Scientia (Annual) - Mercy College, Palakkad

SCIENTIAPeer Reviewed National Science Journal

Volume 13. No.1 u Jan-Dec.2017 u ISSN: 0976-8289

Published by:

MERCY COLLEGEPALAKKAD 678006, KERALA, INDIA

Jan - Dec 2017

SCIENTIA

Volum

e 13. No. 1. ISSN

: 0976-8289

Scientia (Annual)


Scientia (Annual)Jan. - Dec. 2017Volume 13. No. 1ISSN: 0976-8289

ScientiaPeer Reviewed National Science Journal

Published by:

Mercy CollegePalakkad 678 006,

Kerala, India.Govt. Aided Arts and Science College

Affiliated to university of Calicut, re-accredited with ‘A’ grade in third cycle by NAAC

Telephone: 