a phase contrast microscope
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After 24 h of incubation with various concentrations of F12 of AAEA, cells were
examined under a phase contrast microscope. Untreated cells (DMEM vehicle control) had a
normal spindle shape and reached 90% confluence after 24 h of culture. At an F12
concentration of 1500 µg.mL-1, three cell lines (HeLa, MCF-7 and A549) lost their spindle
shape, had lower cell confluence, and there was a lot of debris (Fig.9, Fig.10, Fig.11).
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Fig. 9. Morphological Changes of Human Cancer Cell Line (A549) Treated with F12 of
AAEA (1500μg/mL) and Negative Control under Phase Contrast Microscope at 24 h. The
negative Control showed no change; but treated cells were round, degenerated and dead.
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Control Treatment (1500 µg/mL
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Fig. 10. Morphological Changes - Human Cancer Cell Line (HeLa) morphological changes
at 24 hours, treated with F12 of AAEA (1500µg/mL) and negative control under Phase
Contrast Microscope. The negative Control showed no change, but the treated cells are round,
degenerated, and dead.
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A B
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Control Treatment (1500 µg/mL)
Fig. 11. Morphological changes in the human cancer cell line (MCF-7) Under a phase
contrast microscope, treated with F12 of AAEA (1500µg/mL) and negative Control after 24
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hours. Although the negative control showed no change, the treated cells were round,
degenerated, and dead.
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3.9.3. Apoptosis inducing activity of F12
The percentage of HeLa, MCF-7 and A549 cells undergoing apoptosis was evaluated at
250,500 and 750 μg/mL of F12 fraction (Fig.12). The percentage of Hela cells undergoing
early apoptosis was found to be 45 %, 55 %, and 75 % for concentrations of 250, 500, and
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750 µg/mL respectively. Apoptosis was observed to be 40%, 50%, and 70% for MCF 7 at
concentrations of 250, 500, and 750 µg/mL, respectively. Apoptosis in A549 was seen to be
35%,45%, and 65% for concentrations of 250,500, and 750µg/mL, respectively.
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Fig. 12 The percentage of apoptosis in HeLa, MCF-7, and A549 cells at 250, 500,750 µg/mL
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concentration of F12 fraction

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3.9.4. Lactate dehydrogenase assay
The graphical representation in Figure 13 demonstrated that LDH leakages occurred
from these three cancer lines in dose-dependent manners. HeLa was more sensitive to F12 of
AAEA (750µg/mL) than the other cell lines.
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Fig. 13. Lactate dehydrogenase assay effect of F12 on HeLa, MCF-7and A549 cells at
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different concentration of 250, 500,750µg/mL. SD± (>0.05) bars were inserted

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3.9.5. Effect of F12 fraction on Caspase 3 and Caspase 9 and Bcl-2 protein expression in
HeLa, MCF-7and A549 cells
In order to understand how F12 of AAEA induced apoptosis in cancer cells, we

