SUPPLEMENTARY MATERIAL

Extraction Optimization, Total Phenolic, Flavonoid contents, HPLC-DAD analysis and diverse
Pharmacological evaluations of Dysphania ambrosioides (L.) Mosyakin & Clemants
Tanzeel Zohra1, Muhammad Ovais1, Ali Talha Khalil1, Muhammad Qasim1, Muhammad Ayaz2*,
Zabta Khan Shinwari1, 3

The present study aims to evaluate phytochemical and pharmacological potentials of Dysphania
ambrosioides (L.) Mosyakin & Clemants previously known as Chenopodium ambrosioides L.
Extraction was carried out using 14 solvents with wide range of polarity to find out the best
solvent system for each bioactivity. Total phenolic and flavonoids contents were measured
colorimetrically and polyphenolics were quantified via HPLC-DAD analysis. The samples were
screened for inhibitory potentials against free radicals, leishmania, cancer cell lines, protein
kinase, α-Amylase enzymes and microbial strains. Among all solvents, maximum percentage of
extract was recovered from methanol-water fraction of leaves. HPLC analysis exhibited the
presence of rutin, myricetin and quercetin. In DPPH assay, methanolic leaf extract exhibited IC50
value of 130.7±0.57 µg/mL. Considerable α-amylase inhibitory, cytotoxic, leishmanicidal and
antimicrobial potentials were exhibited by plant samples. D. ambrosioides revealed significant
antioxidant, cytotoxic, antimicrobial and anti-diabetic potentials and thus warrant further detailed
studies to find novel drugs.
Key words: Dysphania ambrosioides L. , HPLC-DAD, Antioxidants, HepG2 cell line,
Cytotoxicity, Leishmania, Protein kinase, Antimicrobial resistance.

4. Experimental

4.1. Chemicals and Reagents

In this current study, all solvents that were used for extraction were of analytical grade. The
solvents included dimethyl sulfoxide (DMSO), chloroform, ethanol, ethyl acetate, acetone,
methanol and n-hexane. The solvents and reagents like potassium acetate, aluminium chloride,
sulfuric acid, potassium ferricyanide, ammonium molybdate, trichloroacetic acid, ferric chloride,
potassium dihydrogen phosphate, dipotassium hydrogen phosphate was purchased from Merck,
Darmstadt, Germany, Folin-Ciocalteu reagent and 2, 2-diphenyl-1-picryhydrazyl (DPPH) were
bought from Sigma–Aldrich (Steinheim, Germany). The Tween-20 was bought from Merck
(Schuchardt, USA), while roxithromycin and cefixime, clotrimazole and amphotericin B,
doxorubicin, vincristine, surfactin, sterile normal saline solution (0.9%), tryptone soy broth
(TSB), Sabouraud dextrose agar (SDA), nutrient agar, sea salt were purchased from Sigma
(Sigma Aldrich, USA), Medium ISP4 (Prepared in lab), and those reagents that were used i.e.
ascorbic acid, gallic acid, rutin, caffeic acid, quercetin, myricetin, catechin, kaempferol were also
gotten from Merck, Darmstadt, Germany.

4.2. Cultures and cell line

4.2.1. Bacterial strains

The bacterial strains and cultures used in biological assays were includes Staphylococcus aureus
(ATCC 6538), Micrococcus luteus (ATCC 10240), Bacillus thuringiensis (ATCC 6633),
Klebsiella pneumonia (ATCC 1705), and Escherichia coli (ATCC 25922).

4.2.2. Fungal strains and cell lines

Fungal strains used as test organism were Fusarium solani (FCBP 0291), Aspergillus flavus
(FCBP 0064), Aspergillus fumigatus (FCBP 66), Mucor species (FCBP 0300) and Aspergillus
niger (FCBP 0198). Hep G2 cancer cell line (RBRC-RCB1648), Leishmania tropica kwh 23
strain, Brine shrimp eggs (Ocean star Int, USA) and Streptomyces 85E strains were used.

4.3. Collection and identification of plant

Dysphania ambrosioides whole plant was collected in November 2014 from Quaid-i-Azam
University campus, Islamabad, Pakistan and verified by Prof. Dr. Mushtaq Ahmad, Department
of Plant sciences, Faculty of Biological Sciences, Quaid-i-Azam University Islamabad. Plant was
selected based on safety profile in humans, cost effectiveness and ease of availability. A voucher
specimen was deposited in MOSAEL Lab’s herbarium (voucher number MOSEAL-345), Quaid-
i-Azam University Islamabad, Pakistan.

4.3.1. Preparation of crude extract

Different parts of D. ambrosioides (leaf, stem and root) were apportioned to remove any
deteriorated or foreign matter followed by rinsing with tap water. Subsequently, they were shade
dried in well-ventilated area at room temperature for four weeks. Plant samples were converted
to powder form with the help of a commercial grinder. The powdered samples were then used for
the extraction. A total of 42 samples were prepared from the subject plant parts (Ali M et al.
2017).

