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International Journal of Natural Products Research


Universal Research Publications. All rights reserved

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

Department of civil Engineering, Government College of Technology,


Coimbatore.India.lakshmipriya95@gmail.com.
2
Centre for Biotechnology, Anna University, Chennai.India.
fabulousswathi@gmail.com
Received 01 September 2015; accepted 21 September 2015

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

delivered mother along with spirituous drink to give relieve


to her postnatal complaints. It is pungent, thermogenic, and
used as a purgative.
In the world market, the tubers are considered as rich
sources of colchicines and gloriosine [8]. Roots are acrid,
anthelmintic, antipyretic, bitter, digestive, expectorant,
highly poisonous and promoting expulsion of the placenta.
Root paste is effective against paralysis, rheumatism, snake
bite and insect bites [9]. Glory lily extract is useful against
many skin diseases. It is used to rectify the many
respiratory disorders. The sap from the leaf tip is used for
pimples and skin eruptions. Tribals of Patalkot apply the
powder of rhizome with coconut oil in skin eruptions and
related diseases. Gloriosasuperbais widely cultivated as an
ornamental for its stunning flowers. The objective of the
present study was to quantify the phytochemical constituent
(Colchicine) of Gloriosasuperba by HPLC, to compare the
Colchicine content in tuber, seeds and leaves and to
evaluate the antibacterial property of tuber, seed and leaf
extracts.
MATERIALS AND METHODS
PLANT MATERIALS:
The fresh leaves, tubers, and seeds of
Gloriosasuperba were collected in the cultivating fields of
Aravakurichi village in Karur district, Tamilnadu.

International Journal of Natural Products Research 2015; 5(3): 34-41

CHEMICALS, SOLVENTS & MEDIUM USED:


Organic solvents used in the present study for
extraction were petroleum ether, dichloromethane,
ammonia, ethanol and methanol were purchased from
Sigma Aldrich. Nutrient agar medium and the nutrient
broth used for culturing the bacteria and for assessing the
antibacterial activity were prepared in the laboratory.
MICROORGANISMS USED:
Bacteria causing infectious diseases both in
animals and human were used in the present study. They
were both Gram positive and Gram negative. One Gram
positive bacteria namely Enterococcus fecalis and two
Gram negative bacteria Klebsiella pneumonia and Proteus
mirablis were used in the present study. All bacterial strains
were obtained from T.Stanesphytopharma testing
laboratory, coimbatore. The cultures were maintained in
nutrient broth in the laboratory.
EXTRACTION PROCESS:
In the present study leaves, tubers and seeds of
Gloriosasuperba were used for evaluating antibacterial
property. The freshly collected leaves, seeds and tubers
were washed thoroughly in tap water. It was then allowed
to air dry for 10 days at room temperature. The thoroughly
dried plant parts were powdered and weighed. This
powdered plant material is used for extraction process. Two
types of extraction procedures were adopted. They are Hot
continuous extraction using Soxhlet apparatus and Cold
extraction using various solvents.
HOT EXTACTION PROCEDURE:
Hot extraction procedure was carried out in
Soxhlet apparatus.5g of finely powdered plant material was
packed & placed in thimble.300 ml methanol was taken in
round bottomed flask as extraction solvent [9].The heating
mantle was set for the temperature of 65C.The extraction
process was continued for around 7 cycles. The sample
packet was then removed from thimble and the solvent is
recovered [10]. Remaining organic phase in the extract
was evaporated to dryness & final yield was measured. It is
then dissolved in methanol to yield crude extract and it
(Fig. 1)

COLD EXTRACTION PROCEDURE:


Powdered plant material of 5g was extracted
twice with 25 ml of petroleum ether with frequent shaking
for 1 h, followed each time by filtration. The solid residues
were air dried and then extracted with 10 ml of
dichloromethane at room temperature for 30 min with
frequent shaking. Then 10% solution of ammonia (0.5 ml)
was added to the mixture with vigorous shaking for 10 min.
The mixture was left undisturbed for 30 min and then
filtered [10] .The residue was washed twice with 10 ml of
dichloromethane and then combined with the filtrate. The
organic phase was evaporated to dryness and then dissolved
in 1 ml of 70% ethanol to yield the test sample [11]. Seeds,
leaves and tubers were used to prepare extract (Fig. 2, 3).

Fig 2. Cold Extraction

Fig 3. Crude Extracts

Fig 1. Hot extraction using Soxhlet apparatus.