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discovered that treatment with F12 of AAEA (750 µg/mL) decreased the expression of
protein of the gene Bcl-2 (Fig.13), while increasing the expression of pro-apoptotic genes
caspase 3 and caspase 9. Furthermore, F12 upregulated the p53 gene (Fig. 14).
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Fig. 14. Protein expression level data of Caspase 3, Caspase 9, P53, Bcl2, treated with F12
(750µg/mL) of AAEAE
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4. Discussion
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Customers are increasingly interested in the antioxidant chemical content and
antioxidant properties of foods, and many mushrooms contain both. The nutritional and
functional value of mushrooms is determined by their antioxidant content. Mushrooms are
high in antioxidant compounds (Carvajal et al., 2012). Some researchers have demonstrated
that Astraeus sp has antioxidant properties (Wani et al.,2010; Biswas et al., 2010; Mandal et
ot
al.,2015). The total phenolic, flavonoids, and ascorbic acids, has also been discovered in
other Astraeus species (Astraeus odoratus and Astraeus pteridis) (Pavithra et al.,2016;
Mallick et al.2016; Arpha et al.,2012; Stanikunaite et al.,2008; Lai et al.,2012; Pimjuk et
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al.,2015; Isaka et al., 2016). Studies on the ascorbic acid, phenol, and flavonoid content of
AAEA extract has been scarce or non-existent. The different parameters of mycochemistry
study of Astraes asiaticus have been successfully analysed in this research article, based on
total phenol, flavonoid, and ascorbic acid antioxidant content, as well as the scavenging
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activity of this mushroom. Antioxidants are crucial substances that aid in boosting the body's
resistance to free radicals. In order to have antioxidant properties, phenolic compounds are
necessary (Velioglu et al.,1998). According to Xu, phenolic compounds are the most
effective anticancer compounds (Xu, 2012). TNF- gene expression and production have been
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found to be inhibited by phenolic compounds (Singh and Vyas, 2011; Paul et al.,2006).
Ascorbic acid was found in low concentrations (1.59 ±0.33 mg AAE/g) in our study, which is
supported by other authors (Mou et al.,2002). The FTIR spectroscopic study provides such
information about the functional group of the extract. The chemical nature of the
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mycochemical compounds found in this mushroom will provide information on the various
functional groups responsible for their anticancer activity. Choong et al. (2011) used FTIR
analysis to show the different functional mycochemical compounds of Ganoderma lucidum
crude extract. Muruganantham et al. (2009) investigated the presence of carboxylic acids,
amines, amides, sulphur derivatives, polysaccharides, nitrates, chlorates, and carbohydrate in
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plant parts such as the leaf, stem, and root of the medicinal plants Eclipta alba and Eclipta
prostrate. FTIR was used by Starlin et al. (2012) to identify amino acids, amides, amines,
carboxylic acid, carbonyl compounds, organic hydrocarbons, and halogens in ethanolic
extracts of Ichnocarpus frutescens. Many authors have proposed that mushrooms with high
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antioxidant activity can help prevent cancer (Ferreira et al., 2009; Kidd, 2000). The
anticancer compound of this AAEA must be identified using the GCMS analysis. The GC-
MS profile of AAEA revealed that 12 compounds out of 61 had a higher proportion with a
percentage peak area greater than one. Silica gel column chromatography was used to elute
different fractions from AAEA extract, and the F12 fraction out of twenty fractions produced
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the best DPPH estimate. F 12 had the highest radical scavenging activity, which was 21.65 ±
0.81 µg/mL. This discovery is supported by other researchers (Keshava et al., 2020; Fang et
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al.,2006).The thin layer chromatogram analysis of F12 revealed eight distinct spots, four of
which matched perfectly with the standard quinine, lupeol, quercetin, and p-Coumaric acid,
implying that the fraction F12 contained an alkaloid, triterpene, flavonoid, and phenol group
Other researchers concur with this finding (Sudirman et al.,2010).The mass spectrum of F12
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revealed six different compounds, of which 3,4,5,6 Tetramethyloctane, Hexadecanoic acid,
and 9,12-Octadecadienoic acid displayed anti-cancerous activity, respectively. 9,12-
Octadecadienoic acid, methyl ester showed antioxidant activity. According to other
researcher these four compounds showed anticancer or antioxidant activity as well (Wang et
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al., 2014; Kristiani et al.,2016). We started to search database (Swiss target prediction, www.
Swiss target pridiction.ch) for the binding affinity cites of these compounds. We discovered
that, 9, 12-Octadecadienoic acid (Z, Z) binds to membrane fatty acid binding protein,
peroxisome proliferator activated receptor, and tyrosyl DNA phosphodiesterase.
Hexadecanoic acid, another important fatty acid compound, is present and has shown
ot
significant results. Human colorectal carcinoma cell cytotoxicity (HCT-116) (Lokesh and
Kannabiran., 2017). According to Harada et al. (2002) n-hexadecanoicacid inhibited the
proliferation of human fibroblast cells by inhibiting DNA topoisomerase-I. According to Lai
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et al. (2008), it binds to the microtubule associated protein 'tau' and inhibits cell division.
In our study, F12 of AAEAE demonstrated potent antiproliferative activity against HeLa,
MCF7, and A549 cell lines. The cytotoxicity assay used in this study revealed a maximum
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inhibition of HeLa, breast, and lung cancer cell lines of 92.03 ± 1.65, 90.38 ± 0.53, 87.51 ±