4.3.2. Maceration
Extraction was carried out using 14 different solvents of wide ranging polarity from non-polar to
highly polar solvents. The solvent systems employed in the current study include the following:
n-hexane (Nh), chloroform (C), ethyl acetate (Eth), acetone (A), methanol (M), ethanol (E),
distilled water (D), ethyl acetate-n-hexane (EthNh), ethanol-n-hexane (ENh), methanol-
chloroform (MC), methanol-ethyl acetate (MEth), methanol-acetone (MA), acetone-distilled
water (AD) and methanol-distilled water (MD). Combinations of solvents were employed in 1:1.
The weighed amount of plant powder (30 g of each) was soaked in different solvents in separate
Erlenmeyer flask and left to macerate for three days by continuous shaking at a frequency of 40
KHz in ultrasonic bath. Each solvent extraction was repeated twice. The crude extracts were
filtered at the end of each extraction by muslin cloth after that by Whatman No.1 filter paper.
The filtrates were vaporized and dried through hot plate and fume hood. The dried crude extracts
were thus obtained and were stored in pre-weighed vials at -20 ºC till further analysis.

4.3.3. Extract recovery

The crude dried extracts were evaluated to determine their percent extract recovery using
formula. % Extract recovery = (X / Y) x 100

X = weight of crude dried extract and y = weight of powdered plant material used for each
extraction

4.4. Phytochemicals screening

4.4.1. Total Phenolic Content (TPC)

The phenolic compounds content present in sample was calculated using Folin Ciocalteu reagent
described previously with slight modifications (Ayaz et al. 2014). Add 20 µL Aliquot of a
sample in respective well plate followed by adding 90 µL Folin Ciocalteu reagent (FCR).
Incubate it for 5 minutes after that add 90 µL of sodium carbonate and again indorsed to stand it
for 30 min incubation at 37 oC. Gallic acid was employed as a positive standard. With the help of
microplate reader in triplicate at 630 nm the absorbance of reaction mixture was recorded
(Biotech USA, microplate reader Elx 800). The calibration curve (y = 0.0136x + 0.0845, R2 =
0.9861) was drawn by application of the gallic acid as a standard. Resulting phenolic content was
expressed as gallic acid equivalent per mg of extract (µg GAE/mg extract).

4.4.2. Total Flavonoid Content (TFC)

Total flavonoid content in different crude extracts of plant material was calculated by slightly
modified colorimetric method which was based on aluminum chloride (Haq et al. 2011). The
estimation was done based on the formation of aluminum chloride complex. Add 20 µL Aliquot
of a sample in respective well plates and then add 10 µL of aluminium chloride, 10 µl of 1 M
potassium acetate and 160 µL of distilled water. Then at room temperature, with the help of
microplate reader in triplicate at 415 nm the absorbance of reaction mixture was recorded
reaction mixture was incubated for about 30 min. Setting quercetin as a standard, the calibration
curve (y = 0.0268x + 0.00764, R2 = 0.9986) was obtained. Resulting flavonoid Content was
expressed as Quercetin equivalent per mg of extract (µg QE/mg extract).

4.5. Quantitative analysis via HPLC-DAD

Chromatographic investigation of D. ambrosioides extracts for polyphenols were carried out via
High-performance liquid chromatography with diode-array detection equipped with C18
analytical column (Agilent chem station Rev. B.02-01-SR1 and 1200 series Agilent binary
bump) attached with diode array detector (DAD; Agilent technologies, Germany). The method
followed was as described by (Jafri et al. 2014) with slight modification. Solutions of each plant
part were prepared in methanol (10 mg/mL) and standard i.e. caffeic acid, apigenin, gallic acid,
rutin, quercetin, catechin, myricetin and kaempferol were also prepared in methanol (50µg/mL)
through sonication and followed by filtration through 0.2 µm sartolon polyamide filter
(Sartorious). All the solutions were prepared before analysis or stored at 4 °C for the later used.
Two types of mobile phases were used for the analysis of polyphenols, one (mobile phase A)
having acetonitrile-methanol-water-acetic acid in 5:10:85:1 ratio and second (mobile phase B)
acetonitrile-methanol-acetic acid in 40:60:1 ratio. The flow rate was kept at 1 mL per minute.
Aliquot of 20 µL of each sample solution was inserted into Zorbax RX-C8 column and the
column was allowed to recondition for 10 min before the next analysis. The gradient volume of
B was 0-50 % in 0-20 min, 50-100 % in 20-25 min and then 100 % from 25-30 min. The
absorption of samples was recorded at different wavelengths e.g. were determined at 257 nm
(gallic acid and rutin), 279 nm (catechin), 325 nm (caffeic acid) and at 368 nm (kaempferol,
quercetin, myricetin).

