POPULATION BIOLOGY/GENETICS
Evaluation of the Colchicine-like Activity of
Gloriosa superba–Extracted Fractions for Mosquito
(Diptera: Culicidae) Cytogenetic Study
W. CHOOCHOTE,1, 2 K. RONGSRIYAM,3 B. PITASAWAT,1 A. JITPAKDI,1
E. RATTANACHANPICHAI,1 A. JUNKUM,1 B. TUETUN,1 AND P. CHAIWONG1
J. Med. Entomol. 41(4): 672Ð676 (2004)
ABSTRACT Four fractions of Gloriosa superba L., i.e., hexane fraction, dichloromethane fraction 1,
dichloromethane fraction 2, and methanol fraction, were investigated for colchicine-like activity using
a mosquito cytogenetic assay. The results revealed that the latter three fractions yielded promisingly
high colchicine-like activity, whereas the hexane fraction yielded very low activity compared with 1%
colchicine in a 0.85% sodium chloride solution. The metaphase rates and average number of metaphase
chromosomes per positive brain ganglion (range) of Aedes aegypti L. larvae after incubation with
0.25Ð2% solutions of dichloromethane fraction 1, dichloromethane fraction 2, 0.5Ð2% solutions of
methanol fraction, and 1% colchicine solution were 90 Ð100% and 7 (2Ð19) to 22 (7Ð 47); 90 Ð100% and
4 (1Ð11) to 30 (4 Ð73); 95Ð100% and 11 (1Ð28) to 17 (2Ð 62); and 100% and 6 (2Ð11), respectively. The
temperature stability tests of the three promising fractions were performed by heating 0.5% working
solution at 121⬚C for 15 min and preparing 0.5% working solution from stock frozen at ⫺20⬚C for 10
mo. These fractions also yielded satisfactory outcomes of metaphase rates and an average number of
metaphase chromosomes per positive brain ganglia compared with 1% colchicine solution.
KEY WORDS Gloriosa superba, fractions, Aedes aegypti, cytogenetics, metaphase chromosomes
COLCHICINE SOLUTION HAS BEEN widely used at a concentration of 0.005Ð1% for metaphase chromosome
preparation in the cytogenetic study of eukaryotic
organisms, e.g., protozoans (Yuh et al. 1997), helminths (Petkeviciute 1996, Hirai et al. 2000), snails
(Petkeviciute et al. 1995), insects (Baimai 1977, Nunamarker et al. 1996), and plants (Vosa 1973, Marks
1976). Spindle formation or microtubule polymerization inhibits arresting mitosis at metaphase (Insel
1996, Haraguchi et al. 1997).
Colchicine is an alkaloid of Colchicum autumnale L.,
a plant that belongs to the Family Liliaceae, and within
this Family, there are at least 11 genera comprising 21
species found indigenously in Thailand (Smitinand
1980). Recently, Jitpakdi et al. (1999) screened seven
plant species of the Family Liliaceae, i.e., Waan haang
chorakhe (Aloe barbadensis Mill.), dried leaf; Nomai
farang (Asparagus officinalis L.), dried root; Prongfaa
(As. plumosus Bak.), dried root; Saamsip (As. racemosus Willd.), dried root; Prik (As. sprengeri Regel),
dried root; Dok mai cheen (Hemerocallis flava L.),
dried ßower; and Dong dueng (Gloriosa superba L.),
dried rhizome, for metaphase chromosome prepara1 Department of Parasitology, Faculty of Medicine, Chiang Mai
University, Chiang Mai 50200, Thailand.
