SUPPLEMENTARY MATERIAL
Chemical composition, antioxidant activities and protein profiling of different parts of
Allamanda cathartica
Amjad Hameed1* Ghazala Nawaz2, and Tahsin Gulzar2
1
Nuclear Institute for Agriculture and Biology (NIAB), P.O. Box 128, Faisalabad, Pakistan.
2
Department of Applied Chemistry, Govt. College (GC) University, Faisalabad, Pakistan.
*email: [email protected]
1
Abstract
The phytochemical screening and protein profiling of Allamanda cathartica
was performed. Biochemical analysis revealed that peroxidase (8730±307
units/g), superoxide dismutase (181±3.79 units/g), catalase (529±28.9 units/g),
protease (3598±79.8 units/g), total phenolic contents (19344±657 µM/g), βesterases (342±46.5 units/g), and the total oxidant status were highest in the
roots as compare to other plant parts. Whereas, total soluble proteins (128±1.54
mg/g), lycopene (5.70±0.61 mg/g), chlorophyll a (161±24.9 µg/g), total
chlorophyll content (267±34.3 µg/g) and total carotenoid content (12.4±1.71
mg/g) were found to be highest in leaves. Moreover, total antioxidant capacity
(5.43±0.29 µM/g) by ABTS method and α-esterase (714.580±23.6 units/g) were
highest in shoots. The protein profiling was performed using SDS-PAGE. In
leaves, 13 peptides with molecular weight (M. wt.) from 27-168 kDa were
detected while in shoot 10 peptides with M. wt. from 30-95 kDa were resolved.
Similarly, in roots, 10 peptides of 30-880 kDa and in flower 7 peptides of 30-88
kDa were detected.
Keywords: SDS-PAGE; flower; stem, roots; leaves; phenolics; pigments
Materials and Methods
Different plant parts i.e.
stem, leaves, roots and flowers of Allamanda cathartica L.
(Voucher number: BISH0000129961) were collected from local nurseries (at Faisalabad,
Pakistan) and plants were identified by authenticated expertises. For different chemical
compositional analysis, plant parts were extracted in different solvents.
Evaluation of antioxidant enzyme activities
Extraction:
For estimation of enzymatic activities plant parts (0.5 g) were ground in cold extraction
buffer specific for different enzymes. Samples were centrifuged at 15,000×g for 10 min at 4
o
C. The supernatant was separated and used for the determination of different enzyme
activities.
Superoxide dismutase (SOD) activity
2
For the estimation of SOD activity, selected parts were homogenized in a medium
composed of 50 mM potassium phosphate buffer (pH 7.0), 0.1mM EDTA and 1mM
dithiothreitol (DTT) as described by Dixit et al. (2001). The activity of SOD was assayed by
measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT)
following the method of Giannopolitis and Ries (1977). One unit of SOD activity was
defined as the amount of enzyme which caused 50% inhibition of photochemical reduction of
NBT.
Peroxidase (POD) activity
For the estimation of POD, selected parts were homogenized in a medium composed
of 50 mM potassium phosphate buffer (pH 7.0), 0.1mM EDTA and 1mM DTT. Activity of
POD was measured using the method of Chance and Maehly (1955) with some modification.
For measurement of POD activity, assay solution (3 mL) contained 50 mM phosphate buffer
(pH 7.0), 200 mM guaiacol, 400 mM H2O2 and 0.1 mL enzyme extract. The reaction was
initiated by adding the enzyme extract. Increase in absorbance of the reaction solution at 470
nm was recorded after every 20 s. One unit POD activity was defined as an absorbance
change of 0.01 units min-1. Enzyme activity was expressed on F. wt. basis.
Catalase (CAT) activity
For the estimation of CAT, selected parts were homogenized in a medium composed
of 50 mM potassium phosphate buffer (pH 7.0) and 1mM dithiothreitol (DTT). CAT was
estimated by the following method described by Beers and Sizer (1952). For measurement of
CAT activity, assay solution (3 mL) contained 50 mM phosphate buffer (pH 7.0), 5.9 mM
H2O2 and 0.1 mL enzyme extract. Decrease in absorbance of the reaction solution at 240 nm
was recorded after every 30 s. An absorbance change of 0.01 units min-1 was defined as one
unit CAT activity. Enzyme activity was expressed on F. wt. basis.
Protease activity
For the estimation of protease activity, selected parts were homogenized in a medium
composed of 50 mM potassium phosphate buffer (pH 7.8). Protease activity was determined
by the casein digestion assay described by Drapeau (1974). By this method one unit is that
amount of enzyme, which releases acid soluble fragments equivalent to 0.001 A280 per
minute at 37 oC and pH 7.8. Enzyme activity was expressed on F. wt. basis.
