Journal of Research in Biology

An International Online Open Access
Original Research paper
Publication group

Exploration of newer substrate for fibrinolytic enzyme production by solid
state fermentation using Penicillium chrysogenum SGAD12
Journal of Research in Biology

Authors: ABSTRACT:
Gopinath SM,
Suneetha TB,
Ashwini patil GM. Rice chaff - a polished substance extracted from Oryza sativa L. cv.
Devamallige was used as a novel substrate for the production of the fibrinolytic
enzyme. This vital enzyme is used in thrombolytic therapy, as a clot buster. The
Institution: production was done by solid state fermentation of rice chaff by Penicillium
Department of chrysogenum SGAD12, locally isolated from vegetable markets. Of the 28 strains
Biotechnology, Acharya isolated and screened, Penicillium chrysogenum SGAD12 was found to give an
Institute of Technology, inhibition zone greater than 2 mm. Hence it was identified as the potential organism
Soldevanhalli, Bengaluru- showing maximum fibrinolytic activity under specified culture conditions. Activity
560090, Karnataka, India. optimization was done under the parameters: Time, Inoculum ratio, Moisture, particle
size. The fibrinolytic activity was favourably maximized at 104 hrs, 7% (v/v) inoculum
ratio, 35-45 % (v/w) moisture content and 500µm particle size.

Corresponding author:
Gopinath SM

Email:
Keywords:
gopinath@acharya.ac.in Rice chaff, Fibrinolytic enzyme, Solid state fermentation, Penicillium
chrysogenum SGAD12.

Web Address: Article Citation:
http://jresearchbiology.com/
Gopinath SM, Suneetha TB, Ashwini patil GM.
Documents/RA0023.pdf.
Exploration of newer substrate for fibrinolytic enzyme production by solid state
fermentation using Penicillium chrysogenum SGAD12.
Journal of research in Biology (2011) 4: 242-245

Dates:
Received: 09 May 2011 /Accepted: 13 May 2011 /Published: 03 Aug 2011

© Ficus Press.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

242-245 | JRB | 2011 | Vol 1 | No 4
Journal of Research in biology
Submit Your Manuscript
An International Open Access Online
Research Journal www.ficuspress.com www.jresearchbiology.com
Gopinath et al.,2011

INTRODUCTION extracts were obtained by filtering through filter
Now-a-days thrombolytic diseases are paper. For 1 g of dry substrate taken 2.8 ml of
responsible for significant incapacitation and filtrate was recovered.
morbidity. Fibrinolytic enzymes have apparent Assay method
significance in thrombolytic therapy in human Fibrin plate analysis method
being (Haber et al, 1989). Despite most of their The original fibrin plate method (Astrup and
wide spread use, the currently available fibrinolytic Mullertz, 1952) with slight modification was used
enzymes have a number of significant limitations for measurement of the fibrinolytic activity of the
(Collen and Lijnen, 1991). Therefore great test preparation along with streptokinase. Petri
attention has been directed towards a search of dishes containing 9 ml of 0.2% fibrinogen solution
thrombolytic agents of various orgins, particularly (pH 7.8) were placed on a horizontal glass plate. To
through microbial activity. each of these Petri dishes, 0.2 ml of plasminogen
Fibrinolytic enzymes occur in bacteria, (10 units) was also added and mixed well. Clotting
earthworm, and snake venom, from fermented was induced by the addition of 0.2 ml of thrombin
foods (Collen and Lejnin, 1993) (Sumi et al, 1987), solution (20 units). In order to speed up the clotting
but upto now there have been very few evidences process, the plates were incubated at 28 ˚C for 20
for production of fibrinolytic enzyme from fungi mins. Plates were prepared afresh every time.
( S. A. El-Aassar et al, 1990)( Sun Tao et al, 1997). Known quantities of the enzyme solution and
Solid state fermentation (SSF) process indicates standards were placed as small droplets on the
significant difference in comparision with surface of the fibrin clot. The plates were incubated
submerged state fermentation (SMF). Its main at 28 ˚C for 2 h and visually inspected for
advantage is that it is a simple technique, it utilizes liquefaction. The area of the digested fibrin was
less amount of water, and it has a low operating considered as a quantitative measure of the
cost and high productivity. Fungi have been widely fibrinolytic activity of the enzyme.
utilized by SSF in the production of enzyme
(Lonsane et al, 1992) (Pandey, 1992). This study RESULTS AND DISCUSSION
reports the production of fibrinolytic enzyme by Enzyme production profile
Penicillin chrysogenum SGAD12, locally isolated Time course
from vegetable markets and new substrate rice chaff Good fungal growth was supported by rice
extracted from Oryza sativa L. cv. Devamallige chaff. The spores germinated within 20 h, giving
collected from local regions of Karnataka, India. rise to mycelium formation, whose density was
seen to increase with time. Visual examination
MATERIALS AND METHODS showed that the substrate was fully impregnated
Penicillium chrysogenum SGAD12, a
fibrinolytic enzyme producer was isolated from soil 60
of vegetable market and was identified. It was
Firinolytic activity (IU/ml)

50
found to produce fibrinolytic enzyme on fibrin
plate. This potential trait was used for further 40
production on solid state fermentation. Culture was 30
maintained on Czepek Dox Agar at 4°C and sub 20
cultured fortnightly. The basic medium contained 10
rice chaff (variety: Devamallige) (20g), KH2PO4
0
(0.5g), MgSO4 .7H2 O (0.5g), MnSO 4 .7H2O
(0.001g), ZnSO4.7H2O (0.002g), FeSO4.7H2O -10
(0.0005g) and 9 ml water. After autoclaving at
0

16

32

48

64

80

96

2
11

121˚C for 30 minutes, the medium was adjusted to
a moisture content of 45% (v/w) and inoculum size Tim e(hours)
of 7% (v/v). It was then inoculated with spore
suspension of 106 spores/ml and incubated at 28˚C. Fig. 1. Time course of enzyme production by
Enzyme Extraction Penicillium chrysogenum SGAD12 on rice chaff
The fresh moldy pith in each flask was (variety: Devamallige). With fermentation temperature
soaked in distilled water and incubated in a rotary 28 ˚C, Inoculums size: 7% v/v. Moisture level: 45%,
shaker at 130 r.p.m at 28 ˚C for 1 hour. The Particle size: mixture of sizes
243 Journal of Research in Biology (2011) 4: 242-245
Gopinath et al.,2011

with mycelium in about 40 h while the

Fibrinolytic Activity(IU/ml)
uninnoculated control plate showed no detectable 70
change. During initial 24 h no fibrinolytic activity 60
was seen, thereafter the enzyme activity increased 50
reaching a maximum at 104 h. With further 40
30
incubation the enzyme activity decreased.
20
Effect of Inoculum Ratio: 10
While optimization for inoculum ratio it was 0
found that 7% to 14% (v/v) (based on the volume of -10
mineral solution) elicited the best enzyme activity, 20 22 24 28 30 34 38 42 45 50 54 58
with 7 %(v/v),as adopted for this experiment,
giving the maximum result (fig 2). Any inoculum Miosture Content(%v/w)
size beyond the optimal range showed lower
activity. An inference can be drawn that larger Fig. 3. Effect of moisture level on the productivity by
inoculum sizes containing more amount of water Penicillium chrysogenum SGAD12on Rice chaff
led to decrease in the enzyme activity. (variety: Devamallige). Fermentation time: 104 hours.
Temperature: 28 ˚C. Particle size: Mixture of all sizes.
Fibrinolytic activity(IU/ml)

70 (fig4). Through visual observation it was inferred
60 that larger particle size provided less surface area
50 hence productivity was less. Whereas significantly
40 smaller particles though gave large surface area but
30 the porosity was decreased to an extent that the
20 filamentous fungi could not reach deep inside to the
10 substrate particles leading to decrease in
0 production.
2

7

12

16

20

24

28

Inoculum ratio(%v/v) 80
activity(IU/ml)
Fibrinolytic

60
Fig. 2. Effect of inoculum size on production by
Penicillium chrysogenum SGAD12 on rice chaff. 40
(variety: Devamallige) Fermentation time: 104 hours.
20
Temperature: 28 ˚C. Particle size: Mixture of different
sizes. 0
Effect of moisture level: 75 100 150 500 1000 >1000
Water has a profound effect on productivity Particle size( µm )
and hence used in limited amount in solid state
fermentation (Lonsane et al, 1992). Moisture level
between 35% - 45% (v/w) (fig3) results in the Fig. 4. Effect of substrate particle size on production
maximum enzyme production. Any value beyond by Penicillium chrysogenum SGAD12. Fermentation
this was unable to give increase in production. A time: 104 hours, temperature: inoculum size-7%
conclusion can be drawn that lower moisture level (v/v), moisture content 45 %( v/w).
leads to dry culture, sparse growth and hence
reduced production. Higher moisture concentration CONCLUSION
also creates an unsuitable environment for solid The above work indicates that Rice chaff
mycelium growth and hyphal diffusion, leading to (variety: Devamallige) can be used as a potential
less production of enzyme. substrate for production of economically important
Effect of substrate particle size: fibrinolytic enzyme with Penicillium chrysogenum
The effect of specific surface area is of high SGAD12. This substrate is easily available and
importance in solid state fermentation. Maximum economically feasible. Penicillium chrysogenum
production was obtained at a particle size of 500µm SGAD12 was identified as the potential organism

Journal of Research in Biology (2011) 4: 242-245 244
Gopinath et al.,2011

showing maximum fibrinolytic activity under El-Aassar SA. 1990. Appl Microbiol Biotechnol
specified culture conditions. Time, Inoculum ratio, 33:26-30.
Moisture content, Particle size; were found to be
ideal parameters for the activity optimization. The Haber E, Quettermous T, Matsueda GR, Runge
fibrinolytic activity was favourably maximized at MS. 1989. Science 243:51-56.
104 hrs, 7% (v/v) inoculum ratio, 35-45 % (v/w)
moisture content and 500µm particle size. Lonsane BK, Castaneda GS, Raimbault M,
Roussos S, Gonzalez GV, Ghildyal NP,
ACKNOWLEDGEMENT Ramakrishna M and Krishnaiah MM. 1992.
The authors are thankful to Visveswaraya Proc. Biochem. 27:259-273.
Technological University, Belgaum for their
financial assistance; and Acharya Institute of Pandey A. (1992) Prac. Biochem.
Technology for all the facilities graciously extended
to carry out the project. Sumi H, Hamada H, Tsushima H, Mihara H and
Muraki H. 1987. Birkhäuser Verlag, CH-4010.
REFERENCES Experentia 43.
Astrup T and Müllertz S. 1952. Archs Biochem.
Biophys 40:346. Sun Tao Li Peng, Liu Beihui, Liu Deming and Li
Zouhu. 1997. Biotechnology Letters , Li Peng, Liu
Collen D and Lijnen HR. 1991. Blood- 78:3114- Beihui, Liu Deming and Li Zouhu . Vol 19(5):465-
3124. 467.

245 Journal of Research in Biology (2011) 4: 242-245
Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

Effects of protein and lipid content of three artificial foods on survival and growth of
common dentex during the on-growing phase (Dentex dentex Linneaus, 1758).
Journal of Research in Biology

Authors: ABSTRACT:
Ali Ait Ali,
Azeddine Abrehouch and The purpose of this work is to highlight the effects of three different foods
Kamal Chebbaki. containing protein and lipids on the survival and growth of common dentex during the
on-growing phase.
Common dentex fingerlings weighting 5-6g were grown for one year in
polyester tanks. Three diets were used (A 1, A2 and A3), respectively, had a protein
Institution:
BP.31. Centre spécialisé en content of 55%, 45%, 33% and lipid level of 10%, 15% 23%. At the end of the
aquaculture de M’diq. experiment, fishes have reached a weight of 310.20 ± 7.76, 406.24 ± 11.01, 230.20 ±
INRH. Morocco. 6.41g, respectively, for A1, A2 and A3. The respective survivals were of 60%, 80% and
88%. The specific growth rates were of 1.04, 1.13 and 1.04 while the respective food
conversion rates were of 1.45, 1.30 and 2.0. Diet containing 45% protein and 15% lipid
gave the highest specific growth rate and the lowest food conversion rate with an
intermediate survival of 80%.
Corresponding author: Results obtained during this phase are encouraged to undertake large-scale
Ali Ait Ali farming in sea cages, knowing that to transfer the fries into cages must have a weight
greater than 6g. This study showed that during one year common dentex reached
commercial size despite a thermal profile whose values have not reached the
optimum temperatures for the majority of sparidae.

Email:
a.aitali@yahoo.fr,
abraze1@yahoo.fr, Keywords:
chebbakikamal@yahoo.fr Common dentex, nutrition, protein, lipid, specific growth rate.

Web Address:
http://jresearchbiology.com/
Documents/RA0053.pdf.
Article Citation:
Ali Ait Ali, Azeddine Abrehouch and Kamal Chebbaki.
Effects of protein and lipid content of three artificial foods on survival and growth of
common dentex during the on-growing phase (Dentex dentex Linneaus, 1758).
Journal of research in Biology (2011) 4: 246-252.

Dates:
Received: 29 Jun 2011 /Accepted: 02 Jul 2011 /Published: 03 Aug 2011

© Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

246-252 | JRB | 2011 | Vol 1 | No 4
Journal of Research in biology
Submit Your Manuscript
An International Open Access Online
Research Journal www.ficuspublishers.com www.jresearchbiology.com
Ait Ali et al.,2011

INTRODUCTION C), salinity (36 - 38‰) and photoperiod conditions
The common Dentex (Dentex dentex) is a were natural. Light intensity was kept around 600
fish with high commercial value. It is considered as ( lx) during the whole experiment. Feeding
a potential candidate for aquaculture, (Conides et al,frequency was twice a day and Dentex’s fingerlings
1996, Conides and Nengas 1998, Rueda and were fed handly to satisfaction.
Martinez 2001). Captive breeding have been Survival, length and weight assessment
undertaken since twenty years ago; high growth Estimation of survival rate was based on
was observed during larval phase (Abellan et al, daily counting of dead fishes and final remaining
1997, Tulli et al, 1997, Koumoundourous et al, number of live fishes at the end of experience.
2004, Ait Ali et al, 2007), fingerlings stage and Common dentex’s fingerlings were measured
larger fishes (Efthimoiou et al, 1994, Company et individually for their total length on the following
al, 1999, Katavic et al, 2000, Koumoundourous et days (d1, d8, d15, d22, d35, d48, d69, d129, d156, d192, d219,
al, 2004, Ait Ali et al, 2008). The Dentex’s d262, d295, d370). They were anaesthetized using
fingerling feeding requirements have been studied phenoxy-2-éthanol (0.1 mL per liter seawater).
(Tibaldi et al, 1996, Cardenete et al, 1997a, b, c, Total length was first assessed using a gradual table
Company et al, 1999) using pellet’s diets for from the snout point until the longest beams point
juveniles weighing 10 - 30 g, but dietary lipid and of the caudal fin when this one is aligned with the
protein still needs improvement. Riera et al, 1993 main axis of the body. Then, individual body
have achieved experimental Dentex’s fingerlings weight was taken using an electronic balance.
grow-out in sea cage feeding on natural food and Fingerlings were thereafter put back in their
obtained adult fish weight of 830 g in 16 - 17 respective rearing tanks.
months, widely superior to growth of cultured Sea Experimental diets and their composition
bass and gilt-head bream. Preliminary studies have Three diets were experimented. A dry pellet
shown that common dentex have rapid growth (A1) which was locally manufactured, an imported
rates, both juveniles and larger fish (Riera et al, dry pellet (A2) basically conceived and used for sea
1993, 1995, Efthimiou et al, 1994, Company et al, bass, and a moist pellet (A3) locally prepared using
1999, Katavic et al, 2000). Nutrient requirements trash fish and fish meal (Table 1).
for growth of dentex’s juveniles (10–30 g initial Sampling and biochemical analysis
live weight) have been studied (Tibaldi et al, 1996, Biochemical analysis was made upon diets.
Cardenete et al, 1997a,b,c, Company et al, 1999) Diet samples were taken one time from locally (A1),
using pellet diets, but little information exists imported dry pellets (A2) and moist pellet (A3)
concerning optimum dietary protein and lipid levels feeding stocks. All samples were frozen before
for fingerlings weighing less than 10 g (Jover et al, freeze-drying.
1998). The aim of this study was to determine the Proteins, humidity and ashes were
optimum of protein and lipid level to have optimum determined using standard method (AOAC 2000).
growth. Total lipids were extracted and measured
gravimetrically according to using dichloromethane
MATERIEL AND METHODS instead of chloroform.
Frie’s culture Statistical analysis
Locally hatchery produced fingerlings of Total length and body weight were
common dentex have been used in this experiment. compared by one-way analysis of variance
They were reared during one year in six fibreglass (ANNOVA) followed by Newman-Keuls test. A
cylinder-conical tanks of volume 2m3. After being significant level of 5% was used for both tests
sorted and selected to constitute homogeneous (Sokal et al, 1981). Statistical study was done by
groups, 25 fishes were initially stocked in each tank STATITFC (ITFC 4.0, 1988); total lengths and
in such way to have equal biomass in the six tanks. weights of fingerlings were submitted to a
Initial average weights of these fingerlings were logarithmic transformation, while the survival rates
ranged between 5.85±0.2 and 6.55±0.2 g. Seawater are treated after an angular transformation.
flow-through system was used with a renewal rate
maintained around 4 - 5 times a day. Oxygenation RESULTS
was provided by two air diffusers and Dissolved Temperature and salinity of breading
Oxygen (DO) was over saturation during the entire Water temperature was included between
period of the experiment. Temperature (17.5 - 22° 17°C and 22°C; salinity was of 36-38 ppm (Fig. 1).
247 Journal of Research in Biology (2011) 4: 246-252
Ait Ali et al.,2011

Table1. Composition of the three experimental diets (A1 and A2 compositions are given following
manufacturers’ technical sheet). DM = dry matter; WM = wet matter.

Diet A1a A2b A3

Ingredients
Dry matter (%) 92 90 66
Fish meal, (%WM) --- --- 34
Fresh fish (%WM) --- --- 48
Sardine oil (%WM) --- --- 14
Premix vitc (%WM) --- --- 4
Crud protein (%DM) 55 45 ---
Crud lipid (%DM) 12 14 ---
Crud fiber (%DM) 1.5 1.2 ---
Ash (%DM) 11 9.8 ---
Carbohydrates(%DM) 14.5 --- ---
Vit A 5000 IU Kg-1 20000 IU Kg-1 ---
Vit D 1000 IU Kg-1 3000 IU Kg-1 ---
Vit E 180 mg Kg-1 250 mg Kg-1 ---
Vit C --- 350 mg Kg-1 ---

Analysed composition (%DM)
Crud protein 55 45 33
Crud lipid 10 15 23
Ash 11.0 9.60 8.50
Energie brute (MJ kg-1) 21.10 21.80 23.00
Prot/énergie (mg/Kcal) 26.06 20.63 14.34

a
Ingredients: fish products, seed oil products and by-product set, cereal seed products, vitamins and minerals,
antioxidants : ethoxyquin.
b
Ingredients: fish meal, soybean, horse bean, pea, fish oil, rapeseed, maize gluten, rapeseed oil, weat, vitamins
and minerals, antioxidants : ethoxyquin.
c
Premix (dose kg-1): Minerals, 75% ; Phosphor, 3 – 4% ; Calcium 25 – 30%, Vitamin A, 2.000000 IU ; Vitamin
D3, 400000 IU ; Thiamine B1, 500 mg ; Riboflavin B2, 1.000 mg; Calcium pantothenate B3, 7500 mg;
Pyridoxine B6, 500 mg; Vitamin B12, 1.5 mg; Tocopherol Acetate E, 2500 mg; Nicotinic Acid PP, 10000 mg;
Cholin (Chloride), 50000 mg.

Survival
Common dentex’s survival for the three
diets were of 60% for A1 diet, 80% for A2 diet and
88% for A3 diet. Mortality starts at d35 because of
flexibacteriosis disease (Fig. 2).
Growth
The initial weight of dentex’s fingerlings
was of 6g. A2 diet, containing 45% CP and 15% CL
gave a best growth results (final live weight and
SGR) followed by A1 and finally A3 which gave the
lowest growth (Table 2). In early, A1 gave a high
growth compared to A2 but from d200 A2 showed
superior growth (Fig.3). Probably it should use in
first stage a protein level of 55% and use after six
months a protein level of 45% during the on-
growing phase of common dentex.
Fig. 1. Temperature and salinity during the on-
growing period

Journal of Research in Biology (2011) 4: 246-252 248
Ait Ali et al.,2011

100 Specific growth rate (SGR) and food conversion
90 ratio were a quadratic function of protein level, of
80 the form SGR = - 0.3212 + 0.066 PL - 0.0007 PL2
(fig. 5); FCR = 8.875 - 0.3183 PL + 0.0033 PL2
Survival rate (%)

70
60 (fig. 6). The optimum protein level was 45%; lipid
50 A1 level had only a linear effect on specific growth rate
40 A2 and food conversion ratio.
30 A3
20 1.15

Specific Growth Rate (SGR)
10
0
1.1
Duration (Days)
Fig. 2. Common dentex’s survival. There is a
significant difference between survivals of the three 1.05 y = -0.000x 2 + 0.066x - 0.321
R² = 1
diets according to Neuwman Keuls test.