4.6. Antioxidant Assays

4.6.1. Free Radical Scavenging Activity

The antioxidant potential of D. ambrosioides extracts were estimated by measuring the change
in color from purple colored DPPH solution to diphenyl picrylhydrazine (Yellow Colored) by
following the standard procedure (Ayaz, Junaid et al. 2015). Briefly, 20 µL Aliquot of sample
was added to respective 96 well plates followed by adding 180 µL of DPPH to each well. The
resultant mixture was subjected to incubation in dark at temperature of 37 oC for one hour. With
the help of micro plate reader, the absorbance of each reaction mixture and the standard was
calculated at 517 nm (Biotech USA, micro plate reader Elx 800). As reference standard, we have
used ascorbic acid and the experiment was performed in triplicate Spectrometric analysis used
for radical scavenging potential of samples and 50 % inhibitory concentration. The samples
which showed greater than 50 % scavenging activity were verified at lower concentrations by
using three-fold dilutions with the final concentration of 200. 66.66, 22.22 and 7.406 ug/mL. The
resultant IC50 value was calculated by using three-fold serial dilutions through table curve
software. Percentage radical scavenging activity of samples was calculated by using following
formula:

% scavenging activity = (1– Absorbance of sample / Absorbance of negative control) *100

4.6.2. Total Antioxidant Capacity

Total antioxidant capacity based on phosphomolybdenum was estimated by mixing of 0.1mL

aliquot of each sample (4 mg/mL) with 1 mL solution of the reagent (0.6 M sulfuric acid, 4 mM
ammonium molybdate and 28 mM sodium phosphate). After that incubate the eppendorf at 90 oC
for 95 minutes in water bath and then it was slowly cooled down at room temperature, then with
the help of PDA spectrophotometer the absorbance was taken at 645 nm (8354 Agilent
Technologies, Germany). All experiments were performed in triplicate (Prieto et al. 1999).
Ascorbic acid was used as positive control and reagent without extract as negative control. The
antioxidant potential of each solvent extract was expressed as ascorbic acid equivalent per
microgram extract (µg AAE/mg extract).

4.6.3. Total Reducing Power

Reducing power reflects the capability to reduce the oxidized species formed during the lipid
peroxidation process. The reducing capacity of different solvent extracts was estimated as
described previously (Jafri, Saleem, Ullah and Mirza 2014) by using ascorbic acid as a reference
standard. Concisely 0.5 mL of phosphate buffer (pH 6.6) and 1 % potassium ferricyanide was
mixed with 0.2 mL of plant extract and after that it was put in incubation for 20 minutes at 50 oC.
Next, 0.5 mL of trichloroacetic acid was added and then the resultant mixture was centrifuged
for 10 minutes at 3000 rpm. Finally, the supernatant was mixed with ferric chloride (0.5 mL)
followed by distilled water (0.1 mL). Absorbance of reaction mixture, blank and standard was
recorded at 700 nm. The high absorbance capacity of reaction mixture indicates higher reduction
potential of the extracts. The results were represented as µg ascorbic acid equivalent per
milligram of extract (µg AAE/mg) after triplicate analysis.

4.7. Antimicrobial Activity

4.7.1. Antibacterial Assay

Antibacterial susceptibility of plant extract was investigated with the help of agar disc diffusion
method (Ayaz, Subhan, Ahmed, Khan, Ullah, Sadiq et al. 2015, Ayaz, Subhan, Ahmed, Khan,
Ullah, Ullah et al. 2015). Five test organism were used i.e. Gram positive strains: S. aureus
(ATCC 6538), M. luteus (ATCC 10240) and B. thuringiensis (ATCC 6633) and Gram negative
strains: K. pneumonia (ATCC 1705), and E. coli (ATCC 25922).An aliquot (10 ml) of sterile
nutrient broth was inoculated with sterile loopful of test organisms spores from stored slants (4
°C) and incubated at 37 oC for 24 hours. Afterwards, turbidity of each inoculum was tuned
accordingly by comparing the 0.5 McFarland turbidity BaSO4 standard to maintain 104 CFU/mL.
About 0.1 mL of every test strain was spread on nutrient agar plates. Sterile filter paper discs of
diameter 6 mm were infused with 5 μL (100 μg) of each test plant extract. Afterwards, discs
were placed on hitherto seeded plates. Disc impregnated with cefixime and DMSO were used as
a positive and negative control. It was then incubated for 24 hours at 37 oC and after that clear
zone of inhibition around the discs was recorded with vernier caliper to the nearest. The assay
was performed in triplicate.

4.7.2. Antifungal Assay

The antifungal activity of extracts was analyzed by agar disc diffusion method (Ayaz, Junaid et
al. 2016, Sadiq et al. 2016) with slight modification according to system feasibility. Five strains
were used as test organism i.e. F. solani (FCBP 0291), A. flavus (FCBP 0064), A. fumigatus
(FCBP 66), Mucor species (FCBP 0300) and A. niger (FCBP 0198). Initially fungal spores from
stock cultures of different fungal strains were refreshed by swabbing on sterile SDA plates and
were put in incubation for 5-7 days at 28 oC. Erstwhile to sensitivity estimation the spores of
fungal strains were harvested in 0.02 % Tween-20 solution and their turbidity of each inoculum
was tuned accordingly by comparing the 0.5 McFarland turbidity BaSO4 standard to maintain 104
CFU/mL. Aliquot of 0.12 mL of each fungal spores was dabbed on plates which contained sterile
SDA. Sterile filter paper discs of diameter 6 mm were infused with 5 μL (100 μg) of each test
plant extract Plates were incubated at 28 oC for 24-48 hour and after that clear zone of inhibition
around the discs was recorded. Clotrimazole and DMSO were used as a positive and negative
control.

