Feb-Apr.2012, Vol.2.No.

2, 792-796

e- ISSN: 2249 1929

Journal of Chemical, Biological and Physical Sciences

An International Peer Review E-3 Journal of Sciences

Available online at www.jcbsc.org

Section B: Biological Sciences
CODEN (USA): JCBPAT
Research Article

Studies on protein profile of some medicinally important

species of Apocyanaceae family using SDS-PAGE
A. John De Britto*, P. Benjamin Jeya Rathna Kumar
and D.Herin Sheeba Gracelin
Plant Molecular Biology Research Unit,Post Graduate and Research Department of Plant
Biology and Biotechnology,St. Xavier's College (Autonomous),
Palayamkottai - 627 002, Tamil Nadu, India.
Received: 18 January 2012; Revised: 16 February 2012; Accepted: 25 February 2012

ABSTRACT
Present study was undertaken to analyze the protein profiles of some medicinally important
speciesofApocyanaceaefamilyusingSDSPAGE.Totalleafproteinswereextractedandseparated
on 10% polycrylamide gels using standard protocols. Protein fragments of various molecular
weights were separated in all selected species. The molecular weight of the protein bands were
calculated using standard curve method. Protein bands were scored for similarity index studies.
ThedendrogramwasconstructedbasedonthissimilarityindextableusingNTSYSPCsoftware.

Keywords: Apocyanaceae, Medicinal plants, SDS-PAGE, Protein profile

INTRODUCTION
Apocynaceae is a large family with about 1500 species found mainly in tropical regions. It includes
many of the most well-known tropical ornamental plants. The sap of most plants is milky latex, which
is often of economic importance for medicinal use. Apocynaceae species used in traditional medicine
reveals that 25 species in 16 genera are of ethno botanical interest. Nineteen species are medicinal,
sixteen of which fall under the subfamily Plumerioideae which usually has indole alkaloids.
The most common category of diseases treated is skin and ectoparasitic diseases followed by
abdominal diseases, diseases of the head, female conditions and venereal diseases. The root is the
most commonly used part of the plant and it is possible that the alkaloids play an important role in the
medicinal value of the plants 1. The species which are selected for this study have the highly
medicinal values. They are morphologically distinct. But sometimes they may be similar in their
genetic level (DNA and protein). Hence this study focused to identify their genotype similarity using
SDS-PAGE analysis. In this technique leaf protein is used as biochemical marker.
792 J. Chem. Bio. Phy. Sci. Sec. B . 2012, Vol.2, N0.2, 792-796

A. John De Britto et al.

Studies on........

MATERIALS AND METHODS

The selected Apocyanaceae species are Thevetia peruviana (Pers.) K. Schum., Nerium indicum Mill.,
Rauvolfia tetraphylla L., Catharanthus roseus (L.) G.Don, Ervatamia coronaria (Jacq.) Stapf. were
identified and collected from in and around Tirunelveli regions. The collected materials are stored in
deep freezer (-700C) for the SDS-PAGE analysis.
Isolation of protein: 250mg of stored plant materials were ground well in a mortar and pestle with
extraction buffer (0.1M Tris-HCl, pH 8.0, 0.01 M MgCl2, 18% (w/v) sucrose, and 40mM
-mercaptoethanol). The homogenate was centrifuged at 10000 rpm for 10 minutes. The supernatant
was taken and the volume was noted. Equal amount of 10% TCA was added and kept in ice for 30
minutes. The mixture was centrifuged at 10000 rpm for 10 minutes. The pellet was collected and
dissolved in 50l of 0.2N NaOH. It was stored at -200C for further analysis.
SDS - PAGE analysis: SDS PAGE of leaf protein was carried out in vertical slab gel discontinuous
buffer system following the method of 2 using 10% acrylamide gel concentration. A total volume of
12l protein extract solution was loaded into each well and electrophoresis was carried out at 100V
until the bromophenol blue dye reaches the bottom of the gel.
Data analysis: Protein bands were scored for similarity index analysis based on the presence and
absence of bands. The dendrogram was constructed based on this similarity index table using NTSYSPC, version 2.013. The molecular weights of the dissociated polypeptides were determined by using
the standard curve. RF value and protein profiling distance (PPD) of each protein bands were
determined.