Esterase activity
3
For the estimation of esterase activity, selected parts were homogenized in a medium
composed of 50 mM potassium phosphate buffer (pH 7.8). The α-esterases and β-esterases
were determined according to the method of Van Asperen (1962) using α–naphthyl acetate
and β–naphthyl acetate as substrates, respectively. The reaction mixture consisted of
substrate solution (30 m M α or β–naphthyl acetate, 1% acetone and 0.04 M phosphate buffer
(pH 7) and enzyme extract. The mixture was incubated for exactly 15 min at 27°C in dark
and then 1 mL of staining solution (1% Fast blue BB and 5% SDS mixed in ratio of 2:5)
was added and incubated for 20 min at 27°C in dark. Amount of α- and β- naphthol produced
was measured by recording the absorbace at 590 nm. Using standard curve, enzyme activity
was expressed as α or β- naphthol produced in µM min-1 per g F. wt.
Total oxidant status (TOS)
TOS was measured using Erel (2005) TOS method, which is based on the oxidation
of ferrous ion to ferric ion in the presence of various oxidative species in acidic medium and
the measurement of the ferric ion by xylenol orange (Harma and Erel 2005). The assay
mixture consisted of sample extract, reagent R1, reagent R2. After 5 minutes, the absorption
of each assay mixture was measured at wavelength 560 nm. A standard curve was prepared
using hydrogen peroxide. The results were expressed in μmol H2O2 equivalent/L.
Total antioxidant capacity (TAC)
The reduction of the blue-green 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate)
radical cation (ABTS•+) by antioxidants to its original colorless ABTS form is the basis of the
ABTS assay. The ABTS•+ is decolorized by antioxidants according to their antioxidant
content (Nenadis et al. 2007). The assay mixture contained reagent R1 (mixture of sodium
acetate buffer solution and glacial acetic acid, pH 5.8), sample extract and reagent R2
(mixture of sodium phosphate buffer solution, glacial acetic acid, hydrogen peroxide and
ABTs). The content of the tube was mixed and allowed to stand for 6 min and the absorbance
was measured at 660 nm.
Inhibition of free radical scavenging activity was calculated using the equation: %
Inhibition = 100 × (absorbance of the control − absorbance of the sample)/absorbance of the
control EC50 value (μg/mL) is the effective concentration at which ABTS•+ is scavenged by
4
50%. A calibration curve of ascorbic acid was established, the antioxidant content of the
plant species extracts were then expressed as mM ascorbic acid equivalent per gram of dry
plant extract.
Measurement of pigments
The chlorophylls a and b and carotenoids (x + c) were extracted in 80% (v/v) aqueous
acetone and vacuum filtered through a Whatman No. 1 filter paper. Pigment measurements
were quantified spectrophotometrically. Absorbances of chlorophylls a and b and carotenoids
(x+c) extracts were determined at wavelengths of 663, 645, 505, 470 and 453 nm
respectively. Concentrations (mg g-1 f. wt.) of pigments were calculated by equations of
Lichtenthaler and Wellburn ( 1983).
Total phenolic contents (TPC)
To carry out the analysis for phenolic contents the micro colorimetric method
mentioned previously (Ainsworth and Gillespie 2007) was applied with some modification in
which Folin-Ciocalteau (F–C) reagent was used. For extraction of TPC, selected parts (0.1 g)
were ground in 85% ice-cold methanol and kept at room temperature for 48h. After 48h
centrifuged at 14,000 rpm for 20 min., the supernatant was separated and used for the
determination of TPC. For estimation of TPC in samples 100 μl of supernatant was mixed
with100 μl of 10 % (v/v) F-C reagent, vortex thoroughly and then 800 μl of 700 mM Na2
CO3 was added. Samples were then incubated at room temperature for 1 hour. Blank
corrected absorbance of samples was measured at 765 nm. A standard curve was prepared
using different concentration of gallic acid and a linear regression equation was calculated.
Phenolic content (gallic acid equivalents) of samples were determined by using linear
regression equation.
Protein profiling using SDS-PAGE
For extraction of total soluble proteins, samples were grounded in 50 mM phosphate
buffer, pH 7.8 followed by centrifugation for 10 min at 14,000 rpm in micro-centrifuge
machine (Sigma 1-14). Total soluble protein concentration in extracts was quantified by dye
binding assay reported by (Bradford 1976). The protein extracts were used for protein
profiling by SDS-polyacrylamide gel electrophoresis following the method of Laemmli
5
(1970). Samples were mixed with cracking solution (10 ml containing 6.5 mL of water 0.01 g
bromphenol blue, 1g SDS, 2 ml 2-mercaptoethanol, 5 g sucrose and 1.5 mL of tris (0.5 M,
pH 6.8) in 4:1 ratio followed by boiling in water bath for 2-3 min to denature the proteins.