4.7.3. Protein Kinase/ hyphae formation Inhibition Assay

This assay was performed by using Streptomyces 85E strain and by following previously
described protocol (Yao et al. 2011) with slight modification .The refreshed culture of
Streptomyces (0.1 mL) was spread to lawn minimal ISP4 plates. The Sterile filter paper discs of
diameter equal to 6 mm were laden with 5 µL (100 µg) of test sample and later on employed on
freshly seeded plates. Disc permeated with surfactin and DMSO infused disc served as positive
and negative control. The plates were incubated for 72 hours at 30 °C to allow the development
of hyphae to take place. After incubation, zone of growth inhibition was measured around each
disc with vernier caliper and recorded. The development of bald zone around the disc suggested
inhibition of phosphorylation (inhibition of spores and mycelia) by the samples. Development of
bald zone represents inhibition of hyphae formation while clear zone indicates cytotoxicity of
samples by killing Streptomyces.

4.8. Cytotoxicity assay

4.8.1. Brine shrimp lethality assay

Lethality test was performed following standard procedure using brine shrimp larvae (Ayaz, M.
Junaid et al. 2016). The following procedure allows the valuation and successively quantification
of proportion of surviving nauplii. The eggs of brine shrimps (Artemia salina) were incubated for
24-48 hrs. at 30 oC in an uneven, perforated, bi-compartment tray filled with artificial sea water.
After incubation, the resulting nauplii were harvested with the help of pipette from the
illuminated compartment. Test sample were tested for lethality estimation at concentrations of
1000, 500, 250 and 125 µg/mL. About 20 nauplii were transferred to each well containing sea
water supplement with dried yeast (6mg/L). The corresponding micro liter of each concentration
was added to each well and final volume in each well was made up to 300 µL with sea water. In
this way, final concentration of DMSO did not exceed 0.5 %. Doxorubicin and DMSO were used
as positive and negative control respectively. Then the uncovered plates were incubated at 30 °C
for 24 hrs in lumified incubator. After incubation, the plates were observed with magnifying
glass and deceased nauplii were count up in each well. LC50 was calculated accordingly for the
extracts with ≥ 50% mortality at highest concentration using table curve software 2D.ver.4.

4.8.2. Anticancer Activity

The cytotoxic potential of extracts towards Hep G2 cancer cell line (RBRC-RCB1648) was
determined with the help of SRB colorimetric assay (Vichai and Kirtikara 2006). An aliquot of
180 µL from culture was then transferred to each well of 96 well polystyrene plated having 20
µL of test samples (consuming 1 % DMSO in PBS) to obtain final concentration i.e. 20 µg/mL.
Doxorubicin (20 - 0.08 µg/mL) and 1 % v/v DMSO in PBS instead of test sample were used as
positive and negative controls correspondingly. The culture plate was then placed in incubation
for 72 hours at 37 °C in a CO2 incubator. The incubation was stopped with the addition of 50 µL
of cold 20% w/v TCA for 1 hour at 4 °C for cell fixation. The fixed cells were then air dried after
washing them four times with tap water and then they were stained with 50 µL of 0.057 % w/v
SRB in 1 % w/v acetic acid at room temperature for 30 minutes. Wells were then placed to dry
overnight after they were washed 4 times with 1% v/v acetic acid. Bound dye was solubilised in
200 µL 10 mM Tris base, pH 10, for 1 hour. In order to determine the percentage of survival
micro plate reader was used to measure the optical density (Biotech USA, microplate reader Elx
800) at 515 nm. In each case, a zero-day control was carried out by adding same number of cells
to sixteen wells, incubating at 37 °C for 1 hour and following the above described procedure.
Percentage of cell growth inhibition was determined using the formula:

% inhibition = 100 – [(ODcells + samples - ODday 0) / (ODcells+1% DMSO - ODday0) x 100]

4.9. Antidiabetic Activity

The antidiabetic potential of plants was determined by amylase inhibition assay following the
slightly modified procedure described by (Williamson et al. 1992). The assay was executed via
96 well polystyrene platted microplate and in each well, 15 µL of phosphate buffer, 25 µL of α
amylase enzyme, 10 µL sample and 40 µl starch was added in successive steps. Incubation was
conceded out at 50 °C for 30 minutes after it 20 µL of 1 M HCl and 90 µL of iodine solution was
added. Blank, positive and negative controls were run in separate wells. Blank solution consists
of buffer, starch and DMSO, while acarbose and DMSO were used as positive and negative
control. Results was examined by using microplate reader and reading was taken at 540 nm. The
result was expressed as antidiabetic potential in term of % enzyme inhibition per mg of plant
extract. Percentage enzyme inhibition was measured by using this formula;

4.10. Antileishmanial Assay

The extracts were screened to assess antileishmanial potential via MTT colorimetric assay using
L. tropica kwh 23 strain culture supplemented with bovine fetal serum as described previously
(Shah et al. 2015). Briefly, 20 µL of test extract having not more than 1% DMSO in PBS was
added to each well at final concentration of 100 µg/mL. Then an aliquot of 180 µL of culture
(seeding density 1×106 promastigotes per ml) was passed on to each 96 well plate followed by
incubation at 24 ˚C for 72 hours. After incubation, 20 µL of pre-filter sterilized MTT solution (4
mg per ml in distilled water) was added to each well. After incubation period (24 ˚C for 4 hours),
supernatant was removed without unsettling colored formazan sediments. DMSO 100 µl was
used to dissolve the sediments and then plate was kept inside for 1 hour to ensure complete
dissolution. The 1 % DMSO in PBS served and amphotericin B at final concentration of 0.33-
0.004 µg/mL used as negative and positive control. The absorbance was recorded at 540 nm by
microplate reader.

