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ISSN: 2249-0353
Original Article
ESTIMATION OF COLCICHINE CONTENT IN TUBER, SEED AND LEAVE
SAMPLES OF GLORIOSA SUPERBA USING HPLC AND THEIR
ANTIBACTERIAL STUDIES ON PATHOGENIC STRAINS.
LAKSHMI PRIYA.T 1, SWATHI.S 2
1
Abstract
In this present study, the presence of the phytochemical compound (i.e.) Colchicine, present in treated tuber, seed and
leaves samples of Gloriosasuperba was confirmed using HPLC. And also the samples were extensively studied for their
antibacterial activity against Gram positive bacteria namely Enterococcus fecalis and two Gram negative bacteria
Klebsiella pneumonia and Proteus mirablis by disk diffusion method. The Gloriosasuperba shows higher inhibition
activity over Enterococcus fecalisfollowed by moderate inhibition on Proteus mirablis and lower inhibition effect over
Klebsiella pneumonia confirming that it has anti-bacterial activity.
2015 Universal Research Publications. All rights reserved
Key words:- Gloriosasuperba, Antibacterial activity, Colchicine,Klebsiella pneumonia, Enterococcus fecalis, Proteus
mirablis.
INTRODUCTION
Glory Lily (Gloriosasuperba) belongs to the family
Liliaceae. It is an ancient medicinal plant in India. India
was the first to use Ayurveda medicine, later as source
colchicines and colchicoside [1]. Colchicine the main
alkaloid of Gloriosa species (Kim et al., 2003) are a useful
agent in the treatment of acute attacks of gout [2], familial
Mediterranean fever [3] and in Behcets disease [4].
Traditionally, colchicine as an experimental tool in the
study of cell division, as it can inhibit mitosis (a type of cell
division), induce polyploidy (cells containing more than
two sets of chromosomes), and has been used in the
treatment of cancer [5]. Haroon et al. (2011) reported that
both gram positive and gram negative bacterial growth was
inhibited by the extracts. It may be due to the reason that
the tubers have constant contact with soil. The plants are
producing large number of organic compounds as
secondary metabolites. These compounds acts as
chemotherapeutic, bactericidal and bacteriostatic. The
tuberous root stocks of glory lily, boiled with Sesamum oil
is applied twice a day on the joints, affected with arthritis
reduces pain [6] Seeds are used for relieving rheumatic
pain and as a muscle relaxant [7]. The tuber is useful in
itching, skin diseases including wounds and ailments
caused by vitiated kapha and vata, can be administered to a
34
35
Fig 4. HPLC
20l was injected at the rate of 1ml/min [13]. and
wavelength of about 245 nm (Fig. 4).
ANTI-BACTERIAL ASSAY:
The screening of the extracts for antibacterial effect was
carried out by determining the zone of inhibition using disc
diffusion method [14-16]. Sterile nutrient agar plates were
prepared and inoculated by spread plate method under
aseptic conditions. The filter paper discs of 6mm diameter
(Whatmans No.1 filter paper) were prepared and sterilized.
The plant extracts to be tested were prepared with various
concentrations at 50mg/ml, 100mg/ml and 150mg/ml and
were added to each disc of holding capacity of 10
microlitres [17]. The sterile impregnated discs with plant
extracts were placed on the agar surface with framed
forceps and gently pressed down to ensure complete
contact of the disc with the agar surface. Control discs of
Gentamycin were prepared and placed on the agar surface.
All the plates were incubated at 37C for 24 hours [18-20]
After incubation, the size (diameter) of the inhibition zones
was measured.
RESULTS
EXTRACTION PROCESS YIELD:
Leaves, tubers and seed samples of Gloriosasuperba were
used for extraction phytochemical (colchicine). Two
extraction process were followed namely hot extraction and
cold extraction [21].Same quantity of sample (say 5g) was
extracted using both process. For hot extraction methanol is
used as solvent. Petroleum ether and dichloromethane were
used for cold extraction process [22].The final yield of hot
extraction was found to be higher than that obtained in cold
extraction. Tuber sample yielded higher extract in both the
process followed by seeds and leaves sample yielded the
least. (Table 1).