1.36% respectively after F12 treatment, and cell morphological changes were observed from
the normal spindle shape to either rounded or irregular shapes; additionally, decreases in
confluence level were observed based on phase contrast microscopy analysis and the F12 of
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AAEA extract occurred apoptosis and gene expression level of Caspase 3,caspase 9, P53,
Bcl2. F12 reduced Bcl-2 protein expression while increasing the expression of pro-apoptotic
genes caspase 3 and caspase 9. Furthermore, F12 increased the expression of the p53 gene.
Similarly, Jedinak et al (2008) found that P. ostreatus altered the morphology of MCF-7
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despite the fact that their study was based on a methanolic extract of the fruit body. Bhat et al.
(2013) prepared gold nanoparticles (AuNPs) from a water-soluble extract of P. florida and
applied them to HeLa and other cell lines for 24h. However, various solvent extracts of other
mushrooms has previously been tested on cancer cell lines. On the A549 cell line, the
methanolic extract of Agaricus lanipes extract demonstrated antiproliferative and strong pro-
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apoptotic activity (Kaygusuz et al.,2017) Antrodia Cinnamonea extract in ethanol
demonstrated anticancer activity in vitro on CL1-0 cells. The extract inhibited migration and
cell motility, suppressed matrix metalloproteinase (MMP)-2 and -9 activity, increased the
level of MMP tissue inhibitors (TIMP-1 and TIMP-2), and inhibited p38, AKT, and JNK1/2
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phosphorylation (Chen et al.,2012) Calvatia gigantea methanolic extract stopped cancer cell
growth in A549 cells. Cell cycle arrest and apoptosis were caused by the extract, which was
mediated by decreased expression of Akt, BCL2, CCND1, CCND2, and CDK4 and increased
expression of caspase-3, caspase-9, BAX, and p53 protein. (Eroğlu et al.,2016) Hexagonia
glebra extracts in ethanol, ethyl acetate, and water displayed anticancer activity in HeLa,
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SiHa, and CaSki cells. In vitro (HeLa, SiHa, and CaSki cells), the fraction stopped cell
division at the G2/M checkpoint and induced apoptosis by increasing the expression of
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caspase-3, caspase-9, and the proapoptotic gene p53. Nonetheless, the antiapoptotic gene
BCL2 expression was reduced (Ghosh et al.,2020). Ethyl acetate fraction from shitake
mushroom accoutred apoptosis and cell line inhibition (Fang et al.,2006). The fraction of
ethyl acetate extract of Astragalus membranaceus showed anticancer activity and apoptosis
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(Park et al., 2018).
5. Conclusion
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In conclusion AAEA extract contains a variety of bioactive compound, functional groups
and anticancer compounds. The partial purification of the AAEA extract yielded the active
F12 fraction, which contained triterpene, phenol, flavonoid, and alkaloid groups. The mass
spectrum of F12 contained six different compounds in which 3,4,5,6 Tetramethyloctane,
Hexadecanoic acid,9,12-Octadecadienoic acid may also have anticancer activity against
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cervical, breast, and lung cancer cell lines in vitro. 9,12-Octadecadienoic acid, methyl ester
demonstrated antioxidant activity. F12 fraction showed apoptosis, LDH effect, and up
regulation of gene expression levels of Caspase 3, Caspase 9, P53, and down regulation of
Bcl2 were observed. As a result, the use of F12 from AAEA extract in cancer treatment will
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be a novel study for future drug development after animal and human trial.
Acknowledgements
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The authors would like to thank the Principal of Ramakrishna Mission Vivekananda
Centenary College for providing the Mycopathology, Biocontrol and Cancer Research Unit,
and Biochemistry laboratory facilities, as well as the Department of Science and Technology
of West Bengal, India, for financial support.
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Conflict of interest
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Authors have no conflict of interest.
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References:
Adamson, J.W. and Longo, D.L., (2005). Anemia and polycythemia. Harrisons Principles
Of Internal Medicine, 16(1), 329.
iew
Anderson, D.M., Biemann, K., Orgel, L.E., Oro, J., Owen, T., Shulman, G.P., Toulmin
III, P. and Urey, H.C., (1972). Mass spectrometric analysis of organic compounds, water
and volatile constituents in the atmosphere and surface of Mars: the Viking Mars
Lander. Icarus, 16(1), 111-138.
v
Arpha, K., Phosri, C., Suwannasai, N., Mongkolthanaruk, W. and Sodngam, S., (2012).
Astraodoric acids A–D: new lanostane triterpenes from edible mushroom Astraeus
re
odoratus and their anti-Mycobacterium tuberculosis H37Ra and cytotoxic
activity. Journal of agricultural and food chemistry, 60(39), 9834-9841.
Ashokkumar, R. and Ramaswamy, M., (2014). Phytochemical screening by FTIR
spectroscopic analysis of leaf extracts of selected Indian medicinal plants. International
journal of Current Microbiology and applied Sciences, 3(1),395-406.
er
Auwal, M.S., Saka, S., Mairiga, I.A., Sanda, K.A., Shuaibu, A. and Ibrahim, A., (2014).
Preliminary phytochemical and elemental analysis of aqueous and fractionated pod
extracts of Acacia nilotica (Thorn mimosa). In Veterinary research forum: an
pe
international quarterly journal, 5(2),95.
Bajpai, V.K., Majumder, R., & Park, J.G. (2016). Isolation and purification of plant
secondary metabolites using column-chromatographic technique. Bangladesh Journal of
Pharmacology, 11(4),844-848.
ot
Bele, A.A., & Khale, A. (2011). An overview on thin layer chromatography. International
journal of pharmaceutical sciences and research, 2(2), 256.
Bhat, R., Sharanabasava, V.G., Deshpande, R., Shetti, U., Sanjeev, G., & Venkataraman,
tn
A. (2013). Photo-bio-synthesis of irregular shaped functionalized gold nanoparticles