4.11. Statistical analysis

The data is analysed by comparing means of three replicates of plant extracts using Complete
Randomized Design (CRD) with Statistix 8.1 software, expressed as mean ± SD (standard
deviation). Correlation analysis was carried out by using the correlation and regression line in
Microsoft Excel Program. LC50 were calculated by table curve 2D ver. 4 software. Graphs are
plotted by Origin 8.5. Software.
 Table S1. % Extract recovery of Dysphania ambrosioides extracts in different extraction
solvents.

Plant Percentage of extract yield

Samples
Leaves Stem Root
Nh 1 ± 1.06 0.7 ± 0.59 0.8± 0.32
C 2.8 ± 1.9 1 ± 1.7 1.2 ± 0.64
Eth 3.6 ± 1.67 1.2 ± 1.34 1.5 ± 0.84
A 1.9 ± 1.03 1.4 ± 1.75 1.3 ± 0.16
E 4.7 ± 0.56 3.4 ± 1.23 1.6 ± 1.03
M 4.9 ± 0.43 4.5 ± 1.01 4.3 ± 1.06
D 10.3 ± 0.52 6.6 ± 0.98 7 ± 1.02
EthNh 1.97 ± 1.03 1.3 ± 1.05 1 ± 1.08
ENh 2.3 ± 0.97 2 ± 0.67 1.1 ± 1.06
EthNh 5.5 ± 1.03 2.7 ± 0.42 3.2 ± 0.87
MEth 3.8± 0.48 3.9± 1.06 1.3 ± 0.97
MA 1.7± 1.05 4.7 ± 1.2 1.5± 1.02
AD 10.8 ± 1.01 7.3 ± 1.08 5.9 ± 1.07
MD 14.1 ± 1.07 9.5 ± 0.3 9 ± 0.34

Values (mean ± standard deviation) was obtained through from triplicate analysis. Nh: n-hexane,
C: Chloroform, Eth: Ethyl acetate, A: Acetone, E: Ethanol, M: Methanol, D: Distilled water,
EthNh: n-hexane: ethyl acetate, ENh: ethanol: n- hexane, MC: methanol: chloroform MEth:
Methanol-Ethyl acetate, MA: methanol: acetone, AD: acetone: water, MD: methanol: water.

Table S2: HPLC-DAD analysis of MEth extracts.

Standard λ (nm) Polyphenols (µg/mg extract)

leaf stem root
Gallic acid 257 - - -
Rutin 257 2.5± 0.49 - -
Caffeic acid 325 - - -
Catechin 279 - - -
Apigenin 325 - - -
Myrecetin 368 2.99± 0.58 0.3± 0.8 0.46± 0.67
Quercetin 368 1.175± 0.3 - 0.43± 1.01
Kaempferol 368 - - -
- = not detected, MEth = methanol ethyl acetate, value was represented as (mean± SD n=3)
Table S3: Brine shrimp lethality, HepG2 cytotoxicity, Streptomyces hyphae formation inhibition and
antileishmanial capability of plant extracts against A. tropica,