2 E-mail: [email protected].
3 Department of Parasitology, Faculty of Medicine, Siriraj Hospital,
Mahidol University, Bangkok 10700, Thailand.
tion in adult mosquitoes using an inoculation technique. The results indicated that the Þltrate of 1Ð15%
solution of dried Gl. superba rhizome powder soaked
in 0.85% sodium chloride solution (NSS) could be
used instead of 1% colchicine in HankÕs balanced salt
solution (HBSS) for the metaphase chromosome
preparation of adult female and male Aedes aegypti L.,
Culex quinquefasciatus Say, Toxorhynchites splendens
(Wiedemann), and Anopheles vagus (Donitz). Subsequently, Choochote et al. (2001) afÞrmed that the
0.5Ð 8% ethanol-extracted Gl. superba solutions could
be used instead of 1% colchicine in HBSS for metaphase chromosome preparation of both fourth-stage
larvae and adult female and male Ae. aegypti. Additionally, the authors mentioned that it could be used
satisfactorily in the metaphase chromosome preparation of other mosquito genera and species [Ae. lineatopennis (Ludlow), Ae. togoi (Theobald), An. aconitus
(Donitz), An. peditaeniatus (Leicester), An. vagus, Tx.
splendens], trematode (Fasciola gigantica Cobbold),
and lymphocytic cultivation for the diagnosis of some
human genetic diseases. As an adjunct to previous
studies, we demonstrate herein the advantage of Gl.
superba–extracted fractions (dichloromethane fraction 1, dichloromethane fraction 2, and methanol fraction) for metaphase chromosome preparation in mosquitoes.
0022-2585/04/0672Ð0676$04.00/0 䉷 2004 Entomological Society of America
July 2004
CHOOCHOTE ET AL.: Gl. superba FOR MOSQUITO CYTOGENETIC STUDY
673
Table 1. Metaphase rates of fourth stage larval brain ganglia of Ae. aegypti after incubation with NSS, 1% colchicine solution, and
0.25–2% solutions of methanol-, hexane-, and dichloromethane-extracted Gl. superba fractions
Experimentsa
NSS (control)
1% Colchicine solution
Hexane fraction
0.25%
0.5%
1%
2%
Dichloromethane fraction 1
0.25%
0.5%
1%
2%
Dichloromethane fraction 2
0.25%
0.5%
1%
2%
Methanol fraction
0.25%
0.5%
1%
2%
Metaphase
rates (no.)
No. mosquitoes having metaphase
chromosome(s) of
Average no.
chromosomes per
positive mosquito
(range)
0
1Ð10
11Ð 40
⬎40
0 (0)
100 (20/20)
20
Ñ
Ñ
18
Ñ
2
Ñ
Ñ
0 (0)
6.75 ⫾ 3.26 (2Ð11)
25 (5/20)
40 (8/20)
50 (10/20)
35 (7/20)
15
12
10
13
5
8
10
7
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
1.80 ⫾ 0.84 (1Ð3)b
1.38 ⫾ 0.74 (1Ð3)b
2.00 ⫾ 1.49 (1Ð5)b
1 ⫾ 0 (1)b
100 (20/20)
100 (20/20)
100 (20/20)
90 (18/20)
Ñ
Ñ
Ñ
2
3
1
3
14
15
16
17
4
2
3
Ñ
Ñ
21.60 ⫾ 11.69 (6Ð46)b
22.85 ⫾ 12.49 (7Ð47)b
22.45 ⫾ 10.00 (8Ð38)b
7.61 ⫾ 5.54 (2Ð19) NS
100 (20/20)
100 (20/20)
90 (18/20)
95 (19/20)
Ñ
Ñ
2
1
1
3
16
18
16
14
2
1
3
3
Ñ
Ñ
28.25 ⫾ 14.99 (6Ð64)b
30.40 ⫾ 16.54 (4Ð73)b
6.83 ⫾ 7.68 (2Ð32) NS
4.16 ⫾ 2.52 (1Ð11)b
90 (18/20)
95 (19/20)
100 (20/20)
100 (20/20)
2
1
Ñ
Ñ
15
10
12
9
3
8
8
10
Ñ
1
Ñ
1
4.56 ⫾ 4.59 (1Ð16) NS
13.32 ⫾ 11.94 (1Ð45)b
11.30 ⫾ 9.67 (1Ð28)b
17.20 ⫾ 14.12 (2Ð62)b
The rates were expressed as at least one metaphase chromosome set per 2,000 cells examined per mosquito brain.
a
Twenty fourth stage larvae for each experiment.
b
P ⬍ 0.05 (t-test, two-sided); NS, P ⬎ 0.05.