Equal volume of samples were loaded in gels and electrophoresis was performed on
constant voltage (120 volts). Protein molecular weight marker (Biobasic Inc. RM-0011) was
also loaded in gels for calculation of approximate molecular weight of protein peptides in the
samples. Consequently, the gel documentation system (UVI-proplatinum) (UVI-tec UK)
(NIB-A (6)-GDS-001) was used to photograph the gel. UVI-proplatinum 1.1 Version 12.9
for windows (copyright® 2004 - 2006) was used to carry out computerized gel analysis.
Table S1: Searched peptides of Allamanda cathartica with known molecular weights
Table S2: Identification of peptides of Allamanda cathartica in database
Figure S1: Protein profiles (left) and histogram (right) of different organs of Allamanda
cathartica. 1: Root, 2: Shoots, 3: leaf, 4: Flowers and M: Protein marker (Bio Basic :
RM0011).
References
Ainsworth EA, Gillespie KM. 2007. Estimation of total phenolic content and other oxidation
substrates in plant tissues using Folin-Ciocalteu reagent. Nature Protocols 2(4):875877.
Beers RF, Sizer IW. 1952. A spectrophotometric method for measuring the breakdown of
hydrogen peroxide by catalase. J Biol Chem 195(1):133-140.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Analytical
biochemistry 72(1):248-254.
Chance B, Maehly A. 1955. Assay of catalases and peroxidases. Methods in enzymology
2:764-775.
Dixit V, Pandey V, Shyam R. 2001. Differential antioxidative responses to cadmium in roots
and leaves of pea (Pisum sativum L. cv. Azad). Journal of Experimental Botany
52(358):1101-1109.
Drapeau G. 1974. Protease from Staphylococcus aureus. Academic Press, NY.
Endress ME, Sennblad B, Nilsson S, Civeyrel L, Chase MW, Huysmans S, Grafstrom E,
Bremer B. A phylogenetic analysis of Apocynaceae s. str. and some related taxa in
Gentianales: a multidisciplinary approach. Second International Rubiaceae
Conference: proceedings.Opera Botanica Belgica; 1996. p. 59-102.
6
Erel O. 2005. A new automated colorimetric method for measuring total oxidant status.
Clinical biochemistry 38(12):1103-1111.
Giannopolitis CN, Ries SK. 1977. Superoxide dismutases I. Occurrence in higher plants.
Plant physiology 59(2):309-314.
Harma M, Erel O. 2005. Oxidative stress in women with preeclampsia. American journal of
obstetrics and gynecology 192(2):656.
He Z, Mao M, Yu H, Li H, Chen X. 2009. Molecular characterization of a distinct
begomovirus infecting Allamanda cathartica in Guangdong, China. Archives of
virology 154(8):1199-1202.
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. nature 227(5259):680-685.
Lichtenthaler HK, Wellburn, A.R. 1983. Determination of total carotenoids and chlorophylls
a and b in leaf extracts in different solvents. Biochemical Society Transactions 11:
591-592.
Mahadani P, Sharma GD, Ghosh SK. 2011. Evaluation of matK sequence for species level
DNA passport in medicinal Rauvolfioideae (Apocynaceae) plants from North East
India. Uniprot; NCBI.
Nenadis N, Lazaridou O, Tsimidou MZ. 2007. Use of reference compounds in antioxidant
activity assessment. Journal of Agricultural and Food Chemistry 55(14):5452-5460.
Sennblad B, Bremer B. 1996. The familial and subfamilial relationships ofApocynaceae
andAsclepiadaceae evaluated withrbcL data. Plant Systematics and Evolution 202(34):153-175.
Surveswaran S, Huang W, Cai Y, Corke H, Sun M. 2008. Phylogenetic analysis of
Apocynaceae based on the sequences of rbcL gene. Uniprot.
Van-Asperen K. 1962. A study of housefly esterases by means of a sensitive colorimetric
method. Journal of Insect Physiology 8(4):401-416.
7
Figure S1: Protein profiles (left) and histogram (right) of different organs of
Allamanda cathartica. 1: Root, 2: Shoots, 3: leaf, 4: Flowers and M: Protein marker (Bio
Basic : RM0011).
8
Table S1: Searched peptides of Allamanda cathartica with known molecular weights
Sr.
#
Accession #
Protein name
1
O19816
Maturase K
Mol.
wt.
(kDa)
58.490
CAA94079
AGB58442
Maturase
Maturase K
55.55*
16.72*
G3KG45
G3KG47
AEN19336
AEN19334
Maturase K
Maturase K
Maturase K
Maturase K
33.48
33.45
30.91*
31.02*
G0KVP1
Ribulose-1,5bisphosphate
carboxylase/oxygenase
large subunit
-do-
49.05
2
AFX62160
Q95612
20.13*
Mol. function
References
RNA binding
(Endress et al. 1996)
Uniprot, NCBI.