35

QUANTIFICATION OF COLCHICINE BY HPLC:


Quantification of Colchicine is done using Waters make
HPLC model 515.Mobile phase used in this study was
Acetonitrile and 3% Acetic acid[12].The array detector
model 2998) used was Photodiode. The sample volume of

International Journal of Natural Products Research 2015; 5(3): 34-41

It has the largest area of 13782344 Au and corresponds to


46.51% of total area. The height of the peak obtained was
888284 Au.
The chromatogram of tubers (Fig. 6 and in Table
3), yielded totally of five peaks. Of the five, fourth peak
obtained during the retention time of 9.613 minutes
corresponds to Colchicine. It has the area of 10181899 Au
which corresponds to 68.46% of the total area. The height
of the peak obtained was 649834 Au.
The chromatogram of leaves (Fig. 7 and depicted
in Table 4), yielded 10 peaks totally. Peak sixth of area
27727 Au and height 3316 Au corresponds to Colchicine.
But the area corresponds only to 0.78%. Thus it has only
low response to colchicine.

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

Fig. 5: Chromatogram of tuber sample

Fig. 6: Chromatogram of seed sample

Fig. 7: Chromatogram of leaves sample


COLCHICINE CONCENTRATION:
The concentration of Colchicine found in 1 ml of
seed, tubers and leaves sample was determined using
HPLC chromatogram [24]. Of the three samples tuber
sample have the highest concentration of colchicines
followed by seeds and very low concentration in leaves
(Table 5).

International Journal of Natural Products Research 2015; 5(3): 34-41

Table 1. Total Yield of Two Extraction Process


S.No

Plant Parts

1
2
3

Leaves
Tubers
Seeds

Final Yield of Extraction Process (mg)


Cold
0.21
0.43
0.39

Table 2. Peak details of tuber sample


S.No
Peak Name
1
Peak 1
2
Peak 2
3
Peak 3
4
Colchicine
5
Peak 5
Table 3 Peak details of seed sample
S.No
Peak Name
Peak 1
1
Peak 2
2

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

Table 4 Peak details of leaves sample


S.No
Peak Name
Peak 1
1
Peak 2
2
Peak 3
3
Peak 4
4
Peak 5
5
Colchine
6
7
Peak 7
8
Peak 8
9
Peak 9
10
Peak 10
Table 5 Colchicine Content
S.No

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

Table 6 Activity of plant extracts against Enterococcus faecalis


S.No

Plant Parts

1
2
3

Leaves
Seeds
Tubers

37

Control
6.7
7.1
8.3

Zone of inhibition (mm)


Concentration (l)
50
100
6.6
7.1
6.3
7.3
7.1
7.4

International Journal of Natural Products Research 2015; 5(3): 34-41

200
7.4
7.6
9.0

Table 7 Activity of plant extracts against Proteus mirablis


S.No

Plant Parts

1
2
3

Leaves
Seeds
Tubers

Control
5.7
7.4
7.9

Zone of inhibition (mm)


Concentration (l)
50
100
5.5
5.9
6.4
7.3
7.0
7.3

200
6.2
7.9
8.4

Zone of inhibition (mm)


Concentration (l)
50
100
3.3
3.9
4.7
5.1
5.2
5.5

200
4.1
5.5
8.1

Table 8 Activity of plant extracts against Klebsiella pneumonia


S.No

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

Figure 8 Activity of tuber extract against Enterococcus


faecalis
Figure 11 Activity against Enterococcus feacalis

Figure 9 Activity of seed extract against Enterococcus


faecalis

38

In case of Proteus mirablis, the inhibition zone formed


(Figs. 12,13,14 and 15) were lesser in diameter than
Enterococcus faecalis. This shows the resistance of this
strain. In this case also the tuber extracts shows greater
activity at the concentration of 200l (Table 7). 100l
concentration of seeds and tubers shows almost same
activity. But leaves extract shows lesser activity than seeds
and tuber extracts. In case of Klebsiella pneumonia, leaves
extract show a least activity when compared to all the three
(Table 8, Figs. 16,17,18,19). Till 100l tuber extract shows
a slight increase in activity but at 200l a drastic increase in
the size of inhibition zone occurs.

International Journal of Natural Products Research 2015; 5(3): 34-41

Figure 12 Activity of tuber extract against Proteus


Mirablis

Figure 16 Activity of tuber extract against Klebseilla


pneumonia

Figure 13 Activity of seeds extract against Proteus


mirablis

Fig 17 Activity of seed extract against Klebseilla


pneumonia

Figure 14 Activity of leaves extract against Proteus


mirablis

Fig 18 Activity of leaves extract against Klebseilla


pneumonia

Fig 19 Activity against Klebsiella pneumonia


Figure 15 Activity against proteusmirablis
International Journal of Natural Products Research 2015; 5(3): 34-41

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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Source of support: Nil; Conflict of interest: None declared

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International Journal of Natural Products Research 2015; 5(3): 34-41