Materials and Methods
Gloriosa superba Extracted Fraction. The rhizomes
of Gl. superba were purchased in Chiang Mai Province, and its voucher specimen (PARA-GL-002) was
deposited at the Department of Parasitology, Faculty
of Medicine, Chiang Mai University, Thailand. The
extraction was performed by macerating 1.5 kg of
dried powdered sample with 5 liters of hexane at room
temperature for 2 d. After Þltering through a Buchner
funnel by suction, and the hexane Þltrate was evaporated by rotary evaporator at 40⬚C and lyophilized to
yield a hexane fraction. The residue after extraction
with hexane was extracted further with 5 liters of
dichloromethane. The dichloromethane solution was
drained and acidiÞed to pH 2Ð3 with 5% HCl to partition between water and dichloromethane. The dichloromethane lower layer was separated, evaporated
at 40⬚C, and lyophilized. The acidulated solution was
basiÞed to pH 10 Ð12 with Na2CO3 at 0⬚C and reextracted with dichloromethane. Separately extracted
solution was evaporated at 40⬚C and lyophilized. The
aqueous layer was discarded. The material obtained
after dichloromethane extraction was extracted with 5
liters of methanol. The resulting solution was Þltered,
evaporated at 60⬚C, and lyophilized. Four fractions
isolated from Gl. superba were kept at ⫺20⬚C before
testing.
A working solution was prepared by diluting the Gl.
superbaÐ extracted fractions with NSS to 0.25Ð2% solutions. For temperature stability tests, the experiments were divided into two sets, i.e., the 0.5% working solution was heated at 121⬚C for 15 min using an
autoclave before use, and the stock of lyophilized
fractions was frozen at ⫺20⬚C for 10 mo before preparation of the 0.5% working solution.
Metaphase Chromosome Preparation. The techniques for chromosome preparations were performed
by following the method described by Choochote et
al. (2001). Brießy, the early fourth-stage larvae of Ae.
aegypti were incubated for 3 h in 0.25Ð2% solutions of
Gl. superbaÐ extracted fractions, 1% colchicine in NSS,
and NSS for experiments, positive control, and negative control, respectively. The larval brains were removed, Þxed, smeared, and stained with 10% Giemsa.
Slides were examined for metaphase rates, and the
number of metaphase chromosomes per 2,000 cells
were counted under a compound microscope using an
oil immersion objective.
Results
Details of metaphase rates and the average number
of metaphase chromosomes per positive larval brain
ganglia after incubation with NSS, 1% colchicine solution, and 0.25Ð2% solutions of methanol-, hexane-,
and dichloromethane-extracted Gl. superba fractions
are shown in Table 1. Pictures of metaphase chromosomes of fourth-stage larval brain ganglia taken after
treatment with 1% colchicine solution and 0.5% solution of methanol- and dichloromethane-extracted Gl.
superba fractions are shown in Fig. 1.
Observations on the slide preparations for brain
ganglia of fourth-stage larvae of Ae. aegypti after incubation with 0.25Ð2% solutions of all Gl. superba–
674
JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 41, no. 4
Fig. 1. Metaphase chromosomes of larval brain ganglia of Ae. aegypti after treatment with 1% colchicine solution or 0.5%
solutions of methanol- and dichloromethane-extracted Gl. superba fractions, stained with conventional Giemsa. (A) Colchicine treatment. (B) Methanol fraction treatment. (C) Dichloromethane fraction 1 treatment. (D) Dichloromethane
fraction 2 treatment.
