Supplementary file
MATERIALS AND METHODS
Preparation of methanol extracts
The biomass from in-vitro propagated plants (8 week old culture, BM-TM medium
supplemented TDZ (6.8 μM) and NAA (1.3 μM) shoots were collected) and wild grown plants
(whole plants) were washed under tap water and dried in oven at 60ºC for two days. The material
was powdered by using electric blender and stored in clean labelled airtight bottles. The powder
(100 g) was extracted by maceration in 300 ml of methanol (100 %) for 3 days with frequent
agitation. The mixture was filtered through Whatman No. 1 filter paper and the filtrate was
concentrated and dried in petri dishes at 60ºC in the oven.
Estimation of total phenolics (TPC) and flavonoids content (TFC)
The total phenolics content of the extracts was determined and calculated as gallic acid
equivalent (GAE) in mg/g DW from the calibration curve according to method described by
Siddhuraju & Becker (2003). The total flavonoids content of sample extract was determined
following a colorimetric method and values were expressed as mg/g rutin equivalent (RE) of
extract according to the method described by Zhishen et al. (1999).
In-vitro antioxidant activity
DPPH radical scavenging activity
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Radical scavenging activity of in-vitro propagated plants and wild grown plants of A.
montana was measured by DPPH radical scavenging method, described by Blois (1958). Plant
extracts at various concentrations were taken and the volume was adjusted to 100 µl with
ethanol. Five ml of 0.1 mmol/l ethanolic solution of DPPH was added and shaken vigorously.
The tubes were allowed to stand for 20 min at 27°C. The absorbance of the sample was measured
at 517 nm. IC50 values of the extract, i.e., the concentration of extract necessary to decrease the
initial concentration of DPPH by 50 were calculated. A lower IC50 value indicates higher
activity.
ABTS cation radical scavenging activity
ABTS cation radical decolorization assay was measured according to the method
described by Re et al (1999). ABTS•+ was produced by reacting 7 mmol/l ABTS aqueous
solution with 2.4 mmol/l potassium persulfate in the dark for 12–16 h at room temperature. Prior
to the assay, this solution was diluted in ethanol (about 1:89, V/V) and equilibrated at 30◦C to
give an absorbance at 734 nm of 0.700 ± 0.02. After the addition of 1 ml diluted ABTS solution
to 10 µl sample or trolox standards (final concentration 0–15 µmol/l) in ethanol, absorbance was
measured at 30°C exactly 30 min after the initial mixing. Solvent blanks were also run in each
assay. The total antioxidant activity (TAA) was expressed as the concentration of trolox having
equivalent antioxidant activity in terms of µmol/l/g sample extract.
Ferric reducing antioxidant power (FRAP) assay
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The ferric reducing antioxidant capacity of in-vitro propagated plants and wild grown
plants of A. montana was estimated by the method described by Pulido et al (2000). FRAP
reagent (900 µl), pre-pared freshly and incubated at 37°C, was mixed with 90 µl of distilled
water and 50 µl test sample (1 mg/ml). The test samples and the reagent blank were incubated at
37°C for 30 min in a water bath. At the end of incubation, the absorbance was taken immediately
at 5 93 nm using a spectrophotometer. Methanolic solutions with known Fe (II) concentration,
ranging from 100 to 2000 µmol/l (as FeSO4·7H2O) were used for the preparation of the
calibration curve. The FRAP values were expressed as mmol/l Fe (II) equivalent/mg extract.
Antimicrobial activity
Test bacteria
The antibacterial activity of in-vitro propagated plants and wild grown plants of A.
montana was evaluated against six pathogenic bacteria such as Escherichia coli (ATCC 25922),
Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 29213), Streptococcus
pnumoniae (ATCC 33400) Klebsiella pnumoniae (ATCC 10031) and Bacillus subtilis (ATCC
6633) procured from the Institute of Microbial Technology (IMTECH) Chandigarh, India. All
the strains were stored in the appropriate medium before use.
Disc diffusion method
Disc diffusion method (Joshi et al. 2010) was used for the evaluation of antibacterial
activity of A. montana methanol extracts of wild-grown plants and in-vitro propagated plants
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using 100 μl of suspension containing 108CFU/ml of bacteria spread on the inoculated agar. A
sterile cotton swab was dipped into the inoculums suspension to remove the excess of fluid.
Whatmann filter paper discs (6 mm diameter) were prepared at the concentration of 25 μg/disc
for wild-grown plants and in-vitro propagated plants extracts and 10 μg/disc reference antibiotic
(Ciprofloxacin). A disc prepared with only the corresponding volume of DMSO was used as
negative control. The petri plates were then incubated and antimicrobial activity was evaluated
by measuring the diameter of the zones of inhibition around the disc. The experiments were
repeated in triplicate and the result was expressed as average value.
Minimum inhibitory concentration
The minimum inhibitory concentration (MIC) of wild-grown plants and in-vitro
propagated plants of A. montana was determined using the micro-dilution assay in 96-well
micro-plates (Siddiqi et al. 2011). Briefly, 500 ml of each re-suspended sample (1.0 mg/ml) in
DMSO (2 %). Serial two-fold dilutions were prepared from the stock solution to give
concentrations ranging from 500 μg to 3.90 μg/ml of the wild-grown plants and in-vitro
propagated plants extracts. The highest concentration of DMSO remaining after dilution (5 %,
v/v) caused no inhibition to bacterial growth. DMSO served as negative control. Streptomycin
and Ciprofloxacin were served as positive controls. An aliquot of 100 μl standardized suspension
of the test bacteria (108 CFU/ml) was transferred to a well of 96 plates. Then, 100 μl diluted
samples were also added to each well and incubated at 37°C for 24h. The MIC was defined as
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the lowest concentration of samples which inhibited the visible growth of tested microorganisms.
For further reconfirmation, 20 μl of MTT reagent (1 mg/ml) was added as an indicator for
microbial growth to each well of the microtitre plates, followed by 20 min incubation at 37°C.
The reagent-suspension colour will remain clear or yellowish indicating complete inhibition
activity as opposed to dark blue for growth (Eloff 1998). The MIC was recorded as the most
repeatable minimum concentration of triplicate.
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