1
450
400 A1
30 40 50 60
350 Protein level (CP)
300 A2
Weight (g)

250 Fig. 5. Plot regression of specific growth rate (SGR)
200 to protein level
A3
150
100 2.2
Food Conversion Ratio (FCR)

50
0 2
0 100 200 300 400
Duration (Days) 1.8 y = 0.003x 2 - 0.318x + 8.875
R² = 1
Fig. 3. Weight growth of common dentex. Bars 1.6
indicates means±SD. There is significant difference
(P<0.01) according to Neuwman Keuls test (n=25). 1.4

1.2
Concerning weight-length relation, correlation of
Pearson was very significant (fig. 4) (n=336) 1
30 40 50 60

400 W(g) = 1E-05TL(mm) 3,0181 Protein level (CP)
R2 = 0,9355
350
Fig. 6. Plot regression of food conversion ratio (FCR)
Body Weight (g)

300 to protein level
250
200 DISCUSSION
150 During this work, the results in terms of
100
survival have been between 60% and 88%, these
results are similar to those obtained by Espinos et
50
al. (2003) using juvenile dentex initial weight of 2.5
0
g for six weeks. These results also are consistent
with those reported by Ait Ali et al. (2008) for fries
Total length (mm) have reached 40 g using two diets (47%, 10% and
45% and 21% protein and lipid) for ten weeks. The
survivals obtained still higher than those obtained
Fig.4. Weigh-length relation of common dentex. by Efthimiou et al.(1994) but lower than for the
249 Journal of Research in Biology (2011) 4: 246-252
Ait Ali et al.,2011

Table2. Biological parameters for the three used diets

Diet A1 A2 A3

Initial weight (g) 6.55±0.20 6.12±0.20 5.85±0.16
Final weight (g) 310.20±7.76 406.24±11.01 230.20±6.41
Survival 60% 80% 88%
FCR 1.45 1.30 2.0
SGR 1.04 1.13 1.04

same species by Company et al.(1999) but for differences between fish dentex (1.5 to 98g) fed
fishes weighing between 10-20 g. Koumoundouros with extruded feed containing 45% or 50% CP.
et al. (2004) worked on fish larger but survivals Differences in the results quoted by various authors
were lowest (On 9300 fishes transferred to sea regarding the optimum protein and lipid can be
cages, only 1346 have survived after a month of explained by the energy level and protein / energy
rearing). In comparison with the results obtained ratio. During this work, the optimum energy that
during this work. gave best results was obtained with food containing
The specific growth rate (SGR) are between 21.10 and 21.80 MJ kg-1 and these results
relatively low, the maximum SGR obtained during are similar to those cited by Espinos et al, 2003 and
this work (1.13) is lesser than the SGR obtained for Tibaldi et al, 1996. During this work, the CP/GE
the same species by Ait Ali et al, 2008 (1.7% 2.4% successful is 20.63 g MJ-1; lower values have
and 2.7% d-1) but to fish smaller, they are also yielded poor results. Espinos et al. (2003) reported
lower than those obtained by Company et al, 1999 that the values of CP/GE ratio lower than 21 g MJ -1
and Cardenete et al, 1997a. These results are gave lowest growth, the same remarks were cited
identical to those reported by Tibaldi et al, 1996. by Tibaldi et al, 1996 for values of CP/GE ratio
However, they are higher than those cited by lower than 23 g MJ-1. The protein and lipid content
Cardenete et al, 1997d. Some lower SGR were which gave best results during this work (45 CP/15
obtained in other species such as sea bass: 07-08% CL) were identical to the results obtained by
d-1, 0.4-0.5% d-1 (Dias et al, 1998; Métailler et al, Company et al, 1999 (46 CP/17 CL). Recent work
1981) and sea bream. More optimum temperature has confirmed that the best protein level for
for this species is not achieved and more experience optimum growth of dentex juveniles seems to be
in the fishes was large (in our case up to 400g) around 50% and 12% (Tomás et al, 2009).
more SGR is down accordingly.
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and final weight. Company et al. (1999) obtained dentex’s fingerlings (Dentex dentex, L). African
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Journal of Research in Biology (2011) 4: 246-252 252
Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

Studies on the insecticidal properties of Chromolaena odorata (Asteraceae)
against the life cycle of the mosquito, Aedes aegypti (Diptera: culicidae).
Journal of Research in Biology

Authors: ABSTRACT:
Rajmohan D and
Logankumar K.

Leaf extract of the species, Chromolaena odorata was evaluated for the egg
Institution:
hatchability, larvicidal and pupicidal activity of mosquito, Aedes aegypti under the
PG and Research
Department of Zoology, room temperature in the laboratory. Dosage value as expressed in ppm was 10 to 140
Kongunadu Arts and for Aedes aegypti. A relationship was observed between the plant extract doses and
Science College, percentage mortality. The percentage of egg hatchability, larval and pupal mortality
Coimbatore-641 029, were found to be increased with increase in the dosage. Based on the probit analysis,
Tamilnadu, India. the LC50 value of egg (99.15), I instar (42.24), IV instar (101.49) and pupae (121.57)
were hence assumed.

Corresponding author:
Rajmohan D

Email: Keywords:
rajmohandevadass@gmail.com Aedes aegypti, Chromolaena odorata, LC50.

Web Address: Article Citation:
http://jresearchbiology.com/ Rajmohan D and Logankumar K.
Documents/RA0065.pdf. Studies on the insecticidal properties of Chromolaena odorata (Asteraceae) against
the life cycle of the mosquito, Aedes aegypti (Diptera: culicidae).
Journal of research in Biology (2011) 4: 253-257

Dates:
Received: 20.Jul 2011 /Accepted: 25 Jul 2011 /Published:

© Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

253-257 | JRB | 2011 | Vol 1 | No 4
Journal of Research in biology
Submit Your Manuscript
An International Open Access Online
Research Journal www.ficuspublishers.com www.jresearchbiology.com
Rajmohan et al.,2011

INTRODUCTION required amount of water, different doses (ppm)
Vector control is a serious concern in were prepared.
developing countries like India due to lack of Eggs of Aedes aegypti were procured from
general awareness, development of resistance and the Research Laboratory of National Institute of
socio-economic reason. The role of mosquitoes is Communicable Diseases (NICD), Mettupalayam,
becoming increasingly important in the recent years Coimbatore and brought to the laboratory and
because of change in ecology caused by human cultured. Eggs, first and fourth larval instars and
intervention. pupae were harvested from the colony and were
Mosquitoes constitute a major public health placed in different concentrations of biocide.
menace as vectors of serious human diseases (EI Twenty individuals were used for each
Hag et al., 1999). Of the various mosquito spread concentration. Eggs, larval instars and pupae were
diseases, dengue fever transmitted by Aedes aegypti checked for mortality every 24 hours. In the case of
is dangerous and is spreading into dengue shock control only carrier solvent was added. Food was
syndrome have taken epidemic form and have been provided in all the test beakers. Each test was
reported from Tamil Nadu, West Bengal, Uttar replicated for five times.The effect of leaf
Pradesh, Gujarat and Delhi (Kebra et al., 1992). In Chromolaena odorata on the egg hatchability,
recent years, scientists try a variety of botanical mortality of first and fourth larval instars and pupal
derivatives to eradicate many harmful insect pests mortality of Aedes aegypti was studied. Following
including mosquitoes. Insecticidal activity of neem 24 hours were corrected for natural response by
has been reported. Vector control is facing a threat Abbott’s formula (Abbott, 1925) as follows:
due to the emergence of resistance to synthetic
insecticides. Insecticides of botanical origin may Corrected percentage kill =
Proportion of test mortality- Proportion of control mortality X 100
serve as suitable alternative biocontrol techniques 1- Proportion of control mortality
in the future. (Nandita Chowdhury et al., 2008).
Aedes are vectors for the pathogens of various Busvin (1971) suggested that the critical
diseases like dengue fever , dengue haemorrhagic doses of susceptibility can be estimated with
fever and yellow fever (Rajmohan and sufficient accuracy from a probit / log concentration
Ramaswamy, 2007). Many authors world wide graph. Based on the log concentration and the
started large screening activity for using extracts of probit mortality percentage values, regression
medicinal and herbaceous plants to control equation was obtained. Using the regression, a
mosquitoes (Halawa, 2001; Das et al., 2003; straight line was fitted. Fitting of regression line
Choochote et al., 2004; Logankumar et al., 2008). and homogeneity of population were also tested
The plant species, Chromolaena odorata is a employing chi-square (χ2) test. By graphical
widely distributed neotropical shrub introduced to interpolation, LC50 values of the leaf extract for 24
many parts of the tropics. It is commonly seen in hours of exposure of egg, first and fourth instar
wastelands and hedges on weed. larvae and pupae of Aedes aegypti and their fiducial
For the present study, this species was screened limits (95% upper fiducial limit and lower fiducial
against egg hatchability, larval and pupal mortality limit) were calculated.
of the mosquito Aedes aegypt. So as to control the
population of Aedes aegypt by an eco-friendly RESULTS AND DISCUSSION
approach. Mortality values of egg, larvae and pupae
treated with different concentrations (ranging from
MATERIALS AND METHODS 10ppm to 140 ppm) of the leaf extract of
Fresh leaves of Chromolaena odorata were Chromolaena odorata at the end of 24 hrs are given
collected from the plants growing in agricultural in Table 1-4 for egg, I instar, IV instar larvae and
lands. Leaves were washed, shade, air dried and pupae of Aedes aegypti. The LC50 values and their
ground in a mixture to form a fine powder. The 25g 95% upper and lower fiducial limits, and chi-square
of the powder was then used for extraction in value of the leaf extract of Chromolaena odorata
acetone in soxhlet apparatus. The extract was for 24h exposure of Aedes aegypti are given in
concentrated on water bath to evaporate the Table 5. Based on the probit analysis the 24 hr LC50
acetone. The filterate was considered as pure value of the leaf extract of Chromolaena odorata
material and redissolved in acetone to form for egg, I instar and IV instar larvae and pupae of
standard formulation. By further dilutions with
254 Journal of Research in Biology (2011) 4: 253-257
Rajmohan et al.,2011

Table 1. Effect of crude sample of Chromolaena odorata against the egg hatchability of Aedes aegypti.
Concentration (ppm)
No of eggs
70 80 90 100 110
exposed
h uh h uh h uh h uh h uh
20 17 3 14 6 11 9 6 14 3 17
20 16 4 12 8 10 10 7 13 2 18
20 17 3 11 9 10 10 5 15 3 17
20 18 2 13 7 11 9 4 16 1 19
20 15 5 11 9 10 10 5 15 4 16
2.6
Mean 16.6 3.4 12.2 7.8 10.4 9.6 5.4 14.6 17.4
1.1
SD 1.14 1.14 1.30 1.30 0.55 0.55 1.14 1.14 1.14
4
Mean % 83 17 61 39 52 48 27 73 87
13
h - hatched, un - unhatched.

Table 2. Effect of crude sample of Chromolaena odorata against the I Instar larvae of Aedes aegypti.
No of larvae Concentration (ppm)
exposed 10 20 30 40 50
Alive dead Alive dead Alive dead Alive dead Alive dead
20 19 1 17 3 12 8 11 9 2 18
20 18 2 15 5 11 9 10 10 4 16
20 19 1 16 4 12 8 10 10 3 17
20 16 4 17 3 13 7 12 8 4 16
20 17 3 15 5 11 9 10 10 5 15
Mean 17.8 2.2 16 4 11.8 8.2 10.6 9.4 6.4 13.6
SD 1.30 1.30 1.00 1.00 0.84 0.84 0.89 0.89 1.14 1.14
Mean % 89 11 80 20 59 41 53 47 32 68

Table 3. Effect of crude sample of Chromolaena odorata against the IV instar larvae of Aedes aegypti.
No of Concentration (ppm)
larvae
exposed 80 90 100 110 120
Alive dead Alive dead Alive dead Alive dead Alive dead
20 19 1 15 5 11 9 8 12 3 17
20 18 2 12 8 10 10 9 11 2 18
20 16 4 11 9 10 10 8 12 1 19
20 19 1 13 7 11 9 7 13 2 18
20 17 3 12 8 12 8 6 14 4 16
Mean 17.8 2.2 12.6 7.4 10.8 9.2 7.6 2.4 2.4 17.6
SD 1.30 1.30 1.52 1.52 0.84 0.84 1.14 1.14 1.14 1.14
Mean % 89 11 63 37 54 46 38 62 12 88
Aedes aegypti was found to be 99.15, 42.24, 101.49 Kalyana Sundaram, 2004; Sun et al., 2006) they are
and 121.57 respectively (Fig.1). These results are in more eco-friendly. The results of the present study,
agreeing with the earlier findings made by many indicate that the leaf extract of the weed species,
workers with botanicals for various properties (for Chromolaena odorata caused low percentage of
oviposition avoidance, larvicidal, Halawa, 2001; egg hatchability and high percentage of larval and
Saleh, 1995, adulticidal, Choochote et al., 2004 and pupal mortality. Hence the large biomass of C.
repellent activities, Choochote et al., 2004; Prakash odarata available in Southern India can be used by
et al., 2000). As the botanical insecticides for the pharmacological industries to obtain effective
including the extract of C. odarata are repellent to control mosquito population is an
biodegradable and harmless to the environment, ecofriendly manner.
pest – specific and relatively harmless to non-target
organisms (Su and Mulla 1998; Sivagnaname and

Journal of Research in Biology (2011) 4: 253-257 255
Rajmohan et al.,2011

Table 4. Effect of crude sample of Chromolaena odorata against the pupae of Aedes aegypti.
No of Concentration (ppm)
larvae
100 110 120 130 140
exposed
Alive dead Alive dead Alive dead Alive dead Alive dead
20 18 2 15 5 12 8 9 11 5 15
20 19 1 14 3 10 10 8 12 3 17
20 18 2 11 9 10 10 9 11 2 18
20 17 3 12 8 10 10 6 14 3 17
20 16 4 11 9 11 9 7 13 4 16
Mean 17.6 2.4 12.6 6.8 10.6 9.4 7.8 12.2 3.4 16.6
SD 1.14 1.14 1.82 2.68 0.89 0.89 1.30 1.30 1.14 1.14
Mean % 88 12 63 34 53 47 39 61 17 83

Table 5. 24 hours LC50 values (ppm) and their 95 % Fiducial (upper and lower) regression equation and Chi-square (c2)
values of the crude extract of Chromolaena odorata for the different developmental stages of Aedes aegypi.
95% Fiducial limit
LC50 (ppm)
Stages c2 X SD SE
(ppm)
Upper Lower
Egg 99.15 103.74 95.65 2.57 52.8 24.79 3.88
I instar 42.24 45.58 39.75 6.66 30.2 13.22 11.52
IV instar 101.49 105.38 107.61 4.58 48.8 25.63 5.25
Pupa 121.57 125.43 117.14 4.25 47.4 24.02 3.11

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256 Journal of Research in Biology (2011) 4: 253-257
Rajmohan et al.,2011

100

90
y = 1.74x - 103.8
Percentage mortality

80

70

60

50

40
`
30

20

10

0

0 10 20 30 40 50 60 70 80 90 100 110 120

Concentration

Egg I Instar

100 90

90 80 y = 1.69x - 155.4
80 y = 1.79x - 130.2

Percentage mortality
70
Percentage mortality

70
60
60
50
50
40
40 ` `
30
30
20
20
10
10
0
0
40 60 80 100 120 140 160
0 20 40 60 80 100 120 140
Concentration
Concentration

IV Instar Pupae
Fig 1. Regression line of log concentration of Chromolaena odorata crude extract vs. percent egg hatchability,
mortality of larvae and puape of Aedes aegypti.

Saleh EH. 1995. Effect of some botanical extracts different strains of Culex pipiens pallens. J. Med.
as potential insecticides for the control of some Entomol., 43:258-261.
mosquitoes in Egypt, 1995. (Ph.D., Dept. Entomol.
Fac. Sci. Cairo. Univ., Egypt.

Sivagnaname N and Kalyanasundaram M. 2004.
Laboratories evaluation of methanolic extract of
Atlantia monophylla (Family: Rutaceae) against
immature stages of mosquitoes and non-target
organisms. Mem. Inst. Oswaldo Cruz, 99:115-118.

Su T and Mulla MS. 1998. Ovicidal activity of
neem products (Azadirachtin) against Culex tarsalis
and Culex quinquefasciatus (Diptera: Culicidae) J.
Amer. Mosq. Contrl. Ass., 14:204-209.

Sun L, Dong H, Guo C. 2006. Larvicidal activity
of extracts of Ginkgo biloba exocarp for three

Journal of Research in Biology (2011) 4: 253-257 257
Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

Seasonal variations in physico-chemical characteristics of Ravivar Peth Lake
at Ambajogai District. Beed Marathwada Region, India.
Journal of Research in Biology

Authors: ABSTRACT:
Raut KS*, Shinde SE**,
Pathan TS*** and
Sonawane DL**.
The present study deals with the assessment of water quality of the Ravivar
Institution:
Peth Lake, Dist. Beed Marathwada Region, India. The physico-chemical characteristics
*Department of Zoology,
L.L.Deshmukh Mahila were studied and analyzed during January – December of the year 2004. Seasonal
College Parli -V. variations at three different sampling sites of the Ravivar Peth Lake were observed
**Department of Zoology, Conductivity, pH, Total Dissolved Solids (TDS), Total Suspended Solids (TSS), Nitrate,
Dr. Babasaheb Ambedkar Sulphate and Phosphate were studied at these studies. The results revealed that the
Marathwada University, condition of this lake in different seasons showed fluctuations in physico-chemical
Aurangabad - 431004, India. parameters and showed pollution status of this lake.
*** Department of Zoology
Kalikadevi Arts, Commerce
and Science College, Shirur
(KA) Dist. Beed (M.S.)
India.

Corresponding author: Keywords:
Raut KS Physico-chemical parameters, seasonal variations, Ravivar Peth Lake.

Email: Article Citation:
kakasahebraut@gmail.com. Raut KS, Shinde SE, Pathan TS and Sonawane DL.
Seasonal variations in physico-chemical characteristics of Ravivar Peth Lake at
Ambajogai Dist. Beed Marathwada Region, India.
Journal of research in Biology (2011) 4: 258-262

Web Address: Dates:
http://jresearchbiology.com/ Received: 13 Jul 2011 /Accepted: 21 Jul 2011 /Published: 16 Aug 2011
Documents/RA0060.pdf.

© Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

258-262 | JRB | 2011 | Vol 1 | No 4
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Raut et al.,2011

INTRODUCTION morning between 8 am to 11 am in the first week of
The life is linked with the quality of every month from January - December 2004. The
environment, hence the biological components of samples were collected in acid washed five liter
freshwater depends solely on physicochemical plastic container from a depth of 5-10 cms below
conditions. Analysis of physico-chemical the surface of water. The samples were analyzed
parameters of water is essential. The changes in the immediately in the laboratory
physico-chemical characteristics adversely affect The physico-chemical characteristics of the
the living things in an environment. These water Lake water like Conductivity. pH, Total Dissolved
bodies pose different problems, pollution being the Solids (TDS), Total Suspended Solids (TSS),
main and it has been rendered unfit for use and nitrate, sulphate and phosphate were determined in
toxic for flora and fauna of the lake. Water bodies monthly variation according to standard methods
situated in the urban areas are under the pressure of (APHA, 2005 and Trivedy and Goel, 1984).
various human activities such as cloth washing,
bathing, and dying of cloths. RESULTS AND DISCUSSION
The quality of surface water including lakes The monthly physico-chemical parameters
and rivers depends on it’s their physical, chemical data of Ravivar Peth Lake at Ambajogai Dist. Beed
and biological properties. The physiochemical Marathwada Region India have been presented
properties give limited picture of water quality at were given below.
particular point of view, while the living organisms PHYSICO-CHEMICAL CHARACTERISTIC
act as continuous monitors of water quality over a Conductivity
period of time. Water quality analysis is important The conductivity is a numerical expression
to preserve and protect the natural ecosystem. of its ability to carry on electric current, which in
Analysis of physico-chemical parameters of water ionic strength as conductivity is a measure of total
is essential, to assess the quality of water for the ions. The ionic strength of a sample depends on
best usage like irrigation, drinking, bathing, fishing, ionization of solutes and other substances dissolved
industrial processing and so on. Water quality deals in it.
with the physical, chemical and biological The electric conductivity ranged from 897.6
characteristics in relation to all other hydrological to 493.9 µmhos/cm. Electric conductivity was
properties. (Shinde et al, 2010). maximum during summer 1680.43±380.80 µmhos/
Keeping this view in mind present study has cm and minimum during winter 1072.62±104.49
been undertaken to assess seasonal mean values and µmhos/cm. The overall mean was 1446.07±489.42
seasonal standard deviation of different parameters µmhos/cm (Table 1). Electric conductivity an
in Ravivar Peth Lake Dam. increases with increase in total dissolve solids.
In India some hydrobiological work on The seasonal variation in the conductivity may be
historic water bodies have been done (Dhere and due to the increase in concentration of salts because
Gaikwad, 2006; Sharma et al, 2007; Pejaver and of discharge of domestic effluents and organic
Gurav, 2008; Ingole et al, 2009; and Shinde et al, matter from the nearby residential areas into the
2010). This essential resource is increasingly scarce Lake. High level of conductivity reflects the
in many parts of the world due to severe pollution status as well as tropic levels of the
impairment of water quality. The increasing aquatic body (Anitha, 2002). In the present
anthropogenic influences in recent years in and investigation, the maximum conductivity was
around aquatic systems and their catchments areas recorded in summer season and minimum during
have contributed to a large extent to deterioration of winter. Chandrashekhar and Kodarkar, (1996)
water quality and declining of water bodies leading reported similar observations in Saroornagar Lake,
to their accelerated eutrophication. Hyderabad.
PH
MATERIALS AND METHODS PH values are expressed as the negative
The water samples for physico-chemical logarithm of hydrogen ion concentration. For acidic
analysis were collected from Ravivar Peth Lake, water, pH varies for 0 to 7 and for alkaline water
geographical coordination 18º 45’ N and 76º 10’ E pH varies from 7-14. It determines the solubility of
Aurangabad, (M.S) India, at 3 different sites viz., chemical nature of most of substances. Medium
Station A (Gaothana Site), Station B (Domestic values are favorable for biological production.
Site) and station C (Temple Site) in the early The pH values ranged from 6.82 to 9.5. The
259 Journal of Research in Biology (2011) 4: 258-262
Raut et al.,2011

maximum pH was recorded in summer 8.54±0.61 In the present investigation the higher values
and minimum was in winter 7.51±0.45 with slight of TDS in monsoon may be due to surface runoff,
increase in monsoon 8.05±0.38. The overall mean precipitation and decaying matter from catchments
was 8.03±0.64 (Table 1). area. Salve and Hiware, (2006) reported seasonal
In the present investigation, the maximum analysis and stated that low total dissolved solids
pH was recorded in summer may be due to recorded in winter season while maximum value in
decreased volume of water by evaporation and monsoon due to addition of solids from surface run
monsoon and minimum in winter season may be off in Wanparakalpa reservoir, Nagapur near Parali
due to short day length and decrease in Vaijanath Dist. Beed, Maharashtra.
photosynthetic activity. Salve and Hiware, (2006) Total Suspended Solids (TSS)
reported the maximum pH in summer and minimum Total suspended solids are the cause of
in winter with slight increase in monsoon. suspended particles into the water body influences
Yeole and Patil, (2005) reported the pH turbidity and transparency.
values in the range of 7.0 to 9.5 in Yedashi Lake. Total suspended solids were ranged from
Bai, (1989) recorded the pH of polluted water in the 35.1 to 182.3 mg/l. Total suspended solids were
range of 8.0 to 9.0. Pawar and Pulle, (2005) maximum during monsoon 146.3±20.87 mg/l and
observed the pH in range of 7.0 to 8.7 and stated were minimum during winter 70.01±22.23 mg/l.
that the pH of water is important for the biotic The overall mean Total suspended solids were
communities because most of the plant and animal 99.79±40.08 mg/l (Table 1).
species can survive in narrow range of pH from In the present investigation, the high values
slightly acidic to slightly alkaline condition. of total suspended solids during monsoon may be
Total Dissolved Solids (TDS) due to siltation, deterioration and heavy
These are composed of inorganic salts like precipitation. Khabade et al, (2002) recorded
calcium, magnesium, potassium, sodium, maximum TDS during monsoon and minimum
bicarbonates, chlorides, sulfates and some heavy during winter and summer at Lodhe water reservoir,
metal compounds; besides organic matter in small Tasgaon. Khanna and Bhutiani, (2003) reported
quantity also contributes the amount of total maximum TDS in monsoon, moderate in summer
dissolved solids in water. and minimum in winter, which supports the
Total dissolve solids were ranged for 493.4 findings of present observations under study.
to 942.3 mg/l. Total Dissolve Solids were Nitrate
maximum during monsoon 840.3±77.55 mg/l and Nitrogen is less soluble in water than
were minimum during winter 564.93±59.10 mg/l. oxygen. But as it constitutes 78% of atmosphere, it
The overall mean Total Dissolve Solids were still accounts for 65% of the dissolved gases at
700.86±128.31 mg/l (Table 1). equilibrium. Nitrogen is important as it is a
necessary element in the structure of protein,
Table-1. Seasonal variations in physico-chemical parameters of Ravivar Peth Lake, Dist. Beed Marathwada
Region, India. (During January - December 2004).

Parameter Range Summer Monsoon Winter Average
Conductivity
897.6 to 493.9 1680.43±380.80 1585.18±610.8 1072.62±104.49 1446.07±489.42
(µmhos /cm)
pH 6.82 to 9.5 8.54±0.61 8.05±0.38 7.51±0.45 8.03±0.64
Total dissolve solids
493.4 to 942.3 697.35±38.71 840.3±77.55 564.93±59.10 700.86±128.31
(mg/l)
Total suspended
35.1 to 182.3 83.04±23.40 146.3±20.87 70.01±22.23 99.79±40.08
solids (mg/l)
Nitrate (mg/l) 8.3 to 21.9 15.28±4.60 12.17±4.50 18.73±3.31 15.39±4.88

Sulphate (mg/l) 20 to 41.71 29.17±7.72 36.56±3.22 30.11±4.51 31.95±6.28

Phosphate (mg/l) 0.23 to 0.887 0.43±0.16 0.77±0.08 0.38±0.13 0.53±0.21

Journal of Research in Biology (2011) 4: 258-262 260
Raut et al.,2011

chlorophyll, RNA and DNA etc. It is essentially there is an entry of detergents in the water body and
required by all living organisms, being a necessary less water quantity and during summer season the
element of biochemical substances. relatively low level of Phosphate have been
The nitrate ranged from 8.3 to 21.9 mg/l. reported which may be attributed to abundance of
Nitrate values were maximum during winter Phytoplankton’s.
18.73±3.31 mg/l and minimum during monsoon Saikh and Yeragi, (2004) have reported that,
12.17±4.50mg/l. The overall mean was 15.39±4.88 increase in phosphate is a result sewage
mg/l (Table 1). contamination record. Urban water bodies subjected
Johnson and Kauser, (2004) stated that to pollution from domestic sewage exhibit high
nitrate in piped municipal water remained constant levels of phosphate and show all the signs of
in summer and monsoon 0.2 mg/l. but increased in eutrophication Adarsh et al, (2006).
winter to 0.9 mg/l. High values of nitrates in winter
may be due to high rain fall observed during winter CONCLUSION
season 665 mm in October 2004 and 902 mm in The present study show detailed physico-
October 2004 and 2005. The increased Nitrate value chemical characteristics and quality of water in
due to Lake Runoff, land drainage and input of Ravivar Peth Lake, Dist. Beed Marathwada Region,
fertilizers from adjacent and agricultural fields and India.
oxidation of ammonia similar results have been The summer, monsoon and winter seasons
reported by Anbazhagan, (1988). shows different seasonal fluctuations in various
Sulphate physico-chemical parameters.
The most abundant form of sulphur is anion Data indicated that among the physico-
sulphate (So4–2). Sulphate is ecologically important chemical parameters of the lake water, the level of
for the growth of plants and its short supply may Conductivity, pH, TDS, TSS, nitrate and sulphate
inhibit the development of planktons. Sulphur is were above the permissible limits prescribed by ISI
also important in protein metabolism. and WHO for drinking water. So the lake water is
Sulphate ranged from 20 to 41.71 mg/l. The not suitable for drinking purpose.
higher values of sulphate were recorded in monsoon It is being polluted by different sources like,
36.56±3.22 mg/l and lower in summer 29.17±7.72 municipal wastes, agricultural wastes, domestic
mg/l. The overall mean was 31.95±6.28 mg/l (Table wastes etc.
1). The continuous biomonitering of Ravivar
In the present investigation, the Sulphate Peth Lake is needed as it affects the flora and fauna
values were maximum during monsoon and of the lake.
minimum during winter. Maximum Sulphate It can be concluded that physico-chemical
concentration during monsoon may be due to the parameters are important to determine the quality of
dilution and utilization of Sulphate by aquatic aquatic environment.
plants. However, the low Sulphate concentration
was noted during winter may be due to ACKNOWLEDGMENT
biodegradation and low water level. Similar results The authors are thankful to Head, Dept of
have been reported by Reddy et al, (2009); Zoology, Dr. Babasaheb Ambedkar Marathwada
Telkhade et al, (2008). University, Aurangabad India for providing
Phosphate laboratory and library Facilities and also thankful to
In water bodies phosphate occurs both in its principal, Dr. R. J. Parlikar madam for kind
inorganic and organic forms as organic assistance and all society members of Late L. D.
phosphorous and orthophosphate, Phosphate plays a Shikshan Prasarak Mandal; Parli Vaijnath Dist.
dynamic role by acting as the limiting nutrient Beed.
presence of phosphate in water and waste water
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Journal of Research in Biology (2011) 4: 258-262 262
Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

Impact of elevation, latitude and longitude on fish diversity in
Godavari River
Journal of Research in Biology

Authors: ABSTRACT:
Sandeep R. Rathod and
Gulab D. Khedkar
India is very rich in biodiversity, India supports about 10% of the world’s
biological diversity, with just 2% of world land area. Therefore India is the seventh
richest biodiversity in the world. The India has Himalaya Mountain, Eastern Ghat,
Institution:
Aquaculture Research Western Ghats Indo-gangetic plain, Deccan plateau, desert, coasts are all present in it.
laboratory, In India many type of river also present, one of the river Godavari River is the second
Deparment of Zoology, largest river in India. Fish biodiversity studies were undertaken during January-2008 to
Dr. Babasaheb Ambedkar Decemeber-2008 to census and commercially important fishes in the Godavari River.
Marthwada The present paper deals with the variety and abundance of fresh water fishes in
University, Aurangabad. Godavari River with reference to the elevation, latitude and longitude. Two sampling
(Maharashtra) India. points were selected with different elevation latitude and longitude. The Results of
the present investigation reveal the occurrence of 53 fish species belonging to 9
orders, 21 families and 37 genera.
Among the collected species two sampling site species composition were so
Corresponding author: different. Species composition in Gangapur dam is lower than the Rajahmundry dam.
Sandeep R. Rathod In species composition order Cypriniformes was most dominant to both sampling site.

Email: Keywords:
rathod.sr@gmail.com Elevation, latitude and longitude Fish biodiversity, Percent wise compositions,
Godavari River.

Web Address: Article Citation:
http://jresearchbiology.com/ Sandeep R. Rathod
Documents/RA0075.pdf. Impact of elevation, latitude and longitude on fish diversity in Godavari River
Journal of research in Biology (2011) 4: 269-275

Dates:
Received: 07 Aug 2011 /Accepted: 11 Aug 2011 /Published: 16 Aug 2011

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Rathod et al.,2011

INTRODUCTION and Jhingran, 1991; K.C. Jayaram, 1999; Sakhare
Fishes form one of the most important and Joshi, 2002; Dutta et al., 2003; Sakhare and
groups of vertebrates, influencing there life in Joshi, 2004; Yadav, 2005; Battul et al., 2007;
various ways, Such as economically and Ashashree et al., 2008.)
nutritionally it very important. Fishes provide Some of them are on the specific ponds,
several by-products to us. Fishes have formed an lakes and the water laying areas fish biodiversity.
important item of human diet from time No more documentation and fishes were recorded
immemorial and are primarily caught for this from the major Indian River ecosystem. We have
purpose. A large amount of phosphorous and other studied on the Contribution and composition of
elements are also present in it. fishes in Godavari River with reference to its
Biodiversity is the degree of variation of life elevation, longitude and latitude.
forms within a given ecosystem, biodiversity is
essential for stabilization of ecosystem protection of MATERIAL AND METHODS
overall environmental quality for understanding Fishes were collected from Godavari River;
intrinsic worth of all species on the earth (Ehrlich et Gangapur Dam Dist. Nashik (M.S) and
al., 1991).The rapid environment change on earth Rajahmundry Dam Dist. Rajahmundry (A.P) India.
therefore has its impact on the biodiversity, that’s We have sampled in three seasons (Monsoon,
why the united nation declares the year 2010 as the winter and summer), Sampling was done with the
international year of biodiversity. India consists of help of local fishermen using different type of nets
six drainage system. These are Indus river system, namely gill nets, cast nets, dragnets, jhel net(Arial
upland cold water bodies, Gangetic river system, net) and tradition net. In rainy session the gill net
Bramhaputra river system, east flowing river varies in mesh sizes. After collection specimen
system, and west flowing system. (Pandey et al, photographs were taken immediately with the help
2007). In this river ecosystem fishes play a very of digital camera.
important role to maintain ecosystem. Fish Fishes were brought to laboratory and
biodiversity of river essentially represents the fish preserved in 10% formalin. Small fishes specimens
faunal diversity and their abundance. River were directly preserve in 10% solution and big
conserves a rich variety of fish species which specimens were injected with 10% formalin in the
support to the commercial fisheries. abdomen of the fish. Keep these specimens in
The Godavari River originates in the separate jars according to the size of species.
Western Ghats near Tribmkeshwar in Nishik The Meristic and morphometric characters
district of Maharashtra. It flows in the east across were measured and identified up to the species
the Deccan plateau through the Maharashtra and level, with the help of standard keys and books
end Bay of Bengal at Yanum, Andhra Pradesh. It (Day, 1978; Jayaram, 1999 and Talwar and
lengths about 1465 km. It is the second largest river Jhingran 1991).
in India. On this river many major irrigation Study area:
projects were constructed. We have selected two Site 1: The Gangapur dam is located near
major dams on Godavari River such as Gangapur Gangawadi village and it is 10 km from the Nashik
dam and Rajahmundry dam for our analysis. city. This earthen dam was constructed in 1954.
Present investigation was undertaken to study the Total length of dam is 3810 m., and maximum
fish diversity of Godavari River. height is 36.56 m. From origin of Godavari river
Indian region fishes are about 2500 species, distance is 28 kms. Distance between Gangapur
freshwater fishes 930 species and remaining 1570 dam to Rajmuhandri dam is 1352kms. (Longitude:
are marine(K.C Jayaram 1999). Present freshwater 74007’E, Latitude: 20000’N and Elevation above
fishes are recorded 801 (Fish base 2004). sea level: 1992ft.).
The ancient Indians classified fish, based on Site 2: Rajahmundri dam is also known as
the shape size and their knowledge from keen Dowleshwaram Dam. It’s just 10 km from the
observation are remarkable as seen from Kautilya’s rajahmundri city. It was built by Sir Alfred cotton
Arthashasthra (Ca300 B.C.), King Someswara’s and constructed 1848-52. (Longitude: 81045’E,
Manasallosa (1127A.D.) In the field of ichthyology Latitude: 17001’N and Elevation above sea level:
there is a valuable contribution by many workers, 74ft.)
(Hamilton Buchanan, 1822; McClelland,1839;
Sykes 1839; T.C. Jerdon 1849; Day 1878; Talwar
270 Journal of Research in Biology (2011) 4: 269-275
Rathod et al.,2011

RESULT AND DISCUSSIONS fraseri, Rasbora Daniconias, Thynicthys
During the study period different fish sandkhol, Osteobrama vigorsii, Nemachilus
varieties can be observed in the Godavari River, Moreh, Nemachilus botia were found less
India. Fishes belonging to nine orders and twenty abundant. Chela laubuca, Puntius dorsalis,
one families were collected during the study period. Puntius filamentosus, were found rare abundant.
Many collected fishes were having economic, Followed by order Perciformes were six species in
medicinal and cultural, ornamental importance and the assemblage composition in which Chanda
sold after collection in the local fish market. In the nama was found most abundant form.
present fish biodiversity study 53 species of 37 Glossogobius giuris giuris were found abundant
different genera 21 families and 9 orders were form. Channa marulius, channa punctatus,
recorded from the Godavari River during January Channa oriantalis, and Parambassis ranga were
2008- December 2009. The members of Order found less abundant. Followed by order
Cypriniformes were dominated with 40 species Siluriformes were four species in the assemblage
followed by Perciformes with 7 species, composition in which Aorichthys aor were found
Siluriformes with 6 species, Beloniformes with five abundant form. Mystus cavasius were found less
species each, Osteoglossiformes with 2 species and abundant. Wallago attu, Heteropneustes fossilis
Synbranchiformes with 1 species. were found rare abundant. Followed by order
In Gangapur Dam there found five orders Synbranchiformes were one species in the
representing by 26 fish species, order assemblage composition in which Macrognathus
Cypriniformes was dominant group with 14 species pancalus were found abundant. Followed by order
in the assemblage composition in which Puntius Osteoglossiformes were one species in the
ticto, Salmostoma navacula, were found most assemblage composition in which Notopterus
abundant. Catla catla, Cyprinus carpio carpio notopterus were found abundant. (Table 1).
Garra mullya were found abundant form. Puntius

Table 1: - The fish biodiversity of Godavari River in Gangapur dam during January 2008 – December 2008.
Family Species Native/Introduce Species
abundance
1 Notopteridae Notopterus notopterus Native ++
2 Cyprinidae Puntius ticto Native +++
3 Cyprinidae Puntius fraseri Native +
4 Cyprinidae Puntius filamentosus Native -
5 Cyprinidae Puntius dorsalis Native -
6 Cyprinidae Salmostoma navacula Native +++
7 Cyprinidae Chela laubuca Native -
8 Cyprinidae Rasbora Daniconias Native +
9 Cyprinidae Thynicthys sandkhol Native +
10 Cyprinidae Osteobrama vigorsii Native +
11 Cyprinidae Catla catla Native ++
12 Cyprinidae Cyprinus carpio carpio Introduced ++
13 Cyprinidae Garra mullya Native ++
14 Balitoridae Nemachilus Moreh Native +
15 Balitoridae Nemachilus botia Native +
16 Chanidiae Chanda nama Native +++
17 Chanidiae Parambassis ranga Native +
18 Gobiidae Glossogobius giuris giuris Native ++
19 Channidae Channa marulius Native +
20 Channidae channa punctatus Native +
21 Channidae Channa oriantalis Native +
22 Bagiridae Aoirichthys aor Native ++
23 Bagiridae Aoirichthys cavasius Native +
24 Siluridae Wallago attu Native -
25 Heteropneustidae Heteropneustes fossilis Native -
26 Mastacembelidae Macrognathus pancalus Native ++
Journal of Research in Biology (2011) 4: 269-275 271
Rathod et al.,2011

Table 2: - The fish biodiversity of Godavari River in Rajahmundri dam during January 2008 – December 2008.
Sr. no Family Species Native/Introduce Species
abundance
1 Notopteridae Notopterus notopterus Native +
2 Notopteridae Notopterus chitala Native +
3 Anguillidae Angulla bengulensis bengalenss Native -
4 Clupeidae Hilsa ilisha Native +
5 Cyprinidae Salmostoma navacula Native ++
6 Cyprinidae Cyprinus carpio carpio Native ++
7 Cyprinidae Osteobrama vigorsii Native +++
8 Cyprinidae osteobrama dayi Native -
9 Cyprinidae Thynnichthys sandkhol Native +
10 Cyprinidae Cirrhinus mirgala Native +
11 Cyprinidae Cirrhinus reba Native +
12 Cyprinidae Puntius Vittatus Native -
13 Cyprinidae Puntius ticto Native +
14 Cyprinidae Puntius fraseri Native +
15 Cyprinidae Puntius filamentosus Native -
16 Cyprinidae Catla catla Native ++
17 Cyprinidae Labeo rohita Native +
18 Cyprinidae Labeo calbasu Native ++
19 Cyprinidae Labeo arzia Native ++
20 Cobitidae Lepidocephalus guntea Native ++
21 Heteropneustidae Heteropneustes fossilis Native -
22 Bagiridae Mystus seenghala Native +
23 Bagiridae Mystus bleekeri Native ++
24 Bagiridae Mystus cavasius Native ++
25 Bagiridae Aorichthys aor Native +
26 Bagiridae Rita rita Native +
27 Siluridae Wallago attu Native ++
28 Siluridae Ompak bimaculatus Native +++
29 Pangasiidae Pangasius pangasius Native +
30 Sisoridae Bagarius bagarius Native ++
31 Schilbeidae Silonia children Native +
32 Nandidae nandus nandus Native -
33 Gobiidae Glossogobius giuris giuris Native ++
34 Chanidiae Chanda nama Native ++
35 Chanidiae Parambassis ranga Native ++
36 Channidae Channa marulius Native ++
37 Channidae channa striatus Native ++
38 Channidae channa punctatus Native ++
39 Channidae channa orentalis Native ++
40 Cichlidae Etroplus suratensis Native +
41 Cichlidae Oreochromis mossambica introduced +
42 Anabantidae Anabus testudineus Native -
43 Mastacembelidae Mastacembelus armatus Native +
44 Mastacembelidae Macrognathus pancalus Native +++
45 Mastacembelidae Macrognathus aculeateda Native +
46 Beonidae Xanthodon cansula Native ++
47 Mugilidae Rhinomugil corsula Native +