extracted fractions indicated that 0.25Ð1% solutions of
dichloromethane fraction 1, 0.25Ð 0.5% solutions of
dichloromethane fraction 2, and 0.5Ð2% solutions of
methanol fraction could be used instead of 1% colchicine solution for metaphase chromosome preparation,
because they provided signiÞcantly higher (P ⬍ 0.05)
average number of metaphase chromosomes per positive larval brain ganglia (dichloromethane fraction
1Ð 0.25%: 21 [6 Ð 46], 0.5%: 22 [7Ð 47], 1%: 22 [8 Ð38];
dichloromethane fraction 2Ð 0.25%: 28 [6 Ð 64], 0.5%:
30 [4 Ð73]; methanol fraction 0.5%: 13 [1Ð 45], 1%: 11
[1Ð28], 2%: 17 [2Ð 62]) than 1% colchicine solution [6
(2Ð11)]. The 2% solution of dichloromethane fraction
1 and 1% solution of dichloromethane fraction 2 provided unsatisfactory outcomes of the average number
of metaphase chromosomes per positive larval brain
ganglia [dichloromethane fraction 1Ð2%: 7 (2Ð19); dichloromethane fraction 2 1%: 6 (2Ð32)], although they
had no statistically signiÞcant differences (P ⬎ 0.05)
from 1% colchicine solution [6 (2Ð11)]. Perhaps most
of the fourth-stage larval brain ganglia were degenerated after a 3-h period of incubation. The 0.25Ð2%
solutions of hexane fraction provided a low yield of
metaphase rates (0.25%, 15%; 0.50%, 12%; 1%, 10%; 2%,
13%) and average numbers of metaphase chromo-
somes per positive larval brain ganglia [0.25%, 1 (1Ð3);
0.5%, 1 (1Ð3); 1%, 2 (1Ð5); 2%, 1 (1)].
Details of metaphase rates and the average number
of metaphase chromosomes per positive larval brain
ganglia after incubation with NSS, 1% colchicine solution, and 0.5% solutions of methanol- and dichloromethane-extracted Gl. superba fractions heated at
121⬚C for 15 min and prepared from stock frozen at
⫺20⬚C for 10 mo are shown in Table 2. Investigation
of these slide preparations revealed that 0.5% solution
of all fractions provided a higher average number of
metaphase chromosomes per positive larval brain ganglia [heated: dichloromethane fraction 1, 26 (13Ð 41),
dichloromethane fraction 2, 36 (12Ð58), methanol
fraction, 7 (1Ð18); frozen: dichloromethane fraction 1,
32 (15Ð59), dichloromethane fraction 2, 28 (14 Ð 44),
methanol fraction, 6 (2Ð12)] than 1% colchicine solution [heated: 5 (1Ð12); frozen: 5 (1Ð13)] in all cases.
Statistical analysis of the average number of metaphase chromosomes per positive larval brain ganglia
among 0.5% solutions of all fractions and 1% colchicine
solution exhibited no signiÞcant differences (P ⬎
0.05) for only 0.5% solution of both heated and frozen
methanol fractions.
July 2004
CHOOCHOTE ET AL.: Gl. superba FOR MOSQUITO CYTOGENETIC STUDY
675
Table 2. Metaphase rates of fourth-stage larval brain ganglia of Ae. aegypti after incubation with NSS, 1% colchicine solution, and
0.5% solutions of methanol- and dichloromethane-extracted Gl. superba fractions heated at 121°C for 15 min and prepared from stock
frozen at ⴚ20°C for 10 mo
0
1Ð10
11Ð 40
⬎40
Average no.