RESULTS AND DISCUSSION

In the present study, Protein profiles of the few medicinal plants in Apocynaceae family were studied
by using SDS-Polyacrylamide gel. In Apocyanaceae family the numbers of proteins bands were
varied from 7 to 13 (Fig.1). More number of peptide bands was produced in R.tetraphylla and less
number of bands was observed in N.indicum. The molecular weights of the bands were calculated
using standard curve. Molecular weight of the five plants varied from 1.26 kD to 89.12 kD. Highest
molecular weight protein was observed inN.indicum (89.12kD). R.tetraphylla has the low molecular
weight (1.26 KD) protein (Table 1).
Fig.1 Total number of protein bands in Apocyanaceae species
14

13
12

No.of protein bands

12

10
10
8

9
7

6
4
2
0

793 J. Chem. Bio. Phy. Sci. Sec. 2012, Vol.2, N0.2, 792-796.

A. John De Britto et al.

Studies on........

Table-1: Molecular weight, Rf values and Protein profiling in Apocyanaceae species

S.
N
o

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23

T.peruviana
Mol.
Wt.
(kDa)

Rf
value
(cm)

63.09
44.67
39.81
25.11
11.22
7.94
2.82
2.24
1.77
-

0.14
0.20
0.27
0.37
0.55
0.61
0.74
0.82
0.89
-

N.indicum

PPD
(cm)
1.0
1.4
1.9
2.5
3.7
4.1
5.0
5.5
6.0
-

R.tetraphylla

C.roseus

E.coronaria

Mol.
Wt.
(kDa)

Rf
value
(cm)

PPD
(cm)

Mol.
Wt.
(kDa)

Rf
value
(cm)

PPD
(cm)

Mol.
Wt.
(kDa)

Rf
value
(cm)

PPD
(cm)

Mol.
Wt.
(kDa)

Rf
value
(cm)

PPD
(cm)

89.12
50.11
44.67
28.18
7.94
5.01
2.51
-

0.08
0.17
0.20
0.31
0.61
0.68
0.80
-

0.6
1.2
1.4
2.1
4.1
4.6
5.4
-

56.23
50.11
30.20
25.11
15.85
11.22
7.94
5.01
3.55
2.51
1.77
1.41
1.26

0.16
0.17
0.29
0.37
0.44
0.55
0.61
0.68
0.73
0.80
0.89
0.94
0.98

1.1
1.2
2.0
2.5
3.0
3.7
4.1
4.6
4.9
5.4
6.0
6.3
6.6

79.43
63.09
50.11
39.81
30.20
25.11
15.85
7.94
3.55
2.51
2.24
1.77
-

0.10
0.14
0.17
0.27
0.29
0.37
0.44
0.61
0.73
0.80
0.82
0.89
-

0.7
1.0
1.2
1.9
2.0
2.5
3.0
4.1
4.9
5.4
5.5
6.0
-

79.43
56.23
44.67
19.05
10.0
7.94
3.55
2.82
2.24
1.41
-

0.10
0.16
0.20
0.41
0.52
0.61
0.73
0.74
0.82
0.94
-

0.7
1.1
1.4
2.8
3.5
4.1
4.9
5.0
5.5
6.3
-

Mol.Wt. Molecular Weight, Rf - Relative mobility, PPD-Protein profiling distance

(from band to base le)

794 J. Chem. Bio. Phy. Sci. Sec. 2012, Vol.2, N0.2, 792-796.

A. John De Britto et al.

Studies on........

The similarity index (Table 2) represents the similarity indices between the species. The dendrogram
(Fig.2) was constructed based on the UPGMA. The dendrogram shows distinct separation of the
collected species into two major groups having 30% similarity. Among the two major clusters, the
species belonging to the lower cluster (LC) is N.indicum. The upper cluster (UC) having T.peruviana,
R.tetraphylla, C.roseus and E.coronaria. Further the upper cluster was grouped into two sub clusters
(UC1 and UC2). UC2 has only the E.coronaria, which have the 37% similarity. UC1 were grouped
into two sub clusters (UC1A and UC1B). UC1A consist only T.peruviana (45% similaity). UC1B
have the R.tetraphylla and C.roseus was having the 64% similarity.