Extra Brine shrimp Hep G2 Protein kinase Antileishmanial

ct Cytotoxicity inhibition Capability
Mortality% LC50 Inhibition IC50 DIZ Bald zone Mortality % LC50
(µg/ml) % (µg/ml (mm) µg/ml
) Clear
zone
Leaves
Nh 97.00±0.57n 187.00±1.1 -- -- -- 10±0.61* 41.2±0.45** > 1000
s
5 ** *
C 57.83±0.44* 865.00±0.5 12±0.4*** > -- -- 10.4±0.89** > 1000
* 7 1000 *
Eth 65.00±0.00* 500.16±1.3 52 ± 1.53* > -- 14 ± -- --
* 0 1000 0.96**
A 27.33±0.33* >1000 34±1.04** > -- 12±0.81* 20.7±0.79** > 1000
** * 1000 * *
E 60.00±1.50* 511.00±0.5 33±0.3*** > -- -- 30.16±0.85* > 1000
* 7 1000 **
M 50.66±1.20* 1001.33±1. 56±2.5* > -- 11±0.76* -- --
** 85 1000 *
D 25.00±2.51* >1000 22±0.65** > -- -- -- --
** * 1000
EthN 44.81±2.46* >1000 17±1.2*** > -- 14±1.04* -- --
h ** 1000 *
ENh 50.00±0.21* 1000.00±0. -- -- -- 13±0.93* -- --
** 57 *
MC 66.33±0.33* 539.66±1.2 -- -- -- 19±1.53 ns -- --
* 0
MEt 37.00±1.15* >1000 -- -- -- 14±0.98* -- --
h ** *
MA 48.00±0.57* >1000 12±1.5*** > -- 8±0.41** -- --
** 1000 *
AD 27.33±1.45* >1000 30±0.9*** > -- -- -- --
** 1000
MD 24.66±1.45* >1000 24 ± > -- -- 35.9±1.3*** > 1000
** 0.69*** 1000
Stem
Nh -- -- -- -- -- 8 ± 27.6±1.02** > 1000
0.61*** *
C -- -- 8±1.54*** > -- -- -- --
1000
Eth -- -- -- -- 9 ± -- --
0.96***
A -- -- 5±2.5*** > -- 8 ± 80.25±0.93 11.00±0.
ns
1000 0.81*** 28
E -- -- 4.1±1.07** > -- -- 40.78±1.04* > 1000
* 1000 **
M -- -- -- -- -- 10±0.76* -- --
**
D -- -- -- -- -- -- 50.13±0.76* > 1000
**
EthN -- -- -- -- -- 9 ± 69.81±0.59* 18.9±0.5
h 1.04*** 7
ENh -- -- -- -- -- 12 ± 34.5±0.63** > 1000
0.93** *
MC -- -- -- -- -- 15 ± -- --
1.53**
MEth -- -- -- -- -- 13 ± 50.07±0.58* > 1000
0.98** **
MA -- -- -- -- -- 8 ± 43.6±0.36** > 1000
0.41*** *
AD -- -- -- -- -- -- -- --
MD -- -- -- -- -- -- -- --
Root
Nh 84.66±3.17n
249.66±0. 7.1±1.53* > 1000 -- -- -- --
s
88 **
C 63.83±1.69* 446.16±0. 4.7±0.92* > 1000 -- 7 ± -- --
* 72 ** 0.34***
Eth 67** 352.00±1. 4.2±0.79* > 1000 -- 14 ± -- --
15 ** 0.48**
A 50*** 1000.00±0 2.8±0.46* > 1000 -- 9 ± -- --
.57 ** 0.27***
E 50*** 1000.33±0 -- -- -- 19 ± 72.6±0.78* 13.6±0.6
ns
.88 1.21 9
M 85ns 224.16±0. -- -- -- 17 ± 1.07 50.03±1.04* > 1000
44 **
D 92ns 215.33±0. -- -- -- -- 31.8±1.06** > 1000
33 *
EthN 70* 200.08±2. 15±1.9*** > 1000 -- 9 ± 0.38 92.51±0.94n 6.8±0.05
s
h 81
ENh 77* 260.00±1. -- -- -- 12 ± 0.69 44.12±0.83* > 1000
73 **
ns
MC 95 206.00±0. -- -- -- 12 ± 0.84 -- --
00
ns
MEth 85 240.00±0. -- -- -- 9 ± 0.47 -- --
57
ns
MA 85 213.00±0. -- -- -- 11 ± 1.21 -- --
00
ns
AD 80 141.50±0. 12±1.53** > 1000 -- -- -- --
 28 *
ns
MD 85 201.08±0. -- -- -- -- 29.9±0.94** > 1000
50 *
P.C 100 5.10±0.00 98.56±0.3 8.1±0. 21.00±0. 27.00±0.0 97.50±0.33 11.55±1.
3 00 00 0 05

Values (mean ± standard deviation) was obtained through from triplicate analysis. Experiments were
conducted at 20-1.25 µg/ml). IC50 values for results of very less clinical significance were expressed
>1000. -- = Not active, firstly the extracts were screened at their higher concentration then extracts
which exhibited more than 50% inhibition were analyzed at their lower concentration for estimation
of their LC50. Negative control: DMSO, P.C: Positive control/ Standard drugs, LC50 of vincristine
and 5- florouraciL (positive control used in anticancer assay) was 5 and 8.1 μg/ml, Doxorubicin
(positive control used in brine shrimp assay) 5.1 μg/ml. Surfactin (20 μg/disc: 27 mm bald zone) was
employed as a positive control in protein kinase inhibition assay. Values (% inhibition) significantly
different i.e. *P < 0.05, **P < 0.01, ***P < 0.001 compared to standard drugs.
Table S4: Antibacterial activity of Dysphania ambrosioides extracts against pathogenic bacteria

Extract Diameter of growth zone inhibition (mm ± SD at 100 µg; MIC: µg/ml)
s
Codes
Gram negative bacteria Gram positive bacteria
K. MIC M. luteus MIC E.coli MIC B. subtilis MIC S. aureus MIC
pneumon µg/ µg/ml µg/ µg/
iae ml ml ml
Leaves
Nh 8 ± 0.61 > 100 7±0.43*** > 6±0.29***> 100 12.4±1.2** 100 7±0.38** > 100
ns
100 * *
C 9 ± 0.69 > 100 --- --- 7±0.45***> 100 11.8±1.03*** 100 --- ---
ns