272 Journal of Research in Biology (2011) 4: 269-275
Rathod et al.,2011

In Rajahmundry Dam there found nine % followed by order Perciformes were
orders representing by 47 fish species, order constituting 22%, Siluriformes constituting 12 %,
Cypriniformes was dominant group with 16 species Osteoglossiformes constituting 8 %, and
in the assemblage composition in which Synbranchiformes constituting 3 % of the total fish
Osteobrama vigorsii were found most abundant. species. (Fig.1)
Lepidocephalus guntea, Labeo arzia, Labeo
calbasu, Catla catla, Cyprinus carpio carpio and 47 species were recorded and identified on the
Salmostoma navacula were found abundant form. Rajahmundry Dam. Among the order
Thynnichthys sandkhol, Cirrhinus mirgala, Cypriniformes were most dominant constituting 43
Cirrhinus reba, Puntius ticto, Puntius fraseri and % followed by order Siluriformes constituting 33
Labeo rohita were found less abundant. Puntius %, Clupeiformes were constituting 7%, Perciformes
filamentosus, Puntius Vittatus, osteobrama dayi was constituting 6%, Beloniformes constituting 4
were found rare abundant. Followed by order %, Synbranchiformes constituting 3 %,
Perciformes were 10 species in the assemblage Anguilliformes constituting 2%, Osteoglossiformes
composition in which Glossogobius giuris giuris, constituting 1 %, and Salmoniformes constituting 1
Chanda nama, Parambassis ranga, Channa % of the total fish species. (Fig.2)
marulius, channa striatus, channa punctatus,
channa orentalis was found abundant. Etroplus When sampling throughout the year, we observed
suratensis, Oreochromis mossambica were found that so many different fish species were being
less abundant. Anabus testudineus was found to be caught in monsoon compared to winter and summer
rare abundant. Next order Siluriformes were 12 seasons. Local Fisherman also undergoes intensive
species in the assemblage composition in which fishing in monsoon seasons as compare to other
Ompak bimaculatus were found most abundant seasons.
form. Mystus bleekeri, Mystus cavasius, Wallago Mahapatra (2003) recorded abundance of catfishes
attu, Bagarius bagarius were found abundant. in Hirakund reservoir. Total of 43 species were
Mystus seenghala Aorichthys aor, Rita rita, present in which 18 were commercially important.
Pangasius pangasius, Silonia children were found Sakhare and Joshi (2003) reported 34 species of
less abundant. Nandus nandus, Heteropneustes fishes in reservoirs of Parbhani Dist. of
fossilis were found rare abundant. Followed by Maharashtra (India). Pisca et al., (2000) reported a
order Synbranchiformes were three species in the genera fish belonging to four orders and 28 species
assemblage composition in which Macrognathus from Ibrahimbagh reservoir of Hyderabad. Sugunan
pancalus were found most abundant. and Yadava, (1992) mentioned 40 fish species from
Mastacembelus armatus Macrognathus Hirakhud reservoir of Orissa forming the
aculeateda were found less abundant. Followed by commercial fishery.
order Osteoglossiformes were two species in the
assemblage composition in which Notopterus
notopterus Notopterus chitala found less abundant.
Followed by order Anguilliformes were one species
in the assemblage composition in which Angulla
bengulensis bengalenss were found rare abundant.
Followed by order Clupeiformes were one species
in the assemblage composition in which Hilsa
ilisha were found rare abundant. Followed by order
Beloniformes were one species in the assemblage
composition in which Xanthodon cansula were
found abundant. Next order Salmoniformes were
one species in the assemblage composition in which
Rhinomugil corsula were found less abundant.
(Table 2)
26 species were recorded and identified on
the Gangapur Dam. Among the order Fig.1. Pie chart image showing Family wise fish
Cypriniformes were most dominant constituting 55 composition, Gangapur Dam (Godavari River) Dist.
Nashik(M.S), India.

Journal of Research in Biology (2011) 4: 269-275 273
Rathod et al.,2011

feature as well as sheep impact and elevation
gradient. Species distribution and abundance
pattern varying from high elevation areas with low
vegetation cover to low elevation areas with higher
vegetation cover.(R.C. Klinger et.al).
In present study the Gangapur Dam
(Longitude: 74007’E, Latitude: 20000’N and
Elevation above sea level: 1992ft.) elevation is high
were less species richness of about 26 species
belonging 10 family included in 5 orders were
recorded. In Rajahmundry Dam (Longitude:
81045’E, Latitude: 17001’N and Elevation above
sea level: 74ft.) elevation is low and species
richness is more it is about 47 species of 20 families
included in 9 orders. On concluding result
Gangapur Dam located at an elevation of about
Fig.2. Pie chart image showing Family wise fish 1992 ft. and species records were found to be26
composition, Rajahmundri dam (Godavari River) Dist. species and the Rajahmundry Dam located at an
Rajahmundri (A.P), India. elevation of about 74 ft. and species records were
found to be 47 species. Altitude as well as latitude
Species diversity is also depending on the is one of the factors which most strongly influence
altitudes because when the altitude increases the the distribution of species.
species richness decreases thus in low latitudes the
species richness increases. The gradient of species ACKNOWLEDGMENTS
richness is asymmetrical about the equator. The The authors are grateful to the Head, Dept of
latitudinal gradients of species richness may be Zoology, Dr. Babasaheb Ambedkar Marathwada
resulted from the energy available to the University, Aurangabad-431004 (M.S) India, and
ecosystems. At lower latitudes, there are higher Aquaculture research laboratory, Department of
amounts of energy available because of more solar Zoology Dr. Babasaheb Ambedkar Marathwada
radiation, more resources (for example, minerals University, Aurangabad-431004 (Maharashtra)
and water); as a result, even higher levels of species India for providing laboratory and library Facilities
richness can be allowed at lower latitudes. during the course of study.
However, there have been relevant studies showing
that species richness and primary productivity are REFERENCES
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Journal of Research in Biology (2011) 4: 269-275 275
Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

Effect of plant growth regulators on in vitro organogenesis in
cultivated tomato (Solanum lycopersicum L.).
Journal of Research in Biology

Authors: ABSTRACT:
Godishala Vikram1,2,
Kairamkonda An efficient and reproducible protocol for organogenesis from cotyledon
Madhusudhan2, explants in cultivated tomato (Solanum lycopersicum L.) cv S-22 is reported. The
Kagithoju Srikanth2,
cotyledon explants of 10-12 days old excised from in vitro grown seedlings were
Mangamoori
cultured on MS medium supplemented with 0.5-5.0 mg/L BAP as a sole growth
Laxminarasu 1 and
Nanna Rama Swamy 2. regulator and also in combination with 0.1 mg/L IAA (Indole-3-acetic acid). Highest
percentage of response for callus induction was recorded in cotyledon explants at 3.0
mg/L BAP where as multiple adventitious shoots were formed at 0.1 mg/L IAA + 2.5-
5.0 mg/L BAP containing medium. Shoots obtained were transferred on to MS
Institution: medium augmented with 0.2-1.0 mg/L GA3 + 3.5 mg/L BAP for shoot elongation. The
1. Center for Biotechnology, medium supplemented with 0.6 mg/L GA 3 in combination with 3.5 mg/L BAP showed
Jawaharlal Nehru the maximum percentage of enhancement of shoot elongation. For in vitro rooting,
Technological University, elongated micro-shoots were transferred onto MS medium supplemented with
Hyderabad, India. 0.5mg/L Indole-3-acetic acid (IAA) / Indole butyric acid (IBA) / Napthalene acetic acid
2. Department of (NAA). Profuse rhizogenesis was observed at 0.5 mg/L IAA compared to NAA / IBA. The
Biotechnology, Kakatiya regenerated plants were acclimatized in the culture room and maintained in the green
University, Warangal – 506 house and transferred to the field. These plants were found to be normal and similar
009, India. to the donar plant. Thus, an efficient and reproducible protocol has been developed in
cultivated tomato cv S-22 which is genotype dependent. This protocol can be used for
Agrobacterium tumefaciens mediated genetic transformation in tomato cv S-22 .
Corresponding author:
Nanna Rama Swamy
Keywords:
Solanum lycopersicum, cotyledon explants, organogenesis, in vitro rooting.
Email: Abbreviations:
swamynr.dr@gmail.com IAA-Indole-3-acetic acid ; IBA-Indole-3-butyric acid; NAA-ά-naphthalene
acetic acid; GA3 -Gibberelic acid.

Article Citation:
Web Address: Godishala Vikram, Kairamkonda Madhusudhan, Kagithoju Srikanth, Mangamoori
http://jresearchbiology.com/
Documents/RA0076.pdf.
Laxminarasu and Nanna Rama Swamy.
Effect of plant growth regulators on in vitro organogenesis in cultivated tomato
Journal of research in Biology (2011) 4: 263-268

Dates:
Received: 04 Aug 2011 /Accepted: 11 Aug 2011 /Published: 16 Aug 2011

© Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

263-268 | JRB | 2011 | Vol 1 | No 4
Journal of Research in biology
Submit Your Manuscript
An International Open Access Online
Research Journal www.ficuspublishers.com www.jresearchbiology.com
Vikram et al.,2011

INTRODUCTION: autoclave for 15-20 minutes. Cotyledon explants
Tomato (Solanum lycopersicum L.) is (1.0 cm2) were excised from 10-12 day old in vitro
considered to be the second important vegetable grown seedlings and inoculated them on MS
crop next to potato (Bhatia et al., 2004). It is also medium supplemented with varying concentrations
considered as a model species for introduction of of BAP and also in combination with IAA. The
agronomically important genes (Wing et al., 1994). micro-shoots were excised and cultured for
Developing a good in vitro regeneration protocol elongation on ½ strength MS, MSO and MS
has been a subject of research because of the medium supplemented with 3.5 mg/L BAP in
commercial value of the crop and its amenability combination with 0.2-1.0 mg/L GA3.For in vitro
for further improvement via genetic manipulation rooting the micro-shoots were transferred on to ½
(Evans 1989). It is one of the most studied higher strength MS, MSO and MS medium supplemented
plants because of its importance as a crop species, with 0.5mgL-1 IAA/IBA/NAA. All the cultures
and of several advantages for genetic, molecular were incubated at 25oC ± 1 under 16 h photoperiod
and physiological studies (Mc Cormick et al.,1986). with light intensity of 50µmol m-2 s-1.
The literature shows that the regeneration protocols In vitro rooted plants derived from cotyledon
have been published in cultivated tomato using explants were washed with sterile distilled water
different explants viz., cotyledons, hypocotyl and and shifted to plastic pots containing sterilized
leaf , different plant growth regulators like BAP, vermiculite: garden soil (1:1). Each plastic pot was
Kinetin, TDZ and Zeatin alone and also in covered with polythene bag to maintain the RH (80-
combination with various concentrations of auxins 90 %) and kept in culture room for 4 weeks. Later
(Zelcer et al., 1984; Park and Son 1988; Hamza these polythene bags were removed and the plants
and Chupeau1993; Ye Li and GL Zhou 1994; were shifted to earthenware pots containing garden
Plastira and Perdikaris 1997; Geetha et al., 1998; soil and maintained in the green house. Later they
Chen et al 1999; Gubis et al., 2003). A good were shifted to field.
regeneration protocol is essential for enhanced Data Analysis:
percentage of the transformants. The success in The data on multiple shoots formation was
tomato regeneration response has been found to be assessed after 6 weeks of culture. The following
genotype dependent, and also depends upon the parameters were evaluated: percentage of response,
orientation of explants and plant growth regulators average number of shoots per explant and mean
used in the culture medium (Praveen and Rama length of shoots. The experiments were repeated at
Swamy 2011). However there is no report on least twice and the data were analyzed statistically.
multiple shoots induction and regeneration from
cotyledon explants in Tomato cv S-22 except on RESULTS AND DISCUSSION:
somatic embryogenesis (Godishala et al., 2011). The cotyledon explants from 10-12 days old
Hence in this communication we report on a simple axenic grown seedlings were cultured on MS
and reproducible regeneration protocol for plantlet medium supplemented with various concentrations
establishment in cultivated tomato cv S-22. of BAP alone (Table 1). Callus induction was
initiated after 2nd week of inoculation at the cut
MATERIALS AND METHODS ends and gradually whole cotyledon turned into
Seeds of cultivated tomato cv S-22 were callus after 4 weeks of culture. Callus was
obtained from M/S Max Agri-Genetics Pvt Ltd, developed on all the concentrations of BAP used
Hyderabad, India. The seeds were washed under from cotyledon explants of tomato cv S-22.
running tap water for10 mins and soaked in sterile Percentage of response was found to be increasing
distilled water for 2 hours. These seeds were with increase in the concentration of the BAP and
surface-sterilized with 0.1 % (w/v) HgCl2 for 2 – 3 maximum percentage of response was recorded at
minutes followed by 3-4 rinses in sterile distilled 3.0 mg/L BAP for callus formation. Further
water. Surface sterilized seeds were germinated on increase in the hormonal concentration was
MS medium (Murashige and Skoog 1962) showing a decline in the callus induction response.
supplemented with 100 mg/l myoinositol and 30 g/l The nature of the callus was also found to be varied.
sucrose. The pH of the media was adjusted to 5.8 Green nodular callus was developed at 2.5-3.5 mg/
either with 0.1 N NaOH or 0.1 N HCl before adding L BAP (Fig.1a).
0.8% (w/v) agar-agar prior to autoclaving. The To know the morphogenetic event, the
medium was sterilized at 121oC under 15 psi in an cotyledon explants were also cultured on MS
264 Journal of Research in Biology (2011) 4: 263-268
Vikram et al.,2011

Table.1. Effect of various concentrations of BAP on shoot buds proliferation efficiency with more
morphogenesis from cotyledon explants in tomato cv S-22 number of multiple shoots formation in tomato cv S
-22.While.
Concentratio % of Morphogenic
n of PGR Response Response Gunay and Rao (1980) have found more
(mg/L) BAP multiple shoots induction on MS medium
augmented with 2.0 mg/L BAP + 0.2 mg/L IAA of
0.5 40 Callusing
tomato cv Rio Grande. According to our
1.0 45 Callusing
1.5 45 Callusing
observation, BAP alone in the medium supported
2.0 50 Nodular Callus for the callus induction and addition of 0.1 mg/L
2.5 60 Green Nodular Callus IAA to the medium along with 2.5-5.0 mg/L BAP
3.0 75 Green Nodular Callus induced multiple adventitious shoots. Similar
3.5 70 Green Nodular Callus results were also obtained from the cotyledon
4.0 65 Brown Callus explants on MS + BAP with IAA in tomato (Oktem
4.5 50 Brown Callus et al., 1999, Fariduddin et al., 2004).
5.0 50 Brown Callus The regenerated micro-shoots when sub-
cultured on ½ strength MS, MS basal media and
medium supplemented with different concentration also the same plant growth regulators combination
of BAP used in combination with 0.1 mg/L IAA and concentration did not support elongation.
(Table 2) .Multiple shoots were induced directly Elongation of micro-shoots was found on all the
from the explants in all the concentrations of BAP concentrations of GA3+3.5 mg/L BAP used (Table
in combination with 0.1 mg/L IAA (Fig.1b) except 3). Maximum percentage of shoot elongation and
at 0.5-2.0 mg/L BAP. Maximum percentage of also longer shoots were recorded at 0.6 mg/L GA3 +
response in cultures with more number of multiple 3.5 mg/L BAP (Fig.1c) without callus formation.
shoots formation was recorded at 3.0-3.5 mg/L For in vitro rooting the elongated shoots
BAP in combination with 0.1 mg/L IAA. But were cultured on ½ strength MS, MSO and MS
multiple shoots induction was found to be reduced medium supplemented with 0.5 mg/L IBA / IAA/
when the concentration of BAP increased beyond NAA (Table 4). Rooting was absent on ½ strength
to 3.5 mg/L BAP (Table 2). MS and MSO media and callus was formed without
Various studies demonstrated that 8-10 days rhizogenesis at the basal region of the shoots. Root
–old cotyledons of tomato were superior to other formation was initiated within 2 weeks of
source of explants, including hypocotyls, stems and incubation in all the auxins used.100 % rooting was
leaves for promoting shoot organogenesis of tomato observed on MS medium supplemented with 0.5
(Hamza and Chupeau1993, Van Roekel et al., 1993, mg/L IAA with profuse rhizogenesis (Fig.1d). In
Ling et al., 1998). Where as in the present vitro rooting was reported without PGRS in tomato
investigation 10-12 day-old cotyledons were cv UC 105 (Mensuali-Sodi et al., 1995). However,
selected as the source of explants which were found the current study could find that cultivation of the
to be superior and showed maximum percentage of micro-shoots on MS medium substituted with
Table.2. Effect of various concentrations of BAP + 0.1 mg/L IAA on organogenesis from
cotyledon explants of tomato cv S-22
Concentration % of Morphogenic Average number Average length
of PGRS (mg/L) Response Response of shoots/ explant (in cm) of shoots
IAA + BAP (±SE)a (±SE)a
0.1+0.5 45 White friable Callus ‫־־־‬ ‫־־־‬
0.1+1.0 45 White friable Callus ‫־־־‬ ‫־־־‬
0.1+1.5 50 White friable Callus ‫־־־‬ ‫־־־‬
0.1+2.0 55 White friable Callus ‫־־־‬ ‫־־־‬
0.1+2.5 65 Multiple Shoots 3.4±0.30 0.8±0.22
0.1+3.0 80 Multiple Shoots 3.8±0.12 1.2±0.32
0.1+3.5 80 Multiple Shoots 4.0±0.20 1.4±0.36
0.1+4.0 75 Multiple Shoots 3.6±0.36 1.6±0.42
0.1+4.5 65 Multiple Shoots 3.5±0.30 1.0±0.33
0.1+5.0 60 Multiple Shoots 3.3±0.26 0.6±0.40
a
Mean ± Standard Error

Journal of Research in Biology (2011) 4: 263-268 265
Vikram et al.,2011

different auxins resulted an effective rooting than based direct regeneration protocol is a pre-requisite
culturing on an auxin-free medium. for Agrobacterium tumefaciens mediated genetic
transformation in the cultivar S-22 for producing
Table.3. Effect of GA3 + 3.5 mg/L BAP on elongation agronomically useful transgenic plants.
of the micro-shoots in tomato cv S-22.
Medium + % of Average length of CONCLUSION.
Growth Response shoots From the above study, it is concluded that
regulators (in cm) (±SE)a the plantlet development was established through
(mg/L) direct organogenesis form cotyledon explants in
MS+0.2 GA3 +
55 2.6±0.30 cultivated tomato cv S 22 on MS medium
BAP
supplemented with BAP(2.5-5.0 mg/L) + 0.1 mg/L
MS+0.4 GA3 +
70 3.0±0.12 IAA. This protocol is simple and reproducible,
BAP
MS+0.6 GA3 + which can be used for Agrobacterium tumefaciens
95 3.8±0.26 mediated genetic transformation in cultivated
BAP
MS+0.8 GA3 + tomato cv S-22.
80 3.6±0.41
BAP
MS+1.0 GA3 + ACKNOWLEDGEMENTS:
66 2.9±0.36
BAP Author is grateful to Sri Ch.Devender
a
Mean ± Standard Error Reddy, Secretary cum Correspondent, Vaagdevi
Institutions and Mr.A.Sheshachalam Principal,
The in vitro regenerated plants were taken Vaagdevi Degree and PG College for their
for hardening by removing the residues of agar encouragement during the research work.
followed by washing with sterile distilled water
under aseptic conditions. Later these were shifted to REFERENCES:
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establishment from cotyledon explants of tomato cv Evans DA. 1989. Somaclonal variation – genetic
S-22. Since cotyledon is a favoured source of basis and breeding applications Trends Genet 5:46-
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267 Journal of Research in Biology (2011) 4: 263-268
Vikram et al.,2011

Fig 1: Callus induction and organogenesis from cotyledon explants in cultivated tomato cv S-22.
a: Callus induction on MS + 3.0 mg/L BAP (after 4 weeks of culture )
b: Multiple shoots induction from cotyledon explant on MS + 0.1 mg/L IAA + 3.5 mg/L BAP.
c: Shoot elongation on MS + 0.6 mg/L GA3 + 3.5mg/L BAP.
d: Profuse rhizogenesis on MS+ 0.5mg/L IAA.
e: Acclimatization of regenerated plant.
f: Regenerated plant growing in the research field.