(Endress et al. 1996) NCBI
NCBI
(Mahadani et al. 2011).
Uniprot
-do(Mahadani et al. 2011).
NCBI
-do(Surveswaran et al. 2008)
Uniprot
Magnesium ion
binding; ribuloseBisphosphate
carboxylase
activity
51.81
-do51.37*
CAA62874
-doACL12036
K7WJ62
3
G3EIS4
G3EIW1
G3EIU6
AEM60211
AEM60189
AEM60226
4
5
6
J9T3X0
G8H2G5
AFR60235
AEQ93983
A7KX24
YP_001974394
ABS32014
A7KX27
YP_001974397
ABS32017
-do-doMADS box transcription
factor
MADS box transcription
factor
MADS box transcription
factor
MADS box transcription
factor
MADS box transcription
factor
MADS box transcription
factor
PsbA
PsbA
PsbA
PsbA
Capsid protein
Capsid protein
Capsid protein
AC1 protein
AC1 protein
AC1 protein
NCBI
(Sennblad
and
Bremer
1996). Uniprot
(Sennblad
and
Bremer
1996). NCBI.
(He et al. 2009). NCBI
48.73*
20.34
20.01
22.56
21.65
20.46
22.99*
21.67*
1.467
1.950
1.43*
2.09*
30.11
28.27*
28.27*
39.93
39.05*
39.49*
9
DNA binding;
sequence-specific
DNA binding
transcription
factor activity
Structural
molecule activity
Endodeoxyribonu
clease activity,
producing 5'phosphomonoeste
rs; structural
molecule activity
Uniprot
-do-doNCBI
-do-do-
(He et al. 2009). Uniprot
-do(He et al. 2009). NCBI
-do(He et al. 2009). Uniprot
(He et al. 2009). NCBI
-do(He et al. 2009). Uniprot
(He et al. 2009). NCBI
-do-
7
8
9
10
A7KX26
YP_001974396
ABS32016
A7KX25
YP_001974395
ABS32015
A7KX28
YP_001974398
ABS32018
AC2 protein
AC2 protein
AC2 protein
AC3 protein
AC3 protein
AC3 protein
AC4 protein
AC4 protein
AC4 protein
15.16*
14.63*
14.63*
16
14.74*
14.74*
9.33*
9.35*
9.35*
Structural
molecule activity
Unknown
-do-doUnknown
-do-do-
(He et al. 2009). Uniprot
(He et al. 2009). NCBI
-do(He et al. 2009). Uniprot
(He et al. 2009). NCBI
-do(He et al. 2009). Uniprot
(He et al. 2009). NCBI
-do-
A7KX23
YP_001974393
ABS32013
AV2 protein
AV2 protein
AV2 protein
13.42*
12.76*
12.76*
Unknown
-do-do-
(He et al. 2009). Uniprot
(He et al. 2009). NCBI
-do-
Note: (*) indicates Mol. wt. in kDa that is calculated by the following formula = no. of amino
acids × 0.11 and -do- represents repetition
10
Table S2: Identification of peptides of Allamanda cathartica in database
Sr.# Protein
peptides
Characterized in literature with known References
family
-
1
2
3
4
AC-L-168
AC-95
AC-88
AC-79
-
5
6
AC-70
AC-50
(Surveswaran et al. 2008).
Ribulose-1,5-bisphosphate
carboxylase/oxygenase large subunit (49.05 Uniprot
(Sennblad
and
Bremer
kDa; 51.81 kDa; 51.37* kDa)
1996). Uniprot
Maturase (55.55* kDa)
7
AC-46
8
9
AC-43
AC-41
10
AC-R-39
Ribulose-1,5-bisphosphate
carboxylase/oxygenase large subunit (48.73*
kDa)
(Sennblad
and
Bremer
1996). NCBI.
(Endress et al. 1996). NCBI
NCBI
(He et al. 2009). Uniprot
AC1 protein (39.93 kDa)
AC1 protein (39.93 kDa; 39.05* kDa and (He et al. 2009). Uniprot
(He et al. 2009). NCBI
39.49* kDa)
(He et al. 2009). NCBI
-
11
AC-L-38
-
12
13
AC-R-37
AC-33
Maturase K (33.48 kDa and 33.45 kDa)
14
AC-30
Maturase K (30.91*kDa and 31.02* kDa)
Capsid protein (30.11 kDa)
15
AC-L-27
Capsid protein (28.27 kDa)
(Mahadani et al. 2011).
Uniprot
(Mahadani et al. 2011).
NCBI
(He et al. 2009). Uniprot
(He et al. 2009). NCBI
Note: (*) indicates Mol. wt. in kDa that is calculated by the following formula = no. of amino
acids × 0.11 and -do- represents repetition, – indicates no information.
11