Eth 12 ± 1.26 100 9±0.76 > 7±0.67***> 100 9.2±0.9*** > 8± > 100
ns
*** 100 100 0.43***
A 8 ± 0.53 > 100 7±0.51 > 7±1.3*** > 100 -- -- 8±0.39** > 100
ns
*** 100 *
E 10 ± 0.87 > 100 6±0.29*** > 7±1.09***> 100 11.2±0.29*** 100 7±0.26** > 100
ns
100 *
M 10 ± 0.79 > 100 10±0.96 > 7.2±0.38**> 100 11.6±1.01*** 100 8±0.49** > 100
ns
*** 100 * *
D 7 ± 0.43* > 100 9±0.64 > -- -- -- -- 6±0.21** > 100
*** 100 *
EthN 9 ± 0.61 > 100 11±1.06 > -- -- 10.2±0.87*** > 8±0.25** > 100
ns
h *** 100 100 *
ENh 10 ± 0.92 > 100 8±0.43 > -- -- -- -- 7±0.28** > 100
ns
*** 100 *
MC 7 ± 0.48* > 100 8±0.33 > -- -- 7.1±0.67*** > 7±0.41** > 100
 *** 100 100 *
MEt 6 ± 0.26* > 100 7±0.38 > 8±0.71***> 100 10.7 ± 0.85 100 7±0.19** > 100
h *** 100 *
MA 10±0. 76 > 100 8±0.53 > -- -- -- -- 8±0.46** > 100
ns
*** 100 *
AD 9 ± 0.59 > 100 8 ± 0.61 > -- -- 9.2±0.45** > 7±0.35** > 100
ns
*** 100 * 100 *
MD 8 ± 0.49 > 100 10±0.82 > 7±0.57***> 100 -- -- 7±0.51** > 100
ns
*** 100 *
Stem
Nh 7 ± 1.2* > 100 --- --- 7±0.9*** > 100 -- -- --- ---
C 7 ± 0.9* > 100 --- --- 8.7±0.65** > 100 -- -- --- ---
*
Eth 7 ± 0.83* > 100 --- --- 6±4.9*** > 100 12.4±1.03** 100 --- ---
*
A 12±0.82 33.33 --- --- 6.7±1.03** > 100 21±1.04*** 11.1 --- ---
ns
*
E 9 ± 0.96 > 100 --- --- 7.1±0.56** > 100 10±1.07*** 100 --- ---
ns
*
M 10 ± 0.94 > 100 --- --- 7.3±0.84** > 100 11.7±0.8*** 100 --- ---
ns
*
D 7 ± 1.2* > 100 --- --- -- -- -- -- --- ---
EthNh 13 ± 1.9 11.11 --- --- -- -- 12±0.47*** 100 --- ---
ns

ENh 11± 1.6 ns > 100 --- --- -- -- -- -- --- ---

MC 10± 0.98 > 100 --- --- -- -- -- -- --- ---
ns

MEt 7± 0.83* > 100 --- --- 7.5±0.78** > 100 10.4±0.73** 100 --- ---
h * *
MA --- --- --- --- -- -- 12±0.63*** 100 --- ---
AD --- --- --- --- -- -- 9±1.01*** > 100 --- ---
MD --- --- --- --- -- -- 8.5±0.83*** > 100 --- ---
Root
Nh 7 ± 0.41*> 100 9±0.71 > -- -- -- -- -- --
*** 100
C 9 ± 0.98 > 100 -- -- -- -- 14±0.6** 33.3 7±0.39** > 100
ns
*
Eth 8 ± 0.57 > 100 -- -- -- -- 7.3±0.45** > 100 -- --
ns
*
A 9 ± 0.71 > 100 -- -- -- -- 12.4±0.65** 100 -- --
ns
*
E 10 ± 0.75 > 100 -- -- -- -- 13±0.93*** 100 -- --
ns

M 9 ± 0.67 > 100 -- -- -- -- 7.1±1.04*** > 100 -- --

ns

D 9 ± 0.51 > 100 -- -- -- -- -- -- 8±0.48** > 100

 ns
*
EthNh 10 ± 0.89 > 100 9±0.57 > -- -- 7.9±0.5*** > 100 8±0.64** > 100
ns
*** 100 *
ENh 10 ± 0.79 > 100 11±1.05 *** >100 -- -- 12.8±1.02** 100 9±0.81** > 100
ns
*
MC 10 ± 0.67 > 100 9±0.77 > -- -- 14.3±0.43** 33.3 -- --
ns
*** 100 *
MEt 9 ± 0.47 > 100 8±0.43 > 8.5±0.6* > 100 11±0.67*** 100 7±0.56** > 100
ns
h *** 100 ** *
MA 8 ± 0.35 > 100 11±0.98 100 7.2±0.9* > 100 10±1.06*** > 100 7±0.54** > 100
ns
*** ** *
AD 9 ± 0.49 > 100 9±0.73 > 7.4±1.02* > 100 7.1±1.2*** > 100 9±0.91** > 100
ns
*** 100 **
MD 9 ± 0.31 > 100 -- -- 7.5±0.49* > 100 7.3±1.05*** > 100 9±0.87** > 100
ns
**

-- = No activity, Values are represented as mean ± standard error of three separate experiments. Growth inhibition
zone of standard drug cefixime was 14 ± 0.7 against S .aureus, 23 ± 1.04 against M. luteus, 12 ± 1.12 mm against
K. pneumonia, 28 ± 1.09 against E.coli and 21 ± 0.9 against B. subtilis; Negative control: DMSO. Zone > 10mm
are further took for MIC determination.