Journal of Research in Biology (2011) 4: 263-268 268
Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

New Report on Hydra viridissima Pallas, 1766 (Cnidaria, Hydrozoa,
Hydridae) from Chingleput Lake, Tamil Nadu- India.
Journal of Research in Biology

Authors: ABSTRACT:
Sonia R and Ramanibai R.

Institution:
Aquatic Biodiversity Unit,
Department of Zoology,
University of Madras, Hydra viridissima Pallas, 1766, a green colour fresh water polyp is well known
Guindy Campus, for over 200 years for its remarkable regenerative capacity. New informations on the
Chennai - 25. Tamil Nadu. genus Hydra collected from Kolavoi Lake, Chingleput is reported here with its
morphometric features.This is the first report on the occurrence of Hydra viridissima
(Pallas, 1766) from the Kolavoi Lake, Chingleput.Tamil Nadu - India.
Corresponding author:
Ramanibai R.

Email:
rramani8@ hotmail.com,
soniadaisy7@gmail.com

Article Citation:
Web Address:
http://jresearchbiology.com/ Sonia R and Ramanibai R.
Documents/RA0071.pdf. Hydra viridissima Pallas, 1766 (Cnidaria, Hydrozoa, Hydridae) from Chingleput Lake,
Tamil Nadu- India.
Journal of research in Biology (2011) 4: 276-278

Dates:
Received: 01 Aug 2011 /Accepted: 11 Aug 2011 /Published: 22 Aug 2011

© Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

276-278 | JRB | 2011 | Vol 1 | No 4
Journal of Research in biology
Submit Your Manuscript
An International Open Access Online
Research Journal www.ficuspublishers.com www.jresearchbiology.com
Sonia et al.,2011

INTRODUCTION: recreation and fishing activities. It is a perennial
Hydra occupy fresh water habitats, belongs lake irrigating about 200h area covering 12 nearby
to the phylum Cnidaria and is an offshoot from villages. The total capacity of the lake is 13.50
sister bilaterians. Hydra polyp exhibit tube shaped Mm3. The water spread area is 8.82 Km2. The
body posses an apical hypostome cultured on the Kolavoi Lake is 3.3 Km long and 1.6 km wide. This
mouth opening and surrounded by a circle of lake is infested by the aquatic vegetation (Bharathi,
tentacles and at the aboral extremity - a basal disc. 2003).
Hydra has the ability to regenerate any missing The hydras were collected using 120 µm
structure after bisection upon regular intake of food mesh size plankton net near the littoral sites where
materials (Simona Chera et al , 2009). aquatic vegetation spread over enormously. At the
Hydras are common in bodies of water in state of free floating, Hydra viridissima can be
every continent except Antarctica (Holstein, 1995). collected using plankton net. In addition, during
They are reported to be absent from oceanic islands post monsoon season ( 30th January 2011) ,20 liters
such as Hawaii and Tahiti (Hickson, 1930; of water were collected from the Lake with the
Mumford, 1940 a). They are small creatures littoral vegetation and transferred to the aquarium
common in lakes, ponds and streams; attached to tank in the laboratory. Then the hydras which are
litters, stones and tree roots. They generally present in the tank were observed under the
resembles marine hydroids but does not undergo all labomed microscope and Morphometric
trace of medusoid stages and bear the gonads measurements and photographs were taken for
directly and solitary under normal conditions. further identification. The pH recorded was 8 which
It exhibits a radial symmetry, reproduces was slightly alkaline and temperature (ºC) observed
asexually by budding (Pennak, 1953) at the base of at the time of sample collection was 22ºC.
the stomach region; successive buds appear as Identification:
spiral sequence in the distal direction. Sexual Only freshly collected polyps were
reproduction is generally related to season (i.e., examined for the study.
temperature dependent). It usually occurs in autumn Taxonomic account:
or early winter in most species but in spring and Hydra viridissima (Pallas, 1766)
early summer in the green hydra. They may be Hydra viridissima are the most widespread
either dioecious or hermaphroditic (Libbie HH , and abundant hydra in Kolavoi lake Chingleput
1959).Its green colour is due to the presence of especially from the Paranur railway station area.
symbiotic green algae—Zoochlorellae—in its Freshly collected polyps were measured between
endodermal cells. (Edmondson, 1959) 0.4 mm to 2 mm in length (Fig. A). The fresh
The modern systematic position of Hydra collections were maintained under laboratory
was drawn out by Schulze (1917) based largely on conditions fed three Ceriodaphnia cornuta
the European fauna. Previous reports were not individuals per day. When appearing relaxed, the
available on the occurrence of the green hydra polyps were measured as 4 to 10 mm in column
particularly in Chingleput Lake, Tamil Nadu – length. They usually possess 6 to 8 tentacles. As
India. Here the systematic position of the green individual polyps grow older and increase in size,
hydra of Chingleput Lake reported for first time. the stalk grows and eventually becomes the size of
Systematic position the upper portion of the column and sometimes
Phylum : Cnidaria larger. Hydra when fed three Ceriodaphnia can be
Class : Hydrozoa able to produce one or two buds per polyp per day
Order : Hydroida (Fig. B & Fig. C). Nematocysts were also found on
Sub order : Anthomedusae
Genus : Hydra / Chlorohydra
Species : viridissima

Materials Examined:
Kolavoi Lake, Chingleput:
Kolavoi Lake is located in the Chingleput
district and it is 58 Km away from the Chennai
City. It is one of the largest lakes of Chingleput A: Hydra – Single Polyp B: Hydra Polyp with bud.
district. People use this lake water for agriculture,
278 Journal of Research in Biology (2011) 4: 276-278
Sonia et al.,2011

the tentacles. Two headed hydra were also seen P (COORDS). Süsswasserfauna von Mitteleuropa.
(Fig. D & Fig. E). Stuttgart: Gustav Fischer Verlag. 1-110.

Libbie HH. 1959. Coelentrata. American Museum
of Natural History, New York, New York. In:
Edmondson,. Fresh water biology.

Mumford EP. 1940. The present status of studies
of faunal distribution with reference to oceanic
islands. Proceedings of the 6th Pacific Science
C: Hydra Polyp with bud D: Two headed Hydra Congress (Berkeley, California) a; 4:241-248.
- Relaxed State.
Pallas PS. 1766. Eleuchus Zoophytorum. Apud
Petrum van cleef, Hagae – Comitum,

Pennak RW. 1953. Fresh water Invertebrates of
the United States. N.Y.: The Ronald Press
Company, Cap. 4:98-113.

Schulze P Neue Beitrage Zu einer. 1917.
Monographie der Gating Hydra. Arch. Biontol.,
E: Two headed Hydra - Relaxed State. 4:29-119.

Hydra viridissima are hermaphroditic in Simona Chera , Wanda Buzgariu , Luiza Ghila
nature. But sexual hydras were not reported from and Brigitte Galliot. 2009. Autophagy in Hydra: A
our sample collections. These hydras are response to starvation and stress in early animal
phototactic and often crowd into one area within a evolution. Biochimica et Biophysica Acta.
culture tank. 16057:12 , 4C:2,3,5,6,8,9,11.
Based on literature records, we conclude that
our report is the first on the occurrence of Hydra
viridissima (Pallas, 1766) from the Kolavoi Lake,
Chingleput.

ACKNOWLEDGEMENT:
We thank UGC - New Delhi for financial
support.

REFERENCES:
Bharathi D Limnological studies of few fresh water
habitats in and around Chennai City. 2003; Ph.D
Thesis submitted to University of Madras, Chennai.

Edmondson WT. 1959. Freshwater Biology, John
Wiley & Sons. New York. 757.

Hickson SJ. 1930. Report on the Hydras collected
during Dr. Hugh Scott’s and Mr. Omer - Cooper’s
expedition to Abyssinia (H.ethiopiae spn.) Proc.
Soc. Lond., 63-64.

Holstein T. 1995. Cnidaria: Hydrozoa. In
BRAUER, A., SCHWOERBEL. J, AND ZWICK,

Journal of Research in Biology (2011) 4: 276-278 278
Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

Studies on phytochemicals, antibacterial and Antioxidant effects of leaf extracts of
Lamprachaenium microcephalum
Journal of Research in Biology

Authors: ABSTRACT:
*MohanaSundaram,
Sivakumar, Karthikeyan,
Magesh, Gopinath, In this investigation, Lamprachaenium microcephalum was analysed for its
Sheeladevi, Thirumalai,
phytochemical constituents qualitatively and quantitatively. The antibacterial property
Pennarasi and Prasanna.
of aqueous, ethanolic and hexane extracts of Lamprachaenium microcephalum was
studied against different bacteria include Escherichia coli, Proteus mirabilis,
Salmonella typhi, Enterobacter aerogenes, and Shigella flexineri and showed growth
Institution: inhibition activity at concentrations in the range of 300mg to 500mg. The antioxidant
Department of effect of those extracts was also studied against α-tocopherol as a control. From the
Biotechnology, results, Alkaloids, flavonoids, saponins, and tannins were revealed to be present in
Karpaga Vinayaga College Lamprachaenium microcephalum. Ethanol extract at the concentration of 500μg/ml
of Engineering and showed 61.16% antioxidant activity against 500 μg/ml of α-tocopherol which showed
Technology, 76.86% as a standard reference.
Maduranthagam,
TamilNadu-603308.
*Lecturer, Department of
Biotechnology,
Karpaga Vinayaga College
of Engineering and
Technology, Keywords:
Maduranthagam, Lamprachaenium microcephalum , phytochemicals, Antibacterial, Antioxidant
Kanchipuram District,
TamilNadu-603308, India.

Corresponding author:
MohanaSundaram

Email: Article Citation:
sbmohan2007@gmail.com MohanaSundaram, Sivakumar, Karthikeyan, Magesh, Gopinath, Sheeladevi,
Thirumalai, Pennarasi and Prasanna.
Studies on phytochemicals, antibacterial and Antioxidant effects of leaf extracts of
Phone No: lamprachaenium microcephalum ..

044-27565486 Journal of research in Biology (2011) 4: 279-284

Web Address: Dates:
http://jresearchbiology.com/ Received: 30 Jul 2011 /Accepted: 11 Jul 2011 /Published:
Documents/RA0070.pdf.

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MohanaSundaram et al.,2011

INTRODUCTION prepared using soxhlet apparatus (Hoffman et al.,
Plants are effective in the treatment of 2004) for about 24h. The extract was distilled and
infectious diseases and many plant extracts have concentrated in vacuo with addition of CaCl2.
been shown to possess antimicrobial properties in Lyophilized aqueous fractions were further used to
vitro (Sofowora, 1983). The increased prevalence of test for the antifungal, antibacterial and antioxidant
antibiotic-resistant bacteria due to the extensive use properties.
of antibiotics may render the current antimicrobial Chemicals and microorganisms:
agents inefficient to control some bacterial diseases Escherichia coli, Proteus mirabilis,
(Tanaka et al., 2006). Herbal medicine is frequently Salmonella typhi, Enterobacter aerogenes and
a part of a larger therapeutic system such as Shigella flexineri were purchased from IMTECH,
traditional and folk medicine. It is necessary to Chandigar, India. Solvent and other chemicals
evaluate, in a scientific base, the potential use of which were used during this study were from
folk medicine for the treatment of infectious Himedia, Merck and s.d. Fine-Chemicals, Mumbai.
diseases produced by common pathogens.
Medicinal plants might represent an alternative METHODS
treatment in non-severe cases of infectious diseases. Phytochemical screening procedures carried
They can also be a possible source for new potent out were adopted from (Oloyed, 2005). This
antibiotics to which pathogen strains are not analysis determines the biologically active
resistant. The search and use of drugs and dietary compounds that contribute to the flavour, colour
supplements derived from plants have been and other characteristics of leaves.
accelerated in recent years. Ethnopharmacologist, Test for alkaloids: About 2 g of the ground sample
botanist, microbiologist and natural product chemist were pounded separately on a mortar. 0.2 g was
are combing the medicinal flora for biological boiled with 5 ml of 2% hydrochloric acid on a
substances that could be developed for the steam bath for 5 min. The mixture was allowed to
treatment of infectious diseases. cool and filtered and the filtrate was shared in equal
Lamprachaenium is a genus of flowering plants in proportion into 3 test tubes and labeled A, B, C.
the Asteraceae family and found in most of the One (1) ml portion of the filtrate was treated with 2
parts of south India. Some rural people of south drops of the following reagents respectively. With
India, including tribals using this plant leaf as Dragendroff’s reagent a red precipitate was shown.
antiseptic agent (Gurudeva MR and With Mayer's reagent a creamy white coloured
Yoganarasimhan SN, 2009). precipitate indicated the presence of alkaloid
The aim of the present investigation is to (Harborne, 1973; Trease and Evans, 1989).
analyze Lamprachaenium microcephalum Test for flavonoids: 0.5 g of the macerated sample
qualitatively to study the phytochemicals and of Lamprachaenium microcephalum was
antibacterial effect of aqueous, ethanolic and introduced into 10 ml of ethyl acetate and heated in
hexane extract of Lamprachaenium microcephalum boiling water for 1 min. The mixture was then
against enteric pathogens with the assessment of its filtered and the filtrate used for the following test. 4
antioxidant potency. ml of the filtrate was shaken with 1 ml of 1%
MATERIALS AND METHODS aluminum chloride solution and kept. Formation of
Preparation of Plant Extract a yellow colour in the presence of 1 ml dilute
The leafs of Lamprachaenium Ammonia solution indicated the presence of
microcephalum used in this study were collected flavonoids (Harborne, 1973).
from Thirukkalukundram village, Kanchipuram Test for saponins: 0.1 g of the sample was boiled
District, TamilNadu, and South India in the month with 5 ml of distilled water for 5 min. Mixture was
of June, 2010. The plant was identified by the filtered while still hot and the filtrate was then used
experts of Centre for Advanced Studies in Botany, for the following tests (Trease and Evans, 1989). To
University of Madras, Guindy campus, Chennai and 1 ml of the filtrates, 2 drops of olive oil was added,
a voucher specimen (No. SAN-2403/10) was the mixture was shaken and observed for the
deposited in our departmental laboratory. The formation of emulsion. 1 ml of the filtrate was
collected plant sample was refluxed in running tape diluted with 4 ml of distilled water. The mixture
water for 1-2 h and shade dried at room temperature was vigorously shaken and then observed on a
for 15 – 20 days. Aqueous, ethanolic and hexane stand for stable froth (Trease and Evans, 1989).
extract of Lamprachaenium microcephalum was Test for tannins: Into 2 g of the ground sample, 5
280 Journal of Research in Biology (2011) 4: 279-284
MohanaSundaram et al.,2011

ml of 45% ethanol was added and boiled for 5 min. of methanol and the content poured at once into the
The mixture was cooled and filtered. 1 ml of the funnel. The filtrate was mixed with 50 ml of water
filtrate was added 3 drops of lead sub acetate and analyzed within an hour. For aqueous
solution. A gelatinous precipitates were observed extractions, 5 ml of water was used for the
which indicates the presence of Tannins. Another 1 extraction and for the rinse and the filtrate was
ml of the filtrate was added 0.5 ml of bromine added to 50 ml of water. 3 ml of 0.1 ml FeCl3 in 0.1
water. A pale brown precipitates were observed NH4Cl was added to 5 ml of the extract and
indicating the presence of Tannins (Trease and followed immediately by timed addition of 3 ml of
Evans, 1989). 0.008 ml K2Fe(CN)6. The absorbance was taken at
Test for glycosides: 2 g of the sample was mixed 720 nm spectrophotometrically (Onwuka, 2005).
with 30 ml of distilled water and it was heated for 5 Antibacterial effect:
min on a water bath, filtered and used as follows: The antibacterial activity of
five ml of the filtrate was added to 0.2 ml of fehling Lamprachaenium microcephalum was evaluated by
solution A and fehling solution B until it turns agar well diffusion method (Chung et al., 1990).
alkaline and heated in a water bath for 2 min. A Muller Hinton agar medium was prepared and
lightish blue colouration was observed (instead of poured into the petridishes. Then it was inoculated
brick red precipitate) which indicates the absence of with a swab of bacterial culture (mid log phase) and
glycosides (Oloyed, 2005). spread throughout the medium uniformly with a
Quantitative Analysis of Phytochemical sterile cotton swab. Using a sterile cork borer
Constituents (10mm diameter) wells were made in the agar
Estimation of alkaloids: 0.5 g of the sample was medium. The test compound was introduced into
dissolved in 96% ethanol-20% H2SO4 (1:1) the wells and all the plates were incubated at 37ºC
mixture. 1 ml of the filtrate was added to 5 ml of for 24 h. The experiment was performed five times
60% tetraoxosulphate (VI), and allowed to stand for under strict aseptic conditions. Sensitivity of the
5 min. Then, 5 ml of 0.5% formaldehyde was added organism was determined by measuring the
and allowed to stand for 3 h. The absorbance was diameter of the zone of inhibition. Each assay was
measured at 565 nm (Harborne, 1976). repeated for five times and the mean value was
Estimation of flavonoids: Flavonoid in the test taken for analyses. The control experiment was
sample was determined by the acid hydrolysis of carried out with the antibiotics such as streptomycin
spectrophotometric method. 0.5 g of processed and chloramphenical (S.Shobana et al, 2009).
plant sample was mixed with 5 ml of dilute HCl Antioxidant effect:
and boiled for 30 min. The boiled extract was The antioxidant activity of aqueous,
allowed to cool and filtered. 1 ml of the filtrate was ethanolic and hexane extracts of Lamprachaenium
added to 5 ml of ethyl acetate and 5 ml of 1% NH3 . microcephalum were determined by ferric
This was then scanned 3 from 420nm-520nm for thiocynate method (Mistuda et al., 1996). 10 mg of
the absorbance. (Harborne,1976). each extract was dissolved separately in 99.5% of
Estimation of saponins: 0.5 g of the sample was ethanol and various concentrations (100, 200, 300,
added to 20 ml of 1N HCl and was boiled for 4 h. 400 & 500μg/ml) were prepared. A mixture of a 2
After cooling it was filtered and 50 ml of petroleum ml of sample in 99.5% ethanol, 2.052 ml of 2.51%
ether was added to the filtrate for ether layer and linoleic acid in 99.5% ethanol, 4 ml of 0.05 M
evaporated to dryness. 5 ml of acetone ethanol was phosphate buffer (pH 7.0) and 1.948 ml of water
added to the residue. 0.4 ml of each was taken into was placed in a vial with a screw cap and placed in
3 different test tubes. 6 ml of Ferrous sulphate an oven at 60oC in the dark. To 0.1 ml of this
reagent was added into them followed by 2 ml of sample solution 9.7 ml of 75% ethanol and 0.1 ml
concentrated H2SO4. It was thoroughly mixed after of 30% ammonium thiocynate was added. After the
10 min and the absorbance was taken at 490 nm addition of 0.1 ml of 2 x 10-2 M ferrous chloride in
(Oloyed, 2005). 3.5% hydrochloric acid to the reaction mixture, the
Estimation of tannins: 5 g of the ground sample absorbance of the red color developed was
was shaken constantly for 1 min with 3 ml of measured in 3 min at 500 nm (Matook and
methanol in a test tube and then poured into a Hashinaga, 2005). The control and standard were
Buchner funnel with the suction already turned on. subjected to the same procedures as the sample,
The tube was quickly rinsed with an additional 3 ml except that for the control, only solvent was added,