Table: 2 Similarity indexes of Apocyanaceae species

T.peruviana
N.indicum
R.tetraphylla
C.roseus
E.coronaria

T.peruviana

N.indicum

R.tetraphylla

1.00
0.25
0.36
0.57
0.42

1.00
0.40
0.32
0.24

1.00
0.64
0.35

C.roseus

1.00
0.36

E.coronaria

1.00

Fig.2: UPGMA Dendrogram of Apocyanaceae species by SDS-PAGE

Similarly, the protein profile of five species (Solanum melongena, S. xanthocarpum, Datura alba,
Lycopersiconesculentum and Capsicum annum) of family Solanaceae through poly acrylamide gel
electrophoresis were investigated by 4. They investigated protein profile revealed that experimental
genus Lycopersicon is very close to the genus Solanum and species Datura alba and Solanum
melongena are more close at molecular level as compared to other species. Similarity index was more
i.e., 22.22% in Capsicum annum and Solanum xanthocarpum which are cultivated and wild
respectively, as compared to two wild species i.e., Datura alba and Solanum xanthocarpum where
similarity index was only 11.11%. The seed protein profile of 42 accessions belonging to 7 species
of 4 different genera (Datura, Hyoscyamus, Withania and Atropa) from the family Solanaceae were
investigated by 5 through poly acrylamide gel electrophoresis. A dendrogram based on UPGMA
revealed the generic status and inter relationship of Hyoscyamus, Atropa, Withania and Datura. The
specimens of Withania somnifera collected from Panjgur (109717, 109718, and 109710) not only
showed the variation morphologically but also based on protein profiles. Based on the total seed
795 J. Chem. Bio. Phy. Sci. Sec. 2012, Vol.2, N0.2, 792-796.

Studies on........

A. John De Britto et al.

protein profile close association is found between Withania/Datura and Atropa/Hyoscyamaus but they
maintain their generic status, as there is no intermixing of species was observed. The taxonomic status
of 14 Turkish endemic species and one variety of the genus Ebenus was analyzed by 6 using seed
proteins using SDS PAGE, the molecular weight ranging between 116 kDa and 21 kDa were detected
in SDS-PAGE. The relative similarity between various taxa estimated by Jaccards similarity index
and clustered in UPGMA.
In conclusion, taxonomic classification of plant species is basically depend on the morphological and
anatomic characters, these features are changeable and sometimes difficult to observe, so it is
necessary to be supported by molecular techniques. This SDS - PAGE study is one of the molecular
marker as a tool to study molecular systematic for identification of genotypes based on proteins

ACKNOWLEDGEMENT
The authors thank Indian Council of Medical Research (ICMR), Government of India, New Delhi
(Ref: No. 59/12/2006/BMS/TRM DT. 26.03.2009) for providing financial support for carry out this
work is gratefully acknowledged.

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2. U.K. Laemmli, Nature, 1970, 227, 680-685.
3. F.J. Rohlf, NTSYS-pc Numerical Taxonomy and Multivariate Analysis System, 2.02.Exeter
Publications, Setauket, New York, 1998.
4. T.M. Bhat, R. Kudesia, Genetic Engineering and Biotechnology Journal, 2011, 20, 20-25.
5. Y.Zubaida, M.Shahid, K.S.Zabta, A.K. Mir, R.Ashiq, Pak. J. Bot., 2008,40, 2289-2297.
6. C.Ayten, A.Leyla, A.Zeki, Pak. J. Bot., 2009, 41, 2477-2486.

*Correspondence Author: A. John De Britto; Plant Molecular Biology Research Unit,

Post Graduate and Research Department of Plant Biology and Biotechnology,
St. Xavier's College (Autonomous), Palayamkottai - 627 002, Tamil Nadu, India.

796 J. Chem. Bio. Phy. Sci. Sec. 2012, Vol.2, N0.2, 792-796.