Table S5: Antifungal activity of Dysphania ambrosioides against filamentous fungi.

Extrac Diameter of growth zone inhibition (mm ± SD at 100 µg; MIC: µg/ml)
t
codes
F. MI A. MI Mucor MI A. MI A. MI
solani C fumigatus C spp. C flavus C niger C
Leaf
Nh -- -- 7 ± 0.37 100 9 ± 0.53 100 -- -- -- --
C 10 ± 100 -- -- 10± 1.87 100 7 ± 0.37 100 -- --
0.81
Eth 12 ± 100 8 ± 0.43 100 10 ± 0.93 100 -- -- -- --
0.89
A -- -- 7 ± 0.97 100 10 ± 0.89 100 -- -- -- --
E 8±0.47 100 10 ± 0.71 100 12 ± 0.94 50 -- -- 9 ± 100
0.67
M -- -- -- -- 9 ± 0.59 100 -- -- --- --
D 7± 100 8 ± 0.57 100 -- -- -- -- 9 ± 100
0.31 0.57
EthN -- -- -- -- 8 ± 0.46 100 -- -- -- --
h
ENh -- -- -- -- 9 ± 0.63 100 -- -- -- --
MC -- -- 8 ± 0.65 100 9 ± 0.71 100 -- -- -- --
MEth -- -- -- -- 13 ± 1.12 100 -- -- -- --
MA -- -- 8 ± 0.43 100 9 ± 0.73 100 -- -- -- --
AD 8 ± 100 7 ± 0.51 100 -- -- 7 ± 0.37 100 -- --
0.49
MD 8 ± 100 8 ± 0.69 100 -- -- -- -- -- --
0.37
Stem
Nh -- -- -- -- -- -- -- -- -- --
C -- 8 ± 0.49 100 -- -- -- -- -- --
Eth -- 7 ± 0.83 100 -- -- -- -- -- --
A -- -- -- -- -- -- -- -- --
E -- -- -- -- -- -- -- 9 ± 100
0.85
M -- -- -- -- -- -- -- -- --
D -- -- -- -- -- -- -- -- --
EthN 7 ± 100 -- -- 7 ± 0.37 100 -- -- -- --
h 0.76
Root
Nh 9 ± 100 -- -- -- -- -- -- -- --
0.71
C -- -- 9 ± 0.49 100 -- -- -- --
Eth -- -- 9 ± 0.83 100 -- -- -- --
A -- -- 10 ± 0.89 100 -- -- -- --
-- = No activity, Values are represented as mean ± standard error of three separate experiments.
Figure S1 (a,b,c): Total phenolic content and flavonoid content determination of leaf, stem and
root extracts of Dysphania ambrosioides. Values (mean ± standard deviation) was obtained
through from triplicate analysis. The Columns with similar alphabets are not significantly
different at P<0.05. Nh: n-hexane, C: Chloroform, Eth: Ethyl acetate, A: Acetone, E: Ethanol,
M: Methanol, D: Distilled water, EthNh: n-hexane: ethyl acetate, ENh: ethanol: n- hexane,
MC: methanol: chloroform MEth: Methanol-Ethyl acetate, MA: methanol: acetone, AD:
acetone: water, MD: methanol: water.
Figure S2 (a,b,c,d): HPLC- DAD profiling of Dysphania ambrosioides methanol: ethyl acetate
extract (MEth) at different wavelength (sig=368nm, sig=257nm, sig=325nm, sig=279nm).
Conditions: Acetonitrile-methanol-water-acetic acid in 5:10:85:1 ratio (Mobile phase A),
acetonitrile-methanol-acetic acid in 40:60:1 ratio (Mobile phase B). Flow rate 1ml/min, injection
volume 20µl. a) standards b) chromatogram of detected leaf polyphenols c) stem d) root
Figure S3 (a,b,c): TAC (Total antioxidant capacity, µg AAE/mg plant extract), TRP (Total
reducing power, µg AAE/mg plant extract) and FRSA (free radical scavenging activity)
determination in leaf, stem and root extract of Dysphania ambrosioides. Values (mean ±
standard deviation) was obtained through from triplicate analysis. The Columns with similar
alphabets are not significantly different at P<0.05. Nh: n-hexane, C: Chloroform, Eth: Ethyl
acetate, A: Acetone, E: Ethanol, M: Methanol, D: Distilled water, EthNh: n-hexane: ethyl
acetate, ENh: ethanol: n- hexane, MC: methanol: chloroform MEth: Methanol-Ethyl acetate,
MA: methanol: acetone, AD: acetone: water, MD: methanol: water.
Figure S4: Percentage of alpha Amylase enzyme inhibition activity determination in Dysphania
ambrosioides extracts. Values (mean ± standard deviation) was obtained through from triplicate
analysis. The Columns with similar alphabets are not significantly different at P<0.05.
Figure S5: Protein kinase inhibition potential of leaf extracts of Dysphania ambrosioides

Figure 6: Antibacterial activity of leaf extracts against M. luteus

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