Supplementary file
MATERIALS AND METHODS
Plant material and extract preparation
Plant material was collected from different growth phases of non-embryonic callus
(NEC), pre-embryoid masses (PEM), somatic embryos at globular stage (SEG), somatic embryos
at heart-shaped stage (SEHS) and cotyledonary embryos (SEC) during somatic embryogenesis.
For each growth stage, five replicates were collected (each 50 g). All samples were washed
under running tap water and dried in oven at 60 °C for 2 days. The material was powdered using
electric blender and stored in clean labeled airtight bottles. The powder (25 g) was extracted by
maceration in 100 ml of methanol (100 %) for 2 days with frequent agitation. The mixture was
filtered through Whatman No. 1 filter paper and the filtrate was concentrated and dried in
Petridishes at 60°C in the oven.
Determination of total phenolic content (TPC)
Total phenolic contents were measured using the method described by Siddhuraju and
Becker 2003. The calibration curve consisted of prepared Gallic acid solutions at concentrations
of 1–15µg. (Y =0.0232, X +0.122; R2 = 0.9989). One hundred microliters of each stage embryos
extract was taken into test tubes and made up to 1 ml with distilled water. Then 0.5 ml Folinciocalteu phenol reagent (1:1 with water) and 2.5 ml sodium carbonate solution (20 %) were
added sequentially in each tube. After that the test tubes were placed in dark for 40 min and the
absorbance was recorded at 725 nm against the reagent blank.
Determination of total flavonoid content (TFC)
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The flavonoid content was determined according to the method described by Zhishen et
al (1999). 0.5 ml of extract aliquot (1 mg/ml) was mixed with 2 ml distilled water and
subsequently with 0.15 ml of 5 % NaNO2 solution. After 6 min, 0.15 ml of 10% AlCl3 was added
and the mixture was allowed to stand for 6 min, and then 2 ml of 4 % NaOH solution was added
to the mixture. Distilled water was immediately added to bring the final volume to 5 ml and then
the mixture was thoroughly mixed and allowed to stand for another 15 min. Absorbance of the
mixture was determined at 510 nm against the water blank. Total flavonoids were measured from
rutin (20–100µg; Y =0.0088, X -0.0477. R2 = 9979) standard curve and expressed as mg rutin
equivalents/g extract.
DPPH free radical scavenging assay
The radical scavenging activity of plant extracts against 1,1-diphenyl-2-picrylhydrazyl
free radical (DPPH●) was determined spectrophotometrically as described by Blois (1958).
Different stages of somatic embryos at various concentrations were taken and the volume was
adjusted to 100µl with ethanol. 5 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%, was calculated. A lower IC 50
value indicates higher activity.
ABTS 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
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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 10µl 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.
FRAP (ferric reducing antioxidant power) assay
The ferric reducing antioxidant capacity of different stage embryos extracts of S.
corymbosa was estimated by the method described by Pulido et al (2000). FRAP reagent (900
µl), prepared freshly and incubated at 37°C was mixed with 90 µl of distilled water and 50µl 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 593 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.
HPLC analysis of the extracts
Swertianin (1), methylswertianin (2) and 1, 2, dihydroxy-6-methoxyxanthone-8-O-β-Dxylopyranosyl (3) content from of non-embryonic callus (NEC), pre-embryoid masses (PEM),
somatic embryos at globular stage (SEG) and somatic embryos at heart-shaped stage (SEHS),
cotyledonary embryos (SEC) were estimation using HPLC was performed as reported by
Mahendran and Narmatha Bai (2014). Briefly, separated using a Waters Acquity HPLC system
(Waters Corporation, Milford, MA) consisting of waters L-7100 HPLC pump, an autosampler,
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an Acquity Tunable absorbance detector and temperature containing module containing (R) C18
column (4.6 mm × 150 mm; 4 μm particle size). Plant extracts were filtered through a 0.2μm
filter and 10 μl of extracts were injected in a HPLC system. The solvents optimized for gradient
elution consisted of (A) acetonitrile and (B) 0.1% acetic acid in Milli Q water. A linear gradient
elution program was applied as follows: 0-4 min : 10% A, 90% B; 4-12 min: 10% A, 90% B; 1216 min: 30%, 70% B; 16-20 min: 40% A, 60% B; 20-24 min: 70% A, 30% B; 24-25 min: 70%
A, 30% B; 25-50 min: 10% A, 90%. The flow rate was maintained at 1.0 ml min-1. Injection
volumes were 10 μl for standards as well as for samples. The detector was set at 254 nm and
instrument operations, data acquisition and processing were performed using EmPower2
chromatographic data software (Waters Corporation, Milford, MA). The compounds peaks from
samples were identified by the comparison of retention times with the corresponding retention
times of standards. Quantification was performed using HPLC and the amounts of secondary
metabolites were calculated using standard curves. All experiments were repeated at least three
times. The results are presented as μg mL-1 of extracts.
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