QUANTIFICATION BY HPLC:
Quantification of colchicine was determined using Waters
make HPLC model 515. The detector used was photodiode
array detector (model:2998) mobile phase used was A:
Acetonitrile and B: 3% Acetic acid[23].The retention time
of standard colchine was found to be 9.63 minutes. The
chromatogram of seed(Fig.5 and in Table 2) yielded totally
of seven peaks during the run time of fifteen minutes. Of
the seven peaks obtained, sixth peak obtained during the
retention time of 9.642 minutes corresponds to Colchicine.
36
Plant Parts
1
2
3
Leaves
Tubers
Seeds
Hot
0.75
1.17
0.94
RT
2.932
3.054
7.835
9.613
10.288
Area
755575
964478
1026028
10181899
1944846
% Area
5.08
6.48
6.90
68.46
13.08
Height
61645
61987
62196
649834
157498
RT
2.939
Area
2540184
% Area
8.57
Height
282231
3.056
3045069
10.28
242868
3
4
Peak 3
Peak 4
3.982
4.790
1556701
3714620
5.25
12.53
110473
124124
Peak 5
7.874
2463204
8.31
101114
6
7
Colchine
9.642
13782344
46.51
888284
Peak 7
10.316
2532413
8.55
223576
% Area
10.18
8.55
8.89
41.01
5.71
0.78
18.66
2.59
0.02
3.60
Height
52614
34137
30460
94457
22515
3316
45609
8950
183
10147
RT
2.919
3.961
6.981
7.150
7.724
9.455
10.543
11.219
11.420
12.086
Area
359802
302240
314220
1449035
201791
27727
654508
91591
579
127134
1
2
Plant Parts
Seeds
Tubers
Colchicine (mg/ml)
2.29
3.38
Leaves
0.14
Plant Parts
1
2
3
Leaves
Seeds
Tubers
37
Control
6.7
7.1
8.3
200
7.4
7.6
9.0
Plant Parts
1
2
3
Leaves
Seeds
Tubers
Control
5.7
7.4
7.9
200
6.2
7.9
8.4
200
4.1
5.5
8.1
Plant Parts
1
2
3
Leaves
Seeds
Tubers
Control
3.7
4.9
5.8
ANTI-BACTERIAL ASSAY:
For this assay [25-27] three bacterial species were
chosen. Of the three one was Gram positive (Enterococcus
faecalis) and other two were Gram negative (Proteus
mirablis, Klebsiella pneumonia). Bacterial strains were
maintained in nutrient agar medium [27-31]. All the three
samples were diluted to 50,100 and 200l concentration
using methanol.
The activity of the samples against Enterococcus
faecalis (Table 6). It is clear, that the tuber sample shows
prominent activity than seed and leaves samples (Figs. 8, 9,
10 and 11). Also the activity of almost all the samples
increases with increase in the concentration. Enterococcus
faecalis shows more susceptibility than other strains.
Figure 10 Activity of leaves extract against Enterococcus
faecalis
38
39
DISCUSSION
In most developing countries of the world, plants are the
main medicinal sources used in treating infectiousdiseases.
The various phytochemical compounds detected are known
to exhibit medicinal activity as well as physiological
activity. Plants are important source of potentially useful
structures for the development of new chemotherapeutic
agents. They have an almost limitless ability to synthesize
aromatic substances, most of which are phenols or their
oxygen-substituted derivatives. The plant is a well-known
ethnomedicinal use in Ayurveda for its colchicine content
which is used to treat arthritis. Phytochemical studies of
tubers or dried roots haveshowed the presence of
colchicines In the present study,the hot and cold extraction
extracts of seeds and tubers samples showed the maximum
yield of phytochemicals. However, the methanol extract of
G.superbashowed maximum growth inhibition on gram
positive and gram negative bacteria. The plants have
beenproducing large number of organic compounds as
secondary metabolites. These compounds acts as
chemotherapeutic, bactericidal and bacteriostatics. The
tuber extracts showed more effect than seeds extract against
all bacteria. It may be due to the reason that the tubers have
strongly contact with soil and microorganisms.
CONCLUSION
Now a days commercial extraction of Colchicine is
obtained mainly from seeds. From the study it can be
concluded that tubers of Gloriosasuperbacan be used as
source for commercial extraction and it is wise to prefer hot
extraction to cold for higher yield. It can also be concluded
that the extracts prepared from the leaves, tubers and seeds
of G.superba are a source of different secondary
metabollites which may act against number of human
pathogens.
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