Journal of Research in Biology (2011) 4: 279-284 281
MohanaSundaram et al.,2011

and for the standard, sample was replaced with the Table 1.2: Quantitative analysis of Lamprachaenium
same amount of α-tocopherol (reference microcephalum for phytochemicals
compound) (Ali Yildirim et al., 2001). The Lamprachaenium
inhibition of lipid peroxidation in percentage (Table S. Name of the microcephalum
3.0) was calculated by following equation: No phytochemical
Dry sample Wet sample
% Inhibition = 1 - (A1/A2) x100 Alkaloids
1. 46.68 ± 3.21 21.39 ± 2.17
(mg/100g)
Where, Flavonoids
A1 - absorbance of the test sample 2. 69.33 ± 4.14 56.18 ± 3.19
(mg/100g)
A2 - absorbance control reaction Saponins
3. 03.77 ± 0.54 01.21 ± 0.84
(mg/100g)
RESULTS & DISCUSSION Tannins
Phytochemical analysis is very useful in the 4. 02.37 ± 0.76 01.67 ± 0.46
(mg/100g)
evaluation of some active biological components of
medicinal plants. The qualitative and quantitative Antioxidants are compounds that protect
analyses were carried out in both dry and wet cells against the damaging effects of reactive
samples. Alkaloids, flavonoids, saponins, tannins, oxygen species, such as singlet oxygen, super
were revealed to be present in Lamprachaenium oxide, peroxyl radicals, hydroxyl radicals and
microcephalum (Table 1.1). This shows high level peroxynitrile. An imbalance between antioxidants
of its possible medicinal and dietary values and reactive oxygen species results in oxidative
(Oloyed, 2005). Although, some of these analyzed stress, leading to cellular damage (Burlon and
constituents of the vegetable species may be Ingold, 1984). Oxidative stresses have been linked
completely harmful to both man and farm animals to cancer, aging, atherosclerosis, inflammation,
and some are species specific as observed in the ischemic injury and neuro degenerative diseases
case of tannins (Odebiyi and Sofowora, 1979). (Parkinson’s and Alzheiner’s) (Palozza, 1998).
Some of these active components have been Flavonoid may help provide protection against
demonstrated to possess anti nutritional effects, these diseases by contributing along with
following their ability to reduce palatability and antioxidant vitamins and enzymes, to the total
digestibility of feedstuff (Odebiyi and Sofowora, antioxidant defense system to the human body.
1979). Epidemiological studies have shown that flavonoids
In Table 1.2, the levels of these and carotenoids intake are inversely related to
phytochemicals (bioactive compounds) were mortality from coronary heart diseases and to the
shown. Generally, the dry sample showed higher incidence of heart attacks (Donald and Cristobal,
levels of these bioactive compounds than the wet 2006).
sample. The reason may be that the bioactive Antibacterial Property
compounds are not volatile compounds and hence From Table 2.0, it is very clear that the
have a high dried weight. High levels of flavonoids aqueous, ethanolic and hexane extracts of
(69.33 ± 4.14 and 56.18 ± 3.19) in Table 1.2 Lamprachaenium microcephalum showed growth
showed that the vegetable is good for the inhibition activity only at higher concentrations in
management of cardiovascular diseases and the range of 300mg to 500mg. E.aerogens and
oxidative stress, since flavonoids are biologic P.mirabilis were sensitive to aqueous extracts of
antioxidants. Lamprachaenium microcephalum rather than
Table 1.1: Qualitative analysis of different extracts of Lamprachaenium microcephalum for phytochemicals
Name of the Different extracts of Lamprachaenium microcephalum
S.No
phytochemical Aqueous extract Ethanolic extract Hexane extract
1. Alkaloids +ve +ve +ve
2. Flavonoids +ve +ve +ve
3. Saponins +ve +ve +ve
4. Tannins +ve +ve +ve
5. Glycosides -ve -ve -ve
+ve – presence -ve - absence
282 Journal of Research in Biology (2011) 4: 279-284
MohanaSundaram et al.,2011

ethanolic and hexane extracts. S.typi showed

E.aerogenes
moderate resistance to all the extracts when

10.3
11.9
14.2
21.4
20.1
NS
NS
compared to others (Table 2.0). It was observed that
in both ethanolic and hexane extracts of
Lamprachaenium microcephalum, bacterial strains
Hexane extract
S.typhi

18 .9
20 .1
are not highly susceptible even at high

10.8
12.3
15.7
NS
NI concentration than aqueous extract.
The antioxidant activity of the aqueous,
P.mirabilis
Table 2.0: Antibacterial activity of aqueous, ethanolic and hexane extracts of Lamprachaenium microcephalum

10.4 ethanolic and hexane extracts of Lamprachaenium
12.8
14.7
20.1
19.9
NI
NI

microcephalum were determined by ferric
thiocynate (FTC) and the values are presented in
Table 3.0. FTC method was used to determine the
E.coli

11.5
13.1
16.2
19.9
19.1
NS

amount of peroxide formed and that react with
NI

ferrous chloride (FeCl2) to form a reddish ferric
chloride (FeCl3) pigment. In this method, the
E.aerogenes

NI – No Inhibition concentration of peroxide decreases as the
11.1
13.4
16.3
19.1
20.7
NS
NS

antioxidant activity of extract increases. Aqueous,
ethanolic and hexane extracts at various
Ethanolic extract

concentration (100,200, 300,400 and 500 in μg/ml),
S.typhi

10.2
11.6
14.4
18.9
19.3

showed antioxidant activities in a concentration
NI
NI

dependent manner. Ethanol extract at the
concentration of 500 μg/ml showed 61.16%, an
P.mirabilis

antioxidant activity at the concentration of 500 μg/
11.2
13.7
16.2
19.5
18.6
NS
NI

ml of α-tocopherol 76.86%, the reference
compound. The aqueous and hexane extracts of
Lamprachaenium microcephalum also have showed
E.coli

12.7
14.6
15.3
20.8
20.1
NS
NS

some significant level of inhibition of lipid
peroxidation. It has been observed that the extract
E.aerogenes

exhibited moderate antioxidant activity.
10.2
14.7
19.7
22.1
19.8
NS
NI

CONCLUSION
Based on the results of the present study, it
Aqueous extract

was commented that different extracts of this plant
S.typhi

11.8
15.6
19.1
20.7
NS
NS
NI

leaf possess antibacterial and antioxidant property.
But, further studies are needed to get the clear
mechanism for such property which would be
P.mirabilis

10.1
12.6
16.1
19.3
20.8
19.6

interesting to learn.
NS

ACKNOWLEDGEMENT
The authors are grateful to Mrs.Meenakshi
E.coli

10.3
13.1
16.5
21.6
22.3
NS
NS

Annamalai, Director, Karpaga Vinayaga College of
NS – Non significant value (<10mm)

Engineering and Technology for the complete
Concentration

support throughout the work with timely and
valuable discussions.
in µg
100
200
300
400
500
100
100
Agent Used for

microcephalum

Chloramphenic
Lamprachaeni

Streptomycin
study

um

al

Journal of Research in Biology (2011) 4: 279-284 283
MohanaSundaram et al.,2011

Table : 3.0 Antioxidant activity of aqueous, ethanolic and hexane extracts of Lamprachaenium microcephalum
% of inhibition of lipid peroxidation
S.No Extract
100µg/ml 200µg/ml 300µg/ml 400µg/ml 500µg/ml
1. Water 12.31 21.12 35.73 45.34 57.75
2. Ethanol 18.17 24.63 43.08 50.67 61.16
3. Hexane 14.71 20.32 28.63 39.34 48.55
4. α-Tocopherol 27.43 39.71 47.87 62.78 76.86

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Antioxidation action of Indole compounds during
284 Journal of Research in Biology (2011) 4: 279-284
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Original Research paper Publication group

Crotalaria pallida extracts as a putative HIV-protease inhibitors
Journal of Research in Biology

Authors: ABSTRACT:
Govindappa M1, Anil
Kumar NV2 and Gustavo
Santoyo3.

In the present investigation, we screened the two solvent (methanol and
Institution:
1
Department of ethanol) extracts of Crotalaria pallida different parts (leaf, flower and stem) for
Biotechnology, Shridevi phytochemical analysis and screening for HIV protease inhibitors. The two solvent
Institute of Engineering & extracts have yielded the presence of major active compounds viz., flavonoids,
Technology, Sira Road, terpenoids, cardiac glycosides, anthraquinones, coumarins, steroids, tannins,
Tumkur-572 106, Karnataka, saponins. For HIV protease inhibitor activity, we used pepsin assay as a substitute for
INDIA, screening HIV protease inhibitors in the present investigation. The methanol and
2
Department of Chemistry, ethanol extracts of stem and flower have showed significant inhibition of protease
Manipal Institute of activity as compared to standard (Pepstatin A).
Technology, Manipal
University, Manipal-576
104, Karnataka.
3
Professor Investigador
Titular “A”, IIQB-UMSNH,
Edificio A1 (B5), Ciudad
Universitaria, Morelia,
Michoacan, Mexico, C.P. Keywords:
58030. Phytochemicals, Crotalaria pallida, HIV protease.

Corresponding author: Article Citation:
Govindappa M Govindappa M, Anil Kumar NV and Gustavo Santoyo.
Crotalaria pallida extracts as a putative HIV-protease inhibitors.
Journal of research in Biology (2011) 4: 285-291
Email:
dravidateja07@yahoo.co.in

Phone No: Dates:
+91-9686114847 Received: 29 Jul 2011 /Accepted: 11 Aug 2011 /Published: 30 Aug 2011

Fax:
+91-816-2212629 © Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
Web Address: creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
http://jresearchbiology.com/ commercial, distribution, and reproduction in all medium, provided the original work is properly
Documents/RA0069.pdf. cited.

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Submit Your Manuscript
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Govindappa et al.,2011

INTRODUCTION other natural products (Chinsembu and Hedimbi,
A number of anti-HIV drugs used in 2010).
conventional AIDS therapy are available in the Crotalaria pallida is a terrestrial, annual,
market, unfortunately the administration of these erect herb, up to 150 cm tall. Taproot white or
compounds clinically to the AIDS patients brown and stem grooved, solid, glabrous. Stipules
exhibited serious side effects (Scinazi et al., 1992). present. Leaves trifoliolate, alternate spiral, stalked,
As the data shows, rapid spread of AIDS epidemic leaflets elliptic or obovate, more than 2 cm long/
and appearance of HIV strain resistance to the wide, hairy on upper surface, margin entire, apex
currently available drugs suggests that an effective obtuse or rounded, base acute, pinnately veined.
and durable chemotherapy is required inheritably Flowers bisexual, grouped together in a terminal
for its treatment. The use of innovative raceme, stalked, petals 5, yellow. Fruit a rounded
combinations of drugs having diverse mechanisms pod. A novel antimicrobial peptide has been
of anti-HIV activity (Tantillo et al., 1994; Balzarini exhibited as a strong antimicrobial agent against
et al., 1996; Lipsky, 1996) and continuous need for human pathogenic micro-organisms (Pellegrini et
alternative inhibitors are also needed to be al., 2009) and lectin (Rego et al., 2002).
developed progressively (Singh et al., 2010). New In the folk and Ayurvedic medicines, C.
anti-HIV agents with such activities may be juncea is used as blood purifier, abortificient,
identified through a variety of approaches, one of is astringent, demulcent, emetic, purgative and in the
through the being screening of natural products. treatment of anaemia, impetigo, menorrhagia and
Plant products have attracted attention as psoriasis (Chauhan and Singh, 2010). Crotalaria
possible anti-HIV drugs, targeted on the specific species possesses anticancer properties (Kumar et
steps of the viral life cycle, such as viral attachment al., 2008). Presence of coumarin has reported from
and entry as well as on essential enzymes and Crotalaria madhurensis (Bhakshu et al., 2008) and
proteins that play a role during viral genome Crotalaria ramosissima (Rao and Narukulla, 2007).
transcription are identified (Matsuse et al., 1999). The literature survey indicates that there are
The approved medications currently in use are no reports available from India regarding the
mainly restricted to target the two viral enzymes, phytochemicals and their anti-HIV properties of C.
Protease and Reverse Transcriptase (RT). These pallida. In the present study was aimed to examine
inhibitors are very expensive and this has led to a the phytochemical analysis of methanol and ethanol
global demand for broader, safer and also cheaper extract of stem, leaves and flower of C. pallida and
medications (Notka et al., 2004). was screened for anti-HIV properties using standard
HIV protease has been suggested as a methods. The findings from this work may add to
potential target for AIDS therapy for a long time the overall value of the medicinal potential of the
(Kramer, 1986; Singh et al., 2010), later it was plant.
shown that a frame-shift mutations in the protease
region of the pol-gene prevented cleavage of the MATERIALS AND METHODS
gag poly protein precursor, which is essential for Collection of plant material
the mutation of the HIV particles. Blockage of HIV The plant was collected in November 2009
protease leads to the formation of immature non from the college campus, Shridevi Institute of
infectious virions (Khol, 1988). The HIV aspartic Engineering & Technology, Sira Road, Tumkur,
protease (HIV-PR) 1 is a key enzyme in the virus Karnataka, India. The plant was identified by their
life cycle and was earlier perceived as a promising vernacular names and later it was compared with
therapeutic target and its inhibition has become the herbarium of Department of Studies in Botany,
successfully used in the treatment of AIDS. Manasa Gangothri, University of Mysore, Mysore
Literature survey shows that there are great and Government Ayurvedic College, Mysore, India.
potential natural products acting as HIV protease Extract Preparation
inhibitors for eg; limonin and nomilin from Citrus Each individual plant parts were used in the
sp. (Battinelli, 2003), maslinic acid from Geum extraction followed by methodology as described
japonicum (Xu, 1996), oxygenated triterpenoids by Martino et al. (2006). Microwave assisted
(Ganoderic acid-α) from Ganoderma lucidum extraction was performed using a closed-vessel
(Mekkawy, 1998), ursolic acid and uvaol from system (GMS 17M 07 WHGX SOLO Microwave).
Crataegus pinatitids (Min, 1999), coumarins from Two grams of powdered different plant parts were
Calophyllum brasiliense (Ricardo et al., 2004) and subjected to extraction in a vessel with of solvent
286 Journal of Research in Biology (2011) 4: 285-291
Govindappa et al.,2011

(ethanol and methanol) up to the volume of 2ml for extracts of C. pallida, the alkaloids, flavonoids,
two cycles of 5 min individually. The resulting terpenoids, saponins, phenols, cardiac glycosides,
extracts were filtered through Whatman filter No.2. steroids, coumarin and tannins were present in all
Clarified supernatant was kept in refrigeration for the solvent extracts. The methanol extract yielded
further studies (phytochemical analysis, coumarin strongly all the phytochemicals compared to
identification and HIV protease inhibition). ethanol (Table 1). In ethanol leaf extracts, it
Phytochemical analysis
Phytochemical analysis was carried out for

Anthraquinones
saponins, flavonoids, terpenoids, steroids, phenol,
alkaloids (Obdoni and Ochuko, 2001), tannins
(Kaur and Arora, 2009) and coumarin

-
-
-

-
-
-
spectrophotometrically as described by Yao et al.
(2008). Wagner’s and Heger’s reagents were used
for alkaloid foam test for saponins, Mg-HCl and Zn
-HCl for flavonoids, Keller-Killani test for cardiac

Coumarins
glycosides, Salkonoski test for terpenoids, acetic

Table 1. Phytochemical analysis of methanol and ethanol extracts of Crotalaria pallida
anhydride and sulphuric acid for steroids, chloride

+
+
+

+
+
+
and gelatin for tannins, ferric chloride for phenol,
hexane and diluted ammonia for anthraquinones
test. All these experiments were carried out for each

Saponins
solvent extracts separately.

+
+
+

+
+
+
Enzyme pepsin inhibition assay
Pepsin has a quite close resemblance in
proteolytic activity with HIV-1 protease one key
enzyme of HIV-1 life cycle as both of them belong Tannin Steroids

+
+
+

+
+
+
to same aspartate enzyme family (Maria et al.,
Phytochemicals

2004). This enzyme was used as substitute of HIV-
1 protease to check out anti-HIV activity of plant
extracts in the present investigation (Singh et al.,
+
+
+

+
+
+
2010).
We followed the method of Aoyagi (1978)
and Singh et al. (2010), for this assay, 50 µg pepsin,
glycosides

800 µg haemoglobin and different parts extracts
Cardiac

Repeated the each experiments thrice, +: presence, -: absent
+
+
+

+
+
+
were taken in 500 µl of reaction mixture. The
mixture was allowed to incubate at 370C, after 20
min, 700 µl of 5% TCA was added to stop the
reaction. It was then centrifuged at 14, 000 g for 5
Flavonoids Terpenoids Phenol

+
+
-
-
-

-

min and the supernatant was collected. Optical
Density (OD) was recorded spectrophotometrically
at 280 nm. Separate blanks were used or both
positive and negative control as well as for sample.
For positive control enzyme and substrate were
+
+
+

+
+
+

taken and followed the above procedure and for
negative control pepstatin A was taken as a well
known inhibitor of both pepsin and HIV-protease.
Each sample was taken in triplicate, so this assay
+
+

+
+

gives reproducible results.
-

-
Methanol extract
Ethanol extract

RESULTS
Phytochemical analysis
Sample

The phytochemical screening showed that
Flower

Flower
Stem

Stem
Leaf

Leaf

the two different solvent ethanol and methanol

Journal of Research in Biology (2011) 4: 285-291 287
Govindappa et al.,2011

showed the absence of flavonoids, the ethanol stem and protease inhibitors have experienced drug
extract showed absence of phenol and ethanol resistance by HIV strains. This imposes the demand
flower and stem extracts showed absence of tannin. for the development of new drugs particularly of
The ethanol stem extract exhibited the absence of plant origin owing to their success as sources of anti
steroids. Methanol leaf extracts showed the absence -HIV drugs. The use of plants for management of
of flavonoids and methanol stem extract exhibited different diseases has become a common practice
the absence of phenol. since olden days in developing countries. Especially
The ethanolic flower and stem extracts in India, most of the people are depending on
exhibited highest amount of coumarins in tested traditional medicines for their primary health care
methods where as methanol flower and stem including the management of HIV/AIDS.
extracts exhibited highest amount of coumarins. Our results have showed protease inhibitor
The ethanol and methanol stem and flower activity and phytochemical screening showed the
extracts exhibited strong inhibition of pepsin presence of many active compounds. The various
enzyme. Strong inhibition was noticed in methanol plant active molecules exhibited as anti-HIV
stem extract followed by ethanol stem extract, properties, such as flavonoids (Mantas et al., 2000),
methanol flower extract and ethanol flower extract terpenoids (Sun et al., 2003), cardiac glyucosides
while other extracts with negligible toxic effect (Prinsloo et al., 2010), tannin (Sakagami et al.,
(Table 2). 1999), steroids (You et al., 2003), saponins
(Konoshima et al., 1995), coumarins (Zhou et al.,
Table 2. Effect of Crotalaria pallida constituents on 2000) and athraquinones are under study (Schinazi
HIV protease inhibition
et al., 1990).
Sample Absorbance at 280 The structure of the dimeric enzyme of HIV-
nm 1-PR superficially resembles that of other aspartic
Control (without any extract/ proteases such as pepsin (Hutchins et al., 1991;
0.4212+0.0085a
inhibitor) Wlodawer and Erickson, 1993). However, whereas
Control with Pepstatin A 0.0016+0.0085 g pepsin exists as a 326-residue monomer, with two
differing domains forming a cleft containing the
Control with methanol leaf
0.2430+0.0085c active site, HIV-1-PR forms a similar groove in the
extract
interface between its two 99-residue subunits. As in
Control with methanol flower
0.0046+0.0085d pepsin, HIV-1-PR has two highly mobile arms of
extract
Control with methanol stem about 10 residues each, which surround and anchor
0.0029+0.0085f the substrate in the region of the active site. The
extract
Control with ethanol leaf flaps themselves are not necessary for enzyme
0.3568+0.0085b activity, although the absence of flaps reduces
extract
Control with ethanol flower d enzymatic activity (Chatfield and Brooks, 1995;
0.0051+0.0085
extract Silva et al., 1996).
Control with ethanol stem e The main aim of our present investigation is
0.0036+0.0085
extract the screening of Crotalaria pallida phytochemical
Repeated the each experiments thrice, +: presence, -: extracts for HIV protease inhibition. The plant has
absent, According to Duncan’s Multiple Range Test potential antimicrobial compounds, two solvent
(DMRT), values followed by different subscripts are extracts of Crotalaria pallida showed a very
significantly different at P<0.05, SE-standard error of
significant inhibition of pepsin enzymatic activity.
the mean.
The previous reports suggested that there are close
structural and functional similarities between pepsin
DISCUSSION and HIV protease. The plant extracts have showed
AIDS-related diseases remain one of the inhibitory activity of pepsin enzyme, may be these
leading causes of death globally. The declines in extracts inhibit the activity of HIV protease. Our
new infections and AIDS-deaths may be attributed results, the methanol and ethanol extracts of stem
to the scale-up of anti-retroviral therapy (ART) and flower have proven potential inhibition of the
programmes, especially in the developing world. pepsin enzyme activity due to the different
Presently, no reliable and friendly treatment can be phytochemical constituents, may be our extracts
claimed to combat this disease. The current anti- have strong inhibitory activity of HIV protease.
HIV drugs have focused on reverse transcriptase Many similar works has been done with plant
288 Journal of Research in Biology (2011) 4: 285-291
Govindappa et al.,2011

extracts (Cos et al., 2004; Debouck, 1992; Aiken Scientific Research 5(3):212-215.
and Chen, 2005; Polya, 2003). The current study
can append one more alternative HIV protease Chinsembu KC and Hedimbi M. 2010.
inhibitor to solve the problem especially arresting Ethanomedical plants and other natural products
the HIV replication. But, it needs further with anti-HIV active compounds and their putative
characterization of active molecules in the extracts, modes of action. International Journal of
purification and mode of action on HIV replication Biotechnology and Molecular Biology Research 1
is needed. (6):74-91.