chromosomes per
positive mosquito
(range)
10 (2/20)
90 (18/20)
100 (20/20)
100 (20/20)
90 (18/20)
18
Ñ
Ñ
Ñ
Ñ
2
18
Ñ
Ñ
18
Ñ
2
19
12
2
Ñ
Ñ
1
8
Ñ
1 ⫾ 0 (1)
5.05 ⫾ 3.20 (1Ð12)
26.70 ⫾ 8.88 (13Ð41)b
36.95 ⫾ 12.98 (12Ð58)b
7.10 ⫾ 4.25 (1Ð18) NS
0 (0/20)
100 (20/20)
100 (20/20)
100 (20/20)
100 (20/20)
20
Ñ
Ñ
Ñ
Ñ
Ñ
18
Ñ
Ñ
18
Ñ
2
16
18
2
Ñ
Ñ
4
2
Ñ
0 (0)
5.05 ⫾ 3.35 (1Ð13)
32.45 ⫾ 11.57 (15Ð59)b
28.05 ⫾ 8.56 (1Ð44)b
6.55 ⫾ 3.02 (2Ð12) NS
Metaphase
rates (no.)
Experimentsa
Heated
NSS (control)
1% colchicine solution
Dichloromethane fraction 1
Dichloromethane fraction 2
Methanol fraction
Frozen
NSS (control)
1% colchicine solution
Dichloromethane fraction 1
Dichloromethane fraction 2
Methanol fraction
No. mosquitoes having metaphase
chromosome(s) of
The rates were expressed as at least one metaphase chromosome set per 2,000 cells examined per mosquito brain.
Twenty fourth-stage larvae for each experiment.
P ⬍ 0.05 (t-test, two-sided); NS, P ⬎ 0.05.
a
b
Discussion
Metaphase chromosome preparation of eukaryotic
organisms is still one of the reliable, traditional tools
used to identify and/or differentiate species, sibling
species, subspecies, chromosomal aberration, etc. ScientiÞcally, the incubation of diagnostic tissues with a
proper concentration of colchicine solution will provide a high chance of obtaining the metaphase chromosomes, because spindle formation or microtubule
polymerization inhibits arresting mitosis at metaphase
(Insel 1996, Haraguchi et al. 1997).
Colchicine is an alkaloid of Colchicum autumnale. It
was isolated from colchicum in 1820 by Pelletier and
Caventou (Insel 1996). It has been used in the metaphase chromosome preparation of wide-range genera
and species of eukaryotic organisms for more than
three decades (Baimai 1969, Fitzgerald and Brehaut
1970, Vosa 1973). At present, the application of indigenous, natural products is one of the popular, alternative ways to replace the synthetic and/or commercial ones. Consequently, Jitpakdi et al. (1999) and
Choochote et al. (2001) have reported the usefulness
of dried Gl. superba rhizome for metaphase chromosome preparations in a wide range of mosquitoes. The
former used 1Ð15% Þltrate of dried powdered Gl. superba rhizome soaked in NSS, whereas the latter used
0.5Ð 8% ethanol-extracted Gl. superba solutions. In addition, the latter authors also mentioned that, by using
their technique, they realized considerable savings.
Our additional work has afÞrmed the use of 0.25Ð1%
solutions of dichloromethane fraction 1, 0.25Ð 0.5%
solutions of dichloromethane fraction 2, and 0.5Ð2%
solutions of methanol fraction of Gl. superbaÐ extracted fractions for metaphase chromosome preparations in Ae. aegypti.
The systematic and continuous studies that evaluated the colchicine-like activity of Gl. superba by Jitpakdi et al. (1999), Choochote et al. (2001), and our
current work have emphasized the beneÞts of Gl.
superba, a common decorative plant widely found in
tropical countries (Smitinand 1980) and used for
metaphase chromosome preparation of eukaryotic organisms. Additionally, the temperature stability of
working solutions of all extracted fractions by heating
working solutions at 121⬚C for 15 min and preparing
working solutions from stock frozen at ⫺20⬚C for several months are important supportive beneÞts. It is
advisable to Þrst determine the optimal concentration
of each of the above-mentioned Gl. superba solutions
suitable for each organism to be tested before routine
application.
Acknowledgments
We thank S. Wudhikarn, Dean of the Faculty of Medicine,
Chiang Mai University, for interest in this research, the Faculty Endowment Fund for Þnancial support, and the Faculty
of Medicine Endowment Fund for Research Publication for
Þnancial support to defray publication costs.
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