using edible mushroom Pleurotus florida and its anticancer evaluation. Journal of
Photochemistry and Photobiology B: Biology, 125, 63-69.
rin
Biswas G, Chatterjee S, Sarkar S., & Acharya K. (2010). Evaluation of antioxidant and
nitric oxide synthase activation properties of Astraeus hygrometricus (Pers.)
Morg.International Journal of Biomedical and Pharmaceutical Sciences ,21-26.
Biswas, G., Sarkar, S., & Acharya, K. (2010). Free radical scavenging and anti-
ep
inflammatory activities of the extracts of Astraeus hygrometricus (Pers.) Morg. Lat Am J

Pharm, 29(4),549-53.
Biswas, G., Sarkar, S., & Acharya, K.(2011). Hepatoprotective activity of the ethanolic
extract of Astraeus hygrometricus (Pers.) Morg. Dig J Nanomater Bios, 6(2), 637-641.
Pr
Biswas, G., &Acharya, K.(2013). Hypoglycemic activity of ethanolic extract of Astraeus
hygrometricus (Pers.) Morg. in alloxan-induced diabetic mice. Int J Pharm Pharm
ed
Sci, 5(1), 391-394..
Bondet, V., Brand-Williams, W., & Berset, C.L.W.T.( 1997). Kinetics and mechanisms
of antioxidant activity using the DPPH. free radical method. LWT-Food Science and
Technology, 30(6), 609-615.
iew
Breene, W.M. (1990). Nutritional and medicinal value of specialty mushrooms. Journal of
food protection, 53(10), 883-894.
Carvajal, A.E.S., Koehnlein, E.A., Soares, A.A., Eler, G.J., Nakashima, A.T., Bracht, A.,
& Peralta, R.M.(2012). Bioactives of fruiting bodies and submerged culture mycelia of
Agaricus brasiliensis (A. blazei) and their antioxidant properties. LWT-Food Science and
v
technology, 46(2), 493-499.
re
Chen, Y.Y., Chou, P.Y., Chien, Y.C., Wu, C.H., Wu, T.S., & Sheu, M.J.( 2012). Ethanol
extracts of fruiting bodies of Antrodia cinnamomea exhibit anti-migration action in
human adenocarcinoma CL1-0 cells through the MAPK and PI3K/AKT signaling
pathways. Phytomedicine, 19(8-9), 68-778.
er
Choong, Y.K., Sun, S.Q., Zhou, Q., Ismail, Z., Rashid, B.A.A., & Tao, J.X.( 2011).
Determination of storage stability of the crude extracts of Ganoderma lucidum using
FTIR and 2D-IR spectroscopy. Vibrational Spectroscopy, 57(1), 87-96.
pe
Das, B., Rajkonwar, J., Jagannath, A., Raul, P.K., & Deb, U.( 2020). Infra-red spectra of
different species of cultivated oyster mushrooms: Possible tool for identifying bioactive
compounds and establishing taxonomic linkage. Def. Life Sci. J, 5, 118-124.
Eccles, S.A., Aboagye, E.O., Ali, S., Anderson, A.S., Armes, J., Berditchevski, F.,
ot
Blaydes, J.P., Brennan, K., Brown, N.J., Bryant, H.E., & Bundred, N.J.( 2013). Critical
research gaps and translational priorities for the successful prevention and treatment of
breast cancer. Breast Cancer Research, 15(5), 1-37.
tn
Ekowati, N., Maharning, A.R., Ratnaningtyas, N.I., Mumpuni, A., & Hikam, A.R.(
2020).November. Effects of Ethyl Acetate Extract of Jew’s Ear Mushrooms (Auricularia
auricula) on Cytotoxic and Apoptosis of Cervical Cancer Cells (HeLa). In IOP
Conference Series: Earth and Environmental Science, 593, 012011.
rin
Eroğlu, C., Seçme, M., Atmaca, P., Kaygusuz, O., Gezer, K., Bağcı, G., & Dodurga,
Y.(2016). Extract of Calvatia gigantea inhibits proliferation of A549 human lung cancer
cells. Cytotechnology, 68(5), 2075-2081.
ep
Fang, N., Li, Q., Yu, S., Zhang, J., He, L., Ronis, M.J., & Badger, T.M. (2006). Inhibition
of growth and induction of apoptosis in human cancer cell lines by an ethyl acetate
fraction from shiitake mushrooms. Journal of Alternative & Complementary
Medicine, 12(2), 125-132.
Pr
Fang, N., Li, Q., Yu, S., Zhang, J., He, L., Ronis, M.J., & Badger, T.M.( 2006). Inhibition
of growth and induction of apoptosis in human cancer cell lines by an ethyl acetate
fraction from shiitake mushrooms. Journal of Alternative & Complementary
Medicine, 12(2), 125-132.
ed
Ferreira, I.C., Barros, L., & Abreu, R.(2009). Antioxidants in wild mushrooms. Current
Medicinal Chemistry, 16(12), 1543-1560.
Finimundy, T.C., Gambato, G., Fontana, R., Camassola, M., Salvador, M., Moura, S.,