ACKNOWLEDGEMENTS Cos P, Maes L, Berghe DV, Hermans N, Pieters
We thank Dr MR Hulinaykar, Managing L and Vlietinck A. 2004. Plant Substances as Anti-
Trustee, Sri Shridevi Charitable Trust (R.) and Dr HIV Agents Selected According to Their Putative
MA Venkatesh, Principal, SIET, Tumkur, India for Mechanism of Action. J. Nat. Prod. 67(2):284-293.
encouragement, Dr P Sharanappa, DOS in
Biosciences, Hemagangothri, University of Mysore, Debouck C. 1992. The HIV-1 Protease as a
Hassan, India for assistance in identifying plant. Therapeutic Target for AIDS. AIDS Research and
Human Retroviruses 8(2):153-164.
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Original Research paper
Publication group

Partial characterization and optimization of protease production from
newly isolated Cohnella thermotolerans from Lonar Lake
Journal of Research in Biology

Authors: ABSTRACT:
Tambekar DH and
Tambekar SD.
Lonar Lake, an impact crater located in the Buldhana district of Maharashtra
State, India is occupied by saline water and harbors various unidentified, unique
haloalkaliphilic bacterial bacillus species which produces thermo-halo-alkaliphilic
Institution: proteases. The present study deals with the isolation, production dynamics,
Post Graduate Department purification, characterization and optimization of a protease from Cohnella
of Microbiology thermotolerans isolated and identified by 16s RNA ribotyping from the Alkaline Lonar
S.G.B. Amravati University Lake. The C. thermotolerans produced protease at maximum rate after 72 h of
Amravati – 444604 (India). incubation at 370C with the agitation speed of 120 rpm and 5% of starter culture. The
best carbon sources for this C. thermotolerans were fructose and maltose and best
nitrogen source was yeast extract, while the most effective inorganic nitrogen source
was ammonium carbonate. Supplementation of the culture medium with amino acid L
-glutamic acid and metal ion Mg2+ improved the protease production substantially.
Under these conditions, newly isolated Cohnella thermotolerans strain was found to
Corresponding author: produce alkaline protease at a maximum rate of optimum pH 10 and temperature at
Tambekar DH 750C.

Keywords:
Email: Alkaline Protease, Cohnella thermotolerans, Environmental factors,
diliptambekar@rediffmail.com Nutritional conditions.

Article Citation:
Web Address:
http://jresearchbiology.com/ Tambekar DH and Tambekar SD.
Documents/RA0073.pdf. Partial characterization and optimization of protease production from newly isolated
Cohnella thermotolerans from Lonar Lake
Journal of research in Biology (2011) 4: 292-298

Dates:
Received: 02 Aug 2011 /Accepted: 11 Aug 2011 /Published: 30 Aug 2011

© Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

292-298 | JRB | 2011 | Vol 1 | No 4
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An International Open Access Online Submit Your Manuscript
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Tambekar et al.,2011

INTRODUCTION MATERIALS AND METHODS
LONAR Lake, an impact crater located in Collection of Lonar lake water and sediment
the Buldhana district of Maharashtra State, India is sample: Totally four sediments and eight water
a circular lake occupied by saline water which was samples were collected in the year 2009 from
formed by meteoritic impact on basaltic rock and it alkaline Lonar Lake. Water samples collected in
has been well known as an inland saline lake with a sterilized plastic cans and sediments in sterilized
considerable amount of sodium carbonate and plastic bags were transferred to laboratory for
chloride (Kanekar et al, 2002). Extracellular isolation and identification of bacteria followed by
enzymes like amylase, lipase, protease and their screening for proteolytic activity. During
cellulases producing Bacillus cereus, Bacillus sample collection the date, time and places were
firmus, Enterococcus caseliflavus, Bacillus noted.
fusiformis, Bacillus cohnii, Bacillus horikoshii were Isolation of Alkaliphiles: About 1.0 g of soil
isolated from water and sediments of alkaline Lonar sample was transferred to 99.0 ml sterilized normal
Lake (Joshi et al, 2008). Proteases are industrially saline in 250 ml conical flask and agitated (100
important enzymes used in the detergent, food, rpm) at 370C for 15 minutes in water bath shaker.
pharmaceutical and leather industries and also have The sample was then heated at 800C for 15 minutes
application in silver recovery from photographic to destroy all the vegetative microbial cells. The
plates and in peptide synthesis which account for suspension was then diluted to 10-7 dilutions. One
about 60% of total industrial enzyme sales ml of each diluted sample was lawn into petri plates
(Horikoshii, 1999). The detergent industry is the containing nutrient agar medium (pH 10) and
largest single market for this enzyme. The enzyme inoculated at 370C for 24 hours.
has better resistance to alkali and some other Screening of bacterial alkaliphiles: Individual
denaturing chemicals in the reaction mixture and bacterial colonies were screened for proteolytic
has a higher affinity towards proteinaceous activities on Skim milk agar medium (skim milk
substrates. It is also a thermostable organism 1%, Peptone 1%, sodium chloride 0.5%, Agar-Agar
growing in naturally alkaline habitats may have 2%, pH 10). The pH of the medium was adjusted to
proteases with special characteristics (Norazizah et 10 with 1N NaOH before sterilization at 121oC for
al, 2005). Therefore, attempt was made to isolate 15 minutes. The inoculated plates were incubated at
new species of bacillus which can produce good 37oC for 72 hrs and observed for zones of
quality of proteases useful in the detergent and clearance, indicating proteolytic activities.
leather industry. A 16S rRNA gene sequence Identification of the proteolytic isolates: The
analysis had been made for identification of the bacterial isolates with prominent zones of clearance
isolated species. on casein agar medium were processed for
Alkaline proteases produced by Cohnella identifications based on morphology, Gram
thermotolerans are of great importance in detergent characteristics, motility, citrate utilization, oxidase,
industry due to their high thermo-stability and pH urease, gelatin liquification, catalase, Vogous-
stability and most important industrial enzymes proskaur, indol tests and acid production from
accounting for about 60% of total enzyme market glucose, arabinose, lactose, mannitol, galactose and
(Kanekar et al, 2002; Borsosi et al, 2005). Very less maltose. The isolates were also tested for their
study had been done on protease from Bacilli of growth at different temperatures and pH. These
Lonar Lake which can withstand high temperature isolates were identified in accordance with the
as well as high pH and has wide applications in methods recommended in Bergey’s Manual of
different industries. As there is large demand of Determinative Bacteriology and Diagnostic
proteases, isolation and production of protease Microbiology. The identified strains were
enzyme is most important to fulfill this demand maintained on nutrient agar slants having pH 10 at
(Srinivasan et al, 2009). The present study aims to 4.0 oC.
deal with the isolation, purification, and Phylogenetic analysis: 16S rRNA sequencing was
characterization and production optimization of a performed at NCCS, Pune. Nearly-full-length 16S
protease from Cohnella thermotolerans isolated rRNA gene sequences were submitted to CHECK-
from the alkaline Lonar Lake. CHIMERA, available on the Ribosomal Database
Project release 10.26, in order to identify chimeras.
Phylogenetic analyses were performed using the
ARB software package. The 16S rRNA gene
293 Journal of Research in Biology (2011) 4: 292-298
Tambekar et al.,2011

phylogenetic analyses were performed by the read at 650 nm (Lalitha et al, 2010).
maximum-likelihood method, using 1,292 Determination of proteolytic activity: Proteases
nucleotide positions. The functional genes were activity was determined by a slightly modified
translated into amino acid sequences, and these method of Yang et al, 2001. The reaction mixture
were included in phylogenetic analyses using the containing 1 ml of 1.0 % casein solution in 0.2 M
neighbor-joining method (Dayhoff PAM model). Glycine-NaOH buffer having pH 10.5 and 1 ml of a
Preparation of crude enzyme extracts: The 100 given enzyme solution was incubated at 40oC for 10
ml Yeast extract casein medium (Glucose 1%, minutes and the reaction was then stopped with 2
Casein 0.5%, yeast extract 0.5%, KH2PO4 0.2%, ml of 10 % tri-chloroacetic acid (TCA). The
K2HPO4 0.2%, MgSO4 0.1%, pH.10.5) was amount of tyrosine liberated was determined as per
dispensed (50 ml each) into two 250 ml capacity tyrosine assay procedure at 650 nm. The proteolytic
conical flasks, after adjusting the pH to 10.5 and unit was defined as the amount of the enzyme that
sterilized in autoclave. After cooling, the broth was released 1ug of tyrosine per minute under the assay
inoculated with Cohnella thermotolerans cultures conditions.
and incubated for 72 h at 370C in shaking incubator. Partial characterization of protease: Partial
After 72h incubation, centrifuged the broth at 5000- characterization of protease was carried out using
8000 rpm for 15 min. The supernatant served as methodology. (Joo et al, 2002).
crude enzyme source. Effect of pH on alkaline protease activity: The
Optimization of crude enzyme protease: The effect of pH on alkaline protease from Bacillus spp.
standard graph of tyrosine was prepared by adding was determined by assaying the enzyme activity at
different concentration of standard tyrosine (1 mg/ different pH values ranging from 7.0 to 10.5 using
ml) into a series of test tubes and made the final the following buffer systems with concentration of
volume in each test tube to 1 ml with distilled each buffer at 0.2 M: phosphate (pH 6-7) tris–HCl
water. Estimation of proteases was carried out with (pH 8-9) and Glycine-NaOH (pH 10-12).
1 ml of casein in a test tube; 1 ml of enzyme source Effect of temperature on alkaline protease
was added and incubated for 10min at room activity: The effect of temperature on alkaline
temperature. After incubation 2 ml of TCA was protease activity was determined by incubating the
added to stop the reaction and centrifuged the reaction mixture (pH 10.5) for 20 minutes at
reaction mixture at 5000-8000 rpm for 15 min. different temperature ranging from 55oC to 90oC.
Supernatant was separated and 1ml of Folin- Effect of substrate on alkaline protease activity:
Ciacalteau reagent and 2 ml of Na2CO3 were added The effect of substrate concentration on alkaline
in 1 ml of supernatant. The reaction mixture was protease activity is determined by incubating the
boiled for 1 min in a boiling water bath and 6 ml of reaction mixture (pH 10.5) for 20 minutes with
distilled water was added to make a final solution to different substrate concentration, ranging from 5
10 ml. In control tube, the reaction was terminated mg/ml to 40 mg/ml.
the reaction at zero time and the absorbance was

Journal of Research in Biology (2011) 4: 292-298 294
Tambekar et al.,2011
Fig. 4: Effect of Temperature on enzyme
Fig. 3: Effect of pH on proteases activity activity
160
140

120
140
100

Tyrosine (µg/ml)
Tyrosine (µg/m)l

80
120
60

40 100

20

0 80

7 7.5 8 8.5 9 9.5 10 10.5 55 60 65 70 75 80 85 90
pH Temperature in degree centigrade

Effect of enzyme on alkaline protease activity: RESULTS AND DISCUSSION
The effect of enzyme concentration on alkaline In the present study, a total of 104 bacterial
protease activity was determined by incubating the isolates were isolated from water and sediment
reaction mixture (pH 10.5) for 20 minutes at sample of Lonar Lake, maintained on slant of
different enzyme concentration ranging from 0.5ml nutrient agar (pH 10.5) and various tests were
to 4ml. The activity of the protease was then performed for identification of bacteria. Out of
measured as per assay procedure. them 67 were from water and 37 from sediments.
Purification of enzymes: Purification of enzyme Then these cultures were inoculated on alkaline
will be made by chilled acetone, isopropyl alcohol skim milk agar at pH 10.5 for studying their
and Ammonium sulphate precipitation method. proteolytic activity and using morphological and
Optimization of environmental and nutritional biochemical characteristics. Out of 104 cultures, 37
conditions for the production of alkaline isolates were identified as Bacillus. Out of 37
protease: Optimization of proteases production was isolates of sediments, only 15 isolates were efficient
studied with the help of fermentor by the in protease production and most efficient bacillus
optimization of medium composition (variation in species were used to study different enzyme
carbon, nitrogen sources and metal ions) and parameter. Among them, a bacterial culture
environmental conditions such pH, temperature, identified as Cohnella thermotolerans by 16S
incubation period etc (Kumar et al, 2002). The rRNA analysis were used for detail study of
present investigation was aimed at optimization of protease production and optimization (Table 1).
medium components which have been predicted to Alkaline protease production was maximum at pH 9
play a significant role in enhancing the production -10.5. Maximum protease production was recorded
of alkaline proteases. Carbon sources chosen for the after 72 h of incubation at 37°C. In the effect of
study were glucose, sucrose, starch, fructose, substrate concentration on enzyme activity of
maltose and lactose. These carbons occur which led protease, the Michalies Menten constant (KM) and
to cell lysis and increased cell sources were used to Maximum velocity (VMax) was found to be 9.09 mg/
replace the carbon source available permeability ml and 0.018 mg/ml by Line weaver-Burk plot. The
due to abrasion by shear forces (Gupta et al, 2002). optimum enzyme concentration required for
Sources of nitrogen include organic nitrogen, maximum activity of protease is 3.5 ml.
inorganic nitrogen and amino acid in which sources Effects of carbon sources on protease
throughout the study were soy tone, soya bean cake, production: It has been reported that pure sugars
beef extract, yeast extract, peptone, ammonium affected protease production considerably.
nitrate, ammonium carbonate, urea, lysine, L- Utilization of pure sugars as carbon and energy
aspartic acid, glutamic acid and glycine. Metal sources was also shown to result in good growth
cations tested to replace metal ion source in the with increased protease production. This
Media were Ca2+, Cu2+, Mg2+ and Mn2+ observation was in agreement with previous studies
(Adinarayana et al, 2003). which suggested that larger amount of enzyme was
synthesized when carbon sources used were poorly
utilized for growth purposes (Ismail and Fattah,
295 Journal of Research in Biology (2011) 4: 292-298
Tambekar et al.,2011

Fig. 5: Effect of pH on protease production Fig. 6: Effect of temperature on protease
production
80 80

70 70
Tyrosine (ug/ml)

Tyrosine (ug/ml)
60
60
50
50
40
40
30

20 30
7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 55 60 65 70 75 80 85 90
pH Temerature in degree centigrade

1999). Various sources of carbon such as glucose, nitrogen. Despite the luxurious bacterial growth the
fructose, sucrose, maltose, starch and lactose were presence of beef extract, peptone and tryptone
used in enhancing the production of alkaline resulted in low protease production. This
proteases. Results obtained showed that starch observation contradicted (Phadatare et al, 1993)
instigated highest protease production compared to which reported that protease production in
other carbon sources at 72 h of incubation due to Conidiobolus coronatus was enhanced by organic
the prolonged incubation time perhaps led to auto nitrogen sources like yeast extract, peptone and
digestion of proteases and proteolytic attack by tryptone.
other proteases. Fructose, maltose and lactose Effects of inorganic nitrogen sources on protease
caused slightly low protease production. production: Inorganic nitrogen sources like
Effect of nitrogen sources on protease ammonium carbonate, ammonium nitrate and urea
production: were tested on the growth and protease production.
Effect of organic nitrogen sources on protease Among them, ammonium carbonate led to high
production: In this study, sources of organic protease activity at 48 and 72 h. Urea did not
nitrogen like soy ton, soya bean cake, beef extract, enhance protease production at early stages of
yeast extract and peptone were used. It was showed incubation but at later stages (48 and 72 h) protease
that soy ton resulted in the highest level of protease production increased. Even though growth was
activity compared to other sources of organic stimulated, only moderate levels of enzyme

Fig 7: Effect of carbon sources on Fig 8: Effect of nitrogen sources on
protease production protease production
90 140
120
Tyrosine (µg/ml)

100
Tyrosine (µg/ml)

60 80
60
40
30 20
0
Ammonium nitrate
Peptone

Ammonium
Soy tone

Soya bean cake

Yeast extract

Urea
Beef extract

carbonate

0
Glucose
Fructose

Maltose
Lactose

Sucrose
Starch

Carbon sources Nitrogen sources

Journal of Research in Biology (2011) 4: 292-298 296
Tambekar et al.,2011

activities were obtained when ammonium nitrate protease at pH 9 and temperature at 800C.
was used as a nitrogen source. This was perhaps In su m mar y, i sol a t ed Cohn el l a
due to the inability of bacteria to utilize ammonia in thermotolerans species from Lonar Lake produce
the media (Ellaiah et al, 2005). alkaline protease and maximum growth at pH 9-
Effects of amino acid on protease production: 10.5. The isolated bacterial C. thermotolerans strain
This observation was corroborated by L- produces the proteases enzyme which was
aspartic acid; glutamic acid and glycine were also theomorphic, alkaliphilic and has potential to
tested as sources of amino acids for protease produce good quality proteases which can use in the
production in Cohnella strain 146. In the presence industry. The C. thermotolerans species were most
of Glycine, protease production was observed to be efficient for protease producing at pH 10.5
high. incubated at 370C for 72h. The protease produced
Effects of metal ions on protease production: The from this species were highly efficient at high
highest level of protease activity was observed in temperature, high salt concentration and tolerate the
the presence of Mg2+ at 72 h incubation. Addition other environmental conditions. This bacterial
of Ca2+, Cu2+ and Mn2+ resulted in high protease species is ubiquitous and non-pathogenic, not
production only at 48 h incubation. It was suggested causing any diseases to human beings and most
that these metal ions increased stability of efficient for protease production among all isolated
proteases, even though effects of the different metal protease-producing bacteria. Protease enzymes have
cations on protease production vary, their presence importance in various industries. Bacillus species
in the culture medium improved the growth of particularly Cohnella thermotolerans, were known
Cohnella strain. Supplementation of culture for their ability to produce proteolytic enzymes with
medium with metal cations improved substantially potential use in industries (Kampfer et al, 2006). In
the protease production of Cohnella addition to the limited number of reports, protease
thermotolerans. This observation strongly production by this microorganism also was shown
suggested the requirement of some metal ions for to be affected by various environmental and
protease production by this organism. These results nutritional conditions. In the present investigation,
were in agreement with the earlier findings which we have determined the optimum parameters for
showed enhancement of protease activity in the maximum production of alkaline protease by the
presence of metal ions and it was suggested that newly isolated thermophilic bacterium Cohnella
these metal ions increased stability of proteases. thermotolerans. This information has enabled the
(Banerjee et al, 1999). ideal formulation of media composition for
Effect of pH and temperature on protease maximum protease production by this organism.
production: This observation was corroborated by
different pH and Temperature range. Cohnella
thermotolerans strain produced maximum alkaline
297 Journal of Research in Biology (2011) 4: 292-298
Tambekar et al.,2011

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Adinarayana K, Ellaiah P, Prasad DS. 2003. microbiology 56:781-786.
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isolated Bacillus subtilis PE-11. AAPS Pharm Sci Kelkar AS. 2002. Optimization of protease activity
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Journal of Research in Biology (2011) 4: 292-298 298
Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

Biochemical responses and proline metabolism in Amaranthus tricolor L.
and Phaseolus vulgaris L. under in vitro NaCl Stress.
Journal of Research in Biology

Authors: ABSTRACT:
Sunukumar SS, Harish SR,
Manoj GS, Remya Salinity is currently the major factor which reduces crop yields. One of the
Krishnan and biological approaches is to use salt tolerant plants. Amaranthus tricolor L. has been
Murugan K.
used as a promising plant to ameliorate the salt affected area. The objective of the
study is to evaluate the effect of NaCl stress on synthesis and catabolism of proline,
soluble proteins, carbohydrates and Na+/K+ ratio in A. tricolor and to compare with the
salt-sensitive species, Phaseolus vulgaris L. The experiments were designed with six
Institution: replications. Seedlings of both species were grown hydroponically with 0, 50, 100,
Plant Biochemistry and 150, 200, 250 and 300 mM NaCl. The in vitro activity of the enzyme pyrroline 5-
Molecular biology Lab, carboxylate synthetase under NaCl stress was higher, while, the activity of proline
Department of Botany, degrading enzyme - proline oxidase showed a reverse trend i.e., low activity in high
University College, NaCl concentrations. Soluble protein content was increased in the shoot of A.tricolor
Thiruvananthapuram, but decreased in P.vulgaris. Roots of both the species showed variation in the protein
Kerala-695 034, India. content. Proline content of shoot and roots increased significantly in all the
treatments in the plants. However, A.tricolor showed a higher level. The total
carbohydrate also showed a similar trend. High level of NaCl decreased the reduced
sugar in shoots and roots of the species. Salt stress increased Na + significantly and
decreased the K+ content in both species. The biochemical variation may be
Corresponding author: interpreted as differential response of the plants to NaCl stress.
Murugan K

Email: Keywords:
harimurukan@gmail.com Amaranthus tricolor, ions, proline, salt stress, soluble proteins.