iew
Hess, J., Henriques, J.A.P., Dillon, A.J.P., & Roesch-Ely, M.( 2013). Aqueous extracts of
Lentinula edodes and Pleurotus sajor-caju exhibit high antioxidant capability and
promising in vitro antitumor activity. Nutrition Research, 33(1), 76-84.
Freshney, R.I.( 2015). Culture of animal cells: a manual of basic technique and
specialized applications. John Wiley & Sons.
v
Fransina, E.G., Tanasale, M.F., Latupeirissa, J., Malle, D. & Tahapary, R.(2019). April.
re
Phytochemical screening of water extract of gayam (Inocarpus edulis) Bark and its
amylase inhibitor activity assay. In IOP Conference Series: Materials Science and
Engineering (Vol. 509, No. 1, p. 012074). IOP Publishing.
er
Ghosh, S.K., Sanyal, T., & Bera, T. (2020). Anticancer activity of solvent extracts of
Hexogonia glabra against cervical cancer cell lines. Asian Pacific Journal of Cancer
Prevention: APJCP, 21(7), 1977..
pe
Gul, R., Jan, S.U., Faridullah, S., Sherani, S. & Jahan, N.( 2017). Preliminary
phytochemical screening, quantitative analysis of alkaloids, and antioxidant activity of
crude plant extracts from Ephedra intermedia indigenous to Balochistan. The Scientific
World Journal, 2017.
ot
Harada, H., Yamashita, U., Kurihara, H., Fukushi, E., Kawabata, J., & Kamei, Y.( 2002).
Antitumor activity of palmitic acid found as a selective cytotoxic substance in a marine
red alga. Anticancer research, 22(5),2587-2590.
tn
Hossain, M.A., AL-Raqmi, K.A.S., Al-Mijizy, Z.H., Weli, A.M., & Al-Riyami, Q.(
2013). Study of total phenol, flavonoids contents and phytochemical screening of various
leaves crude extracts of locally grown Thymus vulgaris. Asian Pacific journal of tropical
biomedicine, 3(9), 705-710.
rin
Isaka, M., Palasarn, S., Srikitikulchai, P., Vichai, V., & Komwijit, S.( 2016). Astraeusins
A–L, lanostane triterpenoids from the edible mushroom Astraeus
odoratus. Tetrahedron, 72(23), 3288-3295.
ep
Jagota, S.K., & Dani, H.M. (1982). A new colorimetric technique for the estimation of
vitamin C using Folin phenol reagent. Analytical biochemistry, 127(1),178-182.
Jagtap, N.S., Khadabadi, S.S., Ghorpade, D.S., Banarase, N.B., & Naphade, S.S.( 2009).
Antimicrobial and antifungal activity of Centella asiatica (L.) Urban,
Pr
Umbeliferae. Research Journal of Pharmacy and Technology, 2(2), 328-330.
Jedinak, A.,Kawasaki, J., Harvey, K., & Slivova,V.(2008). Phellinuslinteussuppresses
growth, angiogenesis and invasive behaviour of breast cancer cells through the inhibition
ed
of AKT signaling. Br. Jr Can, 98, 1348-1356.
Kaygusuz, O., Kaygusuz, M., Dodurga, Y., Seçme, M., Herken, E.N. , &Gezer, K.(
2017). Assessment of the antimicrobial, antioxidant and cytotoxic activities of the wild
iew
edible mushroom Agaricus lanipes (FH Møller & Jul. Schäff.)
Hlaváček. Cytotechnology, 69(1), 135-144.
Keshava, R., Muniyappa, N., & Gope, R. (2020). Bioactivity guided fractionation and
elucidation of anti-cancer properties of Imperata cylindrica leaf extracts. Asian Pacific
Journal of Cancer Prevention: APJCP, 21(3), 707.
v
Kidd, P.M.( 2000). The use of mushroom glucans and proteoglycans in cancer
re
treatment. Alternative medicine review, 5(1), 4-27.
Kristiani, E.B., Nugroho, L.H., Moeljopawiro., & SWidyarini, S.( 2016). Characterization
of volatile compounds of Albertisia papuana Becc root extracts and cytotoxic activity in
breast cancer cell line T47D. Tropical Journal of Pharmaceutical Research, 15(5), 959-
er
964.
Lai, C.S., Mas, R.H., Nair, N.K., Majid, M.I.A., Mansor, S.M., & Navaratnam, V.(2008).
pe
Typhonium flagelliforme inhibits cancer cell growth in vitro and induces apoptosis: an
evaluation by the bioactivity guided approach. Journal of Ethnopharmacology, 118(1),
14-20.
Lai, T.K., Biswas, G., Chatterjee, S., Dutta, A., Pal, C., Banerji, J., Bhuvanesh, N.,
Reibenspies, J.H., & Acharya, K., (2012). Leishmanicidal and anticandidal activity of
ot
constituents of Indian edible mushroom Astraeus hygrometricus. Chemistry &

biodiversity, 9(8), 1517-1524.
Lixia, Z., Cong, F., Songchen, L., Zhenfeng, Z., Lili, J. & Liping, Z. (2011). Chemical
tn
composition and antitumor activity of polysaccharide from Inonotus obliquus. Journal of

Medicinal Plants Research, 5(7), 1251-126.
Lokesh, R., & Kannabiran, K. (2017).Cytotoxic potential of N-hexadecanoicacid
rin
extracted fromKigeliapinnata leaves. Asian J Cell Bio,l12, 20-27.
Mallick, S., Dutta, A., Chaudhuri, A., Mukherjee, D., Dey, S., Halder, S., Ghosh, J.,
Mukherjee, D., Sultana, S.S., Biswas, G., & Lai, T.K. (2016). Successful therapy of
murine visceral leishmaniasis with astrakurkurone, a triterpene isolated from the
ep
mushroom Astraeus hygrometricus, involves the induction of protective cell-mediated

immunity and TLR9. Antimicrobial agents and chemotherapy, 60(5),2696-2708.
Mandal, S., Basu, S., & Maiti, T.(2015). In vivo evaluation of anti-inflammatory and
antioxidant potential of aqueous extract of Astraeus hygrometricus on albino
Pr
rats.European Journal of Pharmaceutical and Medical Research 2,358–361.