Web Address: Article Citation:
http://jresearchbiology.com/
Sunukumar SS, Harish SR, Manoj GS, Remya Krishnan and Murugan K.
Documents/RA0083.pdf.
Biochemical responses and proline metabolism in Amaranthus tricolor L. and
Phaseolus vulgaris L. under in vitro NaCl Stress.
Journal of research in Biology (2011) 4: 299-306

Dates:
Received: 12 Aug 2011 /Accepted: 27 Aug 2011 /Published: 30 Aug 2011

© Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

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Sunukumar et al.,2011

INTRODUCTION higher plant systems (Kavi Kishor et al., 2005).
Agricultural productivity is severely affected Amaranthus tricolor generally grows in the dry arid
by soil salinity because salt levels that are harmful soils of Kerala. Due to its vigorous growth and
to plant growth affect large terrestrial areas of the drought tolerance, this plant has been used as a
world. Efforts to improve crop performance under potential green manure plant. Until now, a few
environmental stresses have not been fruitful studies have been conducted on the mechanism of
because the fundamental mechanisms of stress salt tolerance in this plant. The objective of this
tolerance in plants remain to be completely study is to obtain a better understanding on salt-
understood (Nyagah and Musyimi, 2009). Soil tolerance mechanism in A. tricolor by determining
salinity affects plant growth in a variety of ways, the physiological and biochemical responses of the
reducing water uptake, causing toxic accumulation plant to NaCl in comparison with Phaseolus
of sodium and chloride, and reducing nutrient vulgaris.
availability. Salinity also induces water deficit even
in well-watered soils by decreasing the osmotic MATERIALS AND METHODS
potential of soil solutes, thus making it difficult for Seeds of A. tricolor (obtained from
roots to extract water from their surrounding media Agricultural University, Trichur, Kerala) were
(Jaleel et al., 2007e), Excessive sodium inhibits the soaked in concentrated H2SO4 for 30 min to break
growth of many salt-sensitive plants, which dormancy. The seeds were surface-sterilized with
includes the most crop plants. The typical first NaClO (1.25% active chlorine) under vacuum for
response of all plants to salt stress is osmotic 15 min and rinsed several times with sterile distilled
adjustment. Compatible solute accumulation in the water and soaked in it for 12 h at 25°C. Fifteen
cytoplasm is considered a mechanism to impart salt seeds were placed in a petri dish with two layer
tolerance (Jaleel et al., 2007e) Complex molecular filter papers. Ten ml of 0, 50, 100, 150, 200, 250
responses including the accumulation of compatible and 300 mM NaCl solution was sprayed into the
solutes, the production of stress proteins and the into petri dish. They were then kept at room
expression of different sets of genes are part of the temperature. Germination rate were determined
plant signaling and defense system against salinity. seven days after treatment. The experiment was
It is well known that one of the most common designed with six replications.
responses to salinity is the overproduction and Seeds of A. tricolor were broken their
accumulation of proline, glycine betaine and total dormancy and then surface sterilized as described
sugars. Solute accumulation by cells is one such above. They were then soaked in sterile water at
mechanism (osmoregulation) which contributes to 25°C for one night. Phaseolus vulgaris L. seeds
stabilization of enzyme/protein and turgor were incubated on moist filter paper at 30°C for 2
maintenance in growing organs and has been days in darkness. Germinated seeds were planted in
correlated with productivity under stress (Delauney a container containing vermiculite and grown at 25°
and Verma, 1993). C for 2 days in darkness with aluminum foil cover.
The accumulation of free proline has been Then a half strength of MS medium (1962) was
studied in a number of taxa subjected to added to the vermiculite. After 5 days, seedlings
hyperosmotic stress conditions for over years were transferred to a hydroponic culture with the
(Delauney and Verma, 1993). The accumulation of same nutrient solution. A. tricolor and P. vulgaris
proline under abiotic stress conditions accounts for were grown to the 3rd to 4th and to the 1st to 2nd leaf
few millimolar concentrations, depending on the stage respectively. Salt treatment was given by
species and the extent of stress. Very high adding 50 mM NaCl to the solution. For the
accumulation of cellular proline (up to 80% of the treatments with higher concentrations, plants were
amino acid pool under stress and 5% under normal transferred to 100, 150, 200, 250 and 300 mM at
conditions) due to increased synthesis and two-day intervals. The NaCl solution containing
decreased degradation under a variety of stress nutrient was renewed every 4 days. Plants were
conditions such as salt and drought has been grown in a growth chamber at 25/20°C day/night
documented in many plant species. Although temperatures, with a 14 h photoperiod at 280- 290
proline is known to confer osmotic tolerance during μE/m2/s, at a relative humidity of 70-80%.
stress conditions, its specific role during plant Seedlings were harvested 14 days after starting
growth is not completely clear. The biosynthetic NaCl treatment. All experiments were designed
pathway of proline diverged between bacteria and with six replications.
300 Journal of Research in Biology (2011) 4: 299-306
Sunukumar et al.,2011

Shoots and root were dried at 70oC for 24 h and extracted with 5 mL of Tris-HCl buffer (pH 8.5)
weighed. Sodium, chloride and potassium were grinding medium and centrifuged at 10,000 g for 10
extracted from each sample in a mixture of HNO3, min at 4°C. The supernatant was again centrifuged
HClO4, and distilled water (1: 5: 2.5). The solution at 25,000 g at 20 min at 4°C. A 3-mL assay mixture
was kept for 12 h at 100οC, diluted with 100 mM was prepared by taking 0.1 mL of extract, 1.2 mL
HC1, and analyzed by atomic emission of 50 mM Tris HCl buffer (pH 8.5), 1.2 mL of 5
spectrometry. Soluble proteins were extracted from mM MgCl2, 0.1 mL of 0.5 mM NADP, 0.1 mL of 1
young shoots and roots in an extraction buffer (0.01 mM KCN, 2 0.1 mL of 1 mM phenazine
M Tris-HCl, 10% glycerol, 5% PVP, 1% Triton X methosulphate (PMS), 0.1 mL of 0.06 mM 2, 6-
100, pH = 6.8) and protein assay was carried out dichlorophenol indophenol (DCPIP) and 0.1 mL
according to the method of Bradford, (1976). distilled water instead of PRO. The reaction was
For proline determination, 10 mL of 3% (W/ monitored at 600 nm at 25°C using PRO to initiate
V) aqueous sulfosalicylic acid solution was added reaction; the increase in OD values increase were
to 1 g of fresh shoot and root samples and was noted at 0, 1, 2, 3, 4 and 5 min. PO activity was
homogenized, filtered through one layer of expressed in U (one U = mM DCPIP reduced min-1
Whatmann, No. 1, filter paper, then proline was mg-1 protein).
assayed according to the method of Bates et Five independent determinants from
al ,1973. Total free amino acids were extracted and individual plants were used for statistical analysis.
estimated as per the method of Walter Troll and Student’s t test and analysis of variance (ANOVA)
Keith Cannan (1952). were used for analyzing significant differences
Carbohydrates were extracted from dry between the control and treated plants (P < 0.05).
shoots and roots of plantlets in warm water.
Concentration of total and reduced sugars were RESULTS AND DISCUSSION
determined based on the methods of Dubois, (1956) Total protein and proline content of
and Jeffries,(1998) respectively. A.tricolor and P. vulgaris shoots were increased
Enzyme extraction was based on the method moderately by salt treatment up to 150 mM NaCl
of Chen et al, (2001). Seedlings were homogenized and thereafter declined sharply at higher NaCl
in prechilled mortar and pestle in the extraction concentrations (Data not shown). In the roots the
medium at 4° C. The extraction medium contained protein and proline content increased at all NaCl
100 mM potassium phosphate buffer (pH 7.4), 1 concentrations tested, although a decline was
mM EDTA, 10 mM 2-mercaptoethanol, 1% (w/v) apparent at 300 mM (Table 1 a and b).
PVP, 5 mM MgCl2, and 0.6 M KCl. The Nevertheless, the protein and proline contents of
homogenate was centrifuged at 12000 g for 20 min severely stressed roots were lower than those of
at 4°C, the resulting supernatant was kept at −20°C moderately stressed ones, but still higher than the
and used for enzyme assaying. control. However, seedlings of P. vulgaris wilted or
P5CS activity was assayed according to the died at higher NaCl concentrations (100 mM and
method of modified Vogel and Kopac, (1960) as above). Significant positive relationship was
follows. 0.7 mL reaction medium containing 50 examined between salinity and total protein and
mM Tris (pH 7.5), 2 mM MgCl2, 10 mM ATP, 1.0 proline content (P<0.05).
mM NADH, 50 mM glutamic acid, and 0.1 mL The amount of soluble amino acids
enzyme extract was incubated at 37°C for 30 min. increased in both plants with increasing NaCl
The reaction was then stopped by adding 0.3 mL of concentration. At the highest concentration,
10% (w/v) trichloroacetic acid. Color reaction was although the total amino acid was clearly built up in
developed by incubating with 0.1 mL of 0.5% (w/v) both the shoots and roots of A. tricolor, their
o-aminobenzaldehyde for 1 h. After centrifugation accumulation was more obvious in shoots (Table 1a
at 12000 g for 10 min, the clear supernatant fraction and b). Similarly, the free amino acids
was taken to measure the absorbance at 440 nm. accumulation was also found in P. vulgaris at the
Enzyme activity was calculated using the extinction higher concentration (100 and 150 mM). The data
coefficient of 2.68 / (mM cm). was statistically significant at 1% level.
Proline oxidase (PO) activity was determined We found significant difference in total
according to the method outlined by Huang and carbohydrate and reduced sugar between the two
Cavalieri, (1979). Plant samples (1 g) were species under NaCl treatment. For example, at 150

Journal of Research in Biology (2011) 4: 299-306 301
Sunukumar et al.,2011

Table 1a and b. Amino acid contents (mg/g DW) in NaCl treated A.tricolor and P. vulgaris 14 days after starting
NaCl treatment.
Table 1a
NaCl Roots Shoots
(Mm) Free aminoacids Total proteins Proline Free aminoacids Total proteins Proline
0 2.5 ±0.9 3.6±0.5 0.4±1 3.8.2±1.9 4.1±0.3 0.53±0.01
50 5.9±0.29 4.0±0.4 0.52±0.29 12.7±2 5.2±0.09 1.2±0.03
100 9.2±0.35 4.8±0.25 1.5±0.38 16.9±3.2 6.5±0.26 1.5±1.9
150 11.4±2.9 5.7±0.11 2.2±0.7 23±0.99 7.8±0.38 2.9±2.3
200 12±2.6 6.3±0.23 2.2±0.5 27.8±0.65 8.5±0.4 3.3±3.9

Table 1b
Roots Shoots
NaCl
Total Total
(Mm) Free aminoacids Proline Free aminoacids Proline
proteins proteins
0 3.5 ±0.91 4.0±0.5 0.2±0.45 8.8±1 4.8±2.1 0.5±0.2
50 8.3±0.8 5.4±1.5 0.3±0.12 11.3±2.1 5.6±0.7 0.8±0.34
100 13.1±0.45 6.9±2 0.81±0.33 19.2±2.7 7.0±0.9 0.6±0.5
150 22.4±0.78 7.2±1.7 0.6±0.65 23±2.9 8.0±2.8 0.5±0.8
The data are the mean of six replicates ± S.E. Significant at 0.01 levels. P < 0.05.
mM NaCl total carbohydrate in A.tricolor 18.6 mg observed in P. vulgaris, while it slightly reduced in
g-1 DW while in P. vulgaris was only 8.4 mg g-1 A.tricolor (Fig. 5). The ion analyses in individual
DW. A similar but moderate pattern of leaf showed that Na+ were tended to accumulate in
carbohydrate changes was observed in roots. the older leaves with higher concentration, but roots
Reduced sugar of shoot and root in both the species in P. vulgaris (Fig. 6 a and b).
was decreased when plants are exposed to NaCl ∆1-Pyrroline-5- carboxylate synthetase (P5CS)
stress (Fig. 1 and 2). shows a positive correlation with NaCl
Na+ and Cl¯ contents were enhanced with concentrations up to 150 mM in shoot and root of
increasing salinity in roots and shoot of A.tricolor A.tricolor, but P. vulgaris showed decrease in the
and P. vulgaris (Fig. 3 and 4) However, their activity of P5CS at 100 mM NaCl concentration
accumulation was greater in shoots than in roots in (P< 0.05) (Fig. 7). The trend of change in the
A.tricolor. In the case of P. vulgaris the pattern of activity of P5CS corroborates with the proline
accumulation was different i.e. higher in roots than accumulation in different NaCl treatments in the
in shoots. K+ content on the other hand has been species. The level of proline degrading enzyme,
reduced markedly in both the species. Instability of proline oxidase (PO) activity was inhibited to a
Potassium (K+) content in shoots and roots was large extent in both shoot and roots of A.tricolor by
Shoot Tot. Carbo Shoot Tot. Carbo
Total carbohydrates & Reduced

25 10
Reduced sugar (mg/g DW)

Root Tot. Carbo 9 Root Tot. Carbo
Total carbohydrates &

20 Shoot Red.Sugar 8 Shoot Red.Sugar
sugar (mg/g DW)

7 Root Red.Sugar
Root Red.Sugar
15 6
5
10 4
3
5 2
1
0 0
0 50 100 150 200 0 50 100 150
NaCl concentrations (mM) NaCl concentrations (m M)

Figure 1. Total carbohydrates and reduced sugar in Figure 2. Total carbohydrates and reduced sugar in
shoots and roots from NaCl-treated A.tricolor 18 days shoots and roots from NaCl-treated P.vulgaris 18 days
after starting NaCl treatment. after starting NaCl treatment.
302 Journal of Research in Biology (2011) 4: 299-306
Sunukumar et al.,2011

Shoot (Na+) Root(Na+)
1400 Shoot(Cl-) Root(Cl-)

1200
Na+ and Cl- content

1000

800

600

400

200

0
0 50 100 150 200
NaCl concentration (mM)

+
Figure 3. Ions distribution (Na+ and Cl- content μmol/g Figure 5. Ions distribution (K mol/g DW) in NaCl-
DW) in NaCl-treated A.tricolor 18 days after starting treated A.tricolor and P.vulgaris 18 days after starting
NaCl treatment. NaCl treatment.
NaCl stress (Figure 8), with the lowest activity was vacuole and balancing by compatible solutes within
recorded at 150 mM of NaCl concentrations (50 the cytoplasm was reported as the important salt
mM in P.vulgaris) compared with control. tolerant mechanisms at cellular level (Bremberger
In plants, higher accumulation of Na+ and and Luttge 1992). It may likely indicate that
¯
Cl requires compatible solutes for osmotic A.tricolor has an ability to translocate the ions and
adjustment under salinity stress (Kavi Kishor et al., hold them in the leaves. This physiological process
2005). In this study, accumulation of Na+ and Cl¯ may be important to reduce the salt toxicity in and
found in the species as a response to changing away from the root cells. As a result the plant could
salinity stress levels. However, a different pattern of
Na+ and Cl¯ distribution in each part of the two
species was clearly seen with increasing salinity.
A.tricolor has a higher accumulation of the ions in
the shoots, but P. vulgaris in roots. The ability to
translocate Na+ and Cl¯from roots and transfer them
to shoots is considered to be one of the mechanisms
of salt tolerance in A.tricolor. High accumulation of
the excessive salts in shoots indicates there is some
mechanism to reduce detrimental effect of the ions
in the shoot cells. Localizing Na+ and Cl¯in the
Shoot (Na+) Root(Na+)
1400 Shoot(Cl-) Root(Cl-)

1200
Na+ and Cl- content

1000

800

600

400

200

0
0 50 100 150 200
Figure 6 a and b. Distribution of Na + and Cl- in leaves
NaCl concentrations (mM)
(a and b), and 1st – 6th leaves of A.tricolor 14 days after
Figure 4. Ions distribution (Na+ and Cl- content μmol/g starting NaCl treatment.◊ 0 mM, ■ 50 mM, ▲ 100
DW) in NaCl-treated P.vulgaris 18 days after starting mM, 150 mM, ● 200 mM of NaCl). Vertical bars
NaCl treatment. indicate standard errors (n = 3).

Journal of Research in Biology (2011) 4: 299-306 303
Sunukumar et al.,2011

protein synthesis that is prevented when the stress
becomes too severe. It would be predicted that
plants under stress would have a powerful protein
turnover machinery to degrade stress damaged and
environmentally-regulated proteins. Proline
generally serves as a physiologically compatible
solute that increases in order to maintain a favorable
osmotic potential between the cell and its
surroundings. Proline accumulation under stress
conditions may either be caused by induction or
activation of enzymes of proline biosynthesis or a
decreased proline oxidation to glutamate or
decreased utilisation of proline in protein synthesis,
Figure 7. Pyrroline 5-carboxylate synthetase (P5CS) or enhanced protein turnover (Delauney, and Verna,
activity in shoot and roots of A.tricolor and P.vulgaris 1993). The expression of genes encoding key
18 days after starting NaCl treatments. enzymes of proline synthesis (P5C synthase, P5C
survive even at the higher salinity levels to which reductase,) and proline oxidation (proline
were subjected during this study. dehydrogenase) is controlled by osmotic and
In the present study, accumulation of total salinity stress and precedes the increase or decrease
free amino acids, proteins and proline was observed of proline concentration in plant tissue (Claussen,
in the roots and shoots of both species. However, at 2005). With increasing of NaCl, the proline content
higher NaCl concentrations seedlings of P. vulgaris of two species increased significantly. Proline
died; suggesting that total protein or proline does accumulation in shoots and, mainly, in roots is
not help in reducing salt damage in this plant. On considered as a marker to salt stress that may be
the contrary, these concentrations had no used to select plants with different degrees of
detrimental effect on the seedlings of A.tricolor; tolerance. Proline may also act as a signaling/
this indicates that higher proline accumulation may regulatory molecule able to activate multiple
contribute to the alleviation of NaCl stress in the responses that are component of the adaptation
plant. Our results indicating that increasing or process (Sankar Ganesh et al., 2009).
decreasing of protein content in plants exposed to Change in soluble sugars content under salt
salt stress is relatively genotype dependent. The stress has already been reported for a number of
effect of salt-stress on protein content depended on species (Nyagah and Musyimi, 2009). Higher
the concentration of NaCl. At lower levels of NaCl, amount of carbohydrates may be one of the reasons
there was an increase in protein content, but higher responsible for higher salt tolerance in A.tricolor. In
concentrations caused it to decline in both shoot the preliminary part of this study we have found out
and root (above 250 mM). This suggests that the that A.tricolor has higher seed germination and
initial response to salt stress involves increased better growth under salt stress conditions.
Some halophytes are known to accumulate
salts in their leaves under salinity stress (Greenway
and Munns, 1980). Glycophytes, on the other hand,
respond to salinity basically by ion inclusion. The
majority of these species accumulate high levels of
Na+ in their roots and stems (Flower, 1991). At
lower NaCl concentration, an ability to localize Na+
and Cl- ions in root cells seemed to be important to
alleviate salinity stress in P. vulgaris. At higher
doses of NaCl, however, they could not resist
toxicity of salt resulting in wilting and death of the
plant. The higher Na+ and Cl- accumulation in
leaves of A.tricolor than P. vulgaris may indicate
Figure 8. Proline oxidase (PO) activity in shoot and that leaf cells of A.tricolor has mechanism to
roots of A.tricolor and P.vulgaris 18 days after starting tolerate higher ions concentration. The replacement
NaCl treatment. of K+ by Na+ observed in P. vulgaris was
304 Journal of Research in Biology (2011) 4: 299-306
Sunukumar et al.,2011

considered to be similar with the previous report in CONCLUSION:
tomato in which the replacement occurred The results obtained showed that two
progressively with increasing absorption of Na+, genotypes studied, A.tricolor was the salt-tolerant
and the absorbed Na+ was transported to the stem and P.vulgaris the salt-sensitive species. Sodium
(Besford, 1978). The high concentration of NaCl and Cl- accumulation in the roots as a result of salt
(150 mM) is not a lethal dose of A.tricolor, while stress appeared to play an important role in the
the lowest concentration (50 mM) proved too strong acclimation of the genotype to salt stress,
dose for the shoots of P. vulgaris. Greater suggesting that they could be used as physiological
accumulation of these ions in the older leaves has markers during the screening for salt tolerance.
been reported in several other plant species and this Total protein, carbohydrates, proline and PRO and
function is considered to be effective in avoiding P5CS enzymes responded distinctly to NaCl stress,
huge accumulations of the toxic salts in the growing suggesting divergent of response mechanisms in the
young leaves (Wahome et al, 2001). species. Genetic evaluation of genotypes based on
Under salt stress one of the mechanisms of salt tolerance indices could be exploited in the
salt tolerance is accomplished by uptake and breeding of salt tolerant genotypes.
accumulation of inorganic ions, mainly Na+, K+ and
Cl- (Dionisio-Sese and Tobita, 2000). In our study ACKNOWLEDGEMENTS:
under salinity, A.tricolor accumulated high level of The authors sincerely acknowledge the
Na+ in shoots in comparison with P.vulgaris. The University Grant Commission, New Delhi for the
rise in Na+ concentrations in the shoots lower the financial assistance.
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