Mau, J.L., Lin, H.C., & Song, S.F. (2002). Antioxidant properties of several specialty
mushrooms. Food research international, 35(6), 519-526.
McDonald, S., Prenzler, P.D., Antolovich, M., & Robards, K.(2001). Phenolic content
and antioxidant activity of olive extracts. Food chemistry, 73(1), 73-84.
ed
Meda, A., Lamien, C.E., Romito, M., Millogo, J., & Nacoulma, O.G.( 2005).
Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan
honey, as well as their radical scavenging activity. Food chemistry, 91(3), 571-577.
iew
Muruganantham, S., Anbalagan, G., & Ramamurthy, N.(2009). FT-IR and SEM-EDS
comparative analysis of medicinal plants, Eclipta alba Hassk and Eclipta prostrata
Linn. Romanian J. Biophys, 19(4), 285-294.
Park, H.J., & Park, S.H. (2018). Induction of apoptosis by Ethyl Acetate Fraction of
Astragalus membranaceus in human non-small cell lung cancer cells:-apoptosis induction
v
by Astragalus membranaceus. Journal of Pharmacopuncture, 21(4), 268.
re
Patel, S., & Goyal, A. (2012). Recent developments in mushrooms as anti-cancer
therapeutics: a review. 3 Biotech, 2(1), .1-15.
Paul, A.T., Gohil, V.M., & Bhutani, K.K. (2006). Modulating TNF-α signaling with
natural products. Drug discovery today, 11(15-16), 725-732.
er
Pavithra, M., Sridhar, K.R., Greeshma, A.A., & Tomita-Yokotani, K. (2016). Bioactive
potential of the wild mushroom Astraeus hygrometricus in South-west
India. Mycology, 7(4), 191-202..
pe
Pimjuk, P., Phosri, C., Wauke, T., & McCloskey, S. (2015). The isolation of two new
lanostane triterpenoid derivatives from the edible mushroom Astraeus
asiaticus. Phytochemistry Letters, 14, 79-83..
Sanyal, T., & Ghosh, S.K.(2019). Anti-cancer property of Lenzites betulina (L) Fr. on
ot
cervical cancer cell lines and its anti-tumor effect on HeLa-implanted mice. BioRxiv,
540567.
Singh, V., &Vyas, D.( 2011). Antioxidant potentiality of Pleurotus ostreatus (MTCC142)
tn
cultivated on different agro wastes. Asian Journal of Plant Science & Research.
Stanikunaite, R., Radwan, M.M., Trappe, J.M., Fronczek, F., & Ross, S.A. (2008).
Lanostane-type triterpenes from the mushroom Astraeus pteridis with antituberculosis
activity. Journal of Natural products, 71(12), 2077-2079.
rin
Starlin, T., Ragavendran, P., Raj, C.A., Perumal, P.C., & Gopalakrishnan, V.K.( 2012).
Element and functional group analysis of Ichnocarpus frutescens R.
Br.(Apocynaceae). Int J Pharm Pharm Sci, 4, 343-345.
ep
Sudirman, L.I.( 2010). Partial purification of antimicrobial compounds isolated from

mycelia of tropical Lentinus cladopus LC4. HAYATI Journal of Biosciences, 17(2), 63-
67.
Pr
Sun, Y., & Liu, J.(2009). Purification, structure and immunobiological activity of a
water-soluble polysaccharide from the fruiting body of Pleurotus ostreatus. Bioresource
Technology, 100(2), 983-986.
Talukdar, N., Dutta, A.M., Chakraborty, R., & Das, K. (2017). Screening of
phytochemicals, antioxidant and inhibitory effect on alpha-amylase by ethanolic extract
ed
of Elaeocarpus Ganitrus (Bark). International Journal of Pharmaceutical Sciences and
Research, 8(12), 5270-5275.
Usman, H., Abdulrahman, F., & I Usman, A.( 2009). Qualitative phytochemical screening
and in vitro antimicrobial effects of methanol stem bark extract of Ficus thonningii
iew
(Moraceae). African Journal of Traditional, Complementary and Alternative
Medicines, 6(3).
Velioglu YS, Mazza G, Gao L, Oomah BD. (1998).Antioxidant activity and total
phenolics in selected fruits, vegetables, and grain products. J. Agric. Food Chem., 46:
4113-4117.
Vorauer, K., Steindl, F., Jungbauer, A., Hahn, R., & Katinger, H. (1996). Cytokine
v
activity assay by means of proliferation measured in plane convex microtiter
wells. Journal of biochemical and biophysical methods, 32(2), 85-96.
re
Wang, C., Dong, R., Wang, X., Lian, A., Chi, C., Ke, C., Guo, L., Liu, S., Zhao, W., Xu,
G., & Li, E.( 2014). Exhaled volatile organic compounds as lung cancer biomarkers
during one-lung ventilation. Scientific reports, 4(1),1-8.
er
Wani, BA., Bodha, RH., &Wani, AH. (2010). Nutritioal and medicinal importance of
mushrooms. Journal of Medicinal Plants Research, 4(24),2598-2604.
World Health Organization (WHO), 2020.World Cancer report
pe
2020,(https://www.who.int/news-room/fact-sheets/detail/cancer )
Xu, T. (2012). Anti-cancer effects of phenolic-rich extracts of button mushrooms
(Agaricus bisporus).
ot
tn
rin
ep
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2.1 Solvent Extraction

evaporator (Superfit, Model: PBV-7D) at 500 C, lyophilized (Biobase, Model: BK-FD10P)

2.2 Mycochemical Screening of AAEA extract

2.2.1. Test for Flavonoid

0.2 g of AAEA extract was confiscated and dissolved in 2 mL of chloroform and 3

2.2.3. Test for cardiac glycosides

2.2.4. Test for steroid

2.2.5. Alkaloid test

alkaloid emanation was observed (Auwal et al., 2014).

2.2.6. Tests for carbohydrate

investigated at the interface. (Auwal et al., 2014).

2.2.7. Test for phlobatannins

precipitate (Auwal et al., 2014).

2.2.8.Test for Phenol

2.3.2 Totality of flavonoid (TFC) content assay

2.3.3. Ascorbic acid content assay

2.4. Fourier Transform Infrared Spectrophotometer (FTIR) analysis of AAEA extract

2.5 GCMS analysis of AAEA extract

2.5.1. Preparation of the AAEA

GC-MS analysis was carried out on a GC system comprising a Gas Chromatograph

interfaced to a Mass Spectrometer (GC-MS) instrument; Agilent Technologies, GC-6860N

Purification of AAEA extract was carried out by column chromatography (Bajpai et

2.6.1. Charging of the Column

and the removal of air bubbles, resulting in a static bed.

2.7.Antioxidant test of each fraction

2.7 .TLC Profiling of F12 of AAEA extract

Retention factor (Rf) =

b = Distance travelled by solvent

2.8. Mass sprectrum analysis of F12

At = Absorbance of the test.

2.9.3. Cell morphology examined under a phase contrast microscope

2.9.4. Apoptosis analysis

2.9.6. Western blot analysis for study of expression of genes

Biotechnology, USA) were studied.

Fig. 1. A brief summary of the general approaches in extraction, functional group

3.4. FTIR spectral data interpretation of AAEA extract

Fig. 3. FTIR spectrum of AAEA extract

2 2926.2 2850-3000 S C-H stretch Alkanes and Alkyls

5 1460 1600-1450 m-s ring C-C stretch Aromatic compound

10 978.6 980-960 S =C-H bend Alkens group

12 722.6 725-715 W - (CH2)n bend Alkanes and Alkyls

Fig. 4. GC-MS chromatogram of the AAEA extract

Table 3 GCMS profiling of AAEA extract

S/N of compound Apex RT Start RT End RT Area % Area Height %Height

4 10.8 10.7 11.15 46684.75 15.35 3321.27 11

33 24.17 24.12 24.21 106.096 0.03 82.244 0.27

36 24.49 24.44 24.54 285.437 0.09 68.851 0.23

40 25.36 25.3 25.77 4495.222 1.48 258.873 0.86

44 26.89 26.85 26.93 35.277 0.01 27.51 0.09

49 28.83 28.8 28.95 232.886 0.08 42.56 0.14

6 21.3 2.52 cis-9,10-Epoxyoctadecan-1-ol 284 C18H36O2

8 22.67 2.83 3-Ethyl-3-undecanol 200 C13H28O

12 25.05 2.17 Heptaethylene glycol 326 C14H30O8

3.7. Analytical TLC of the F 12 of AAEA extract

Sample/ Stationary Mobile Run time No of spots Rf value

Ethyl 0.87 0.45

Quinine SILICA gel- Chloroform 20 1 1 0.37 0.37

3.8. Mass spectrum analysis of F 12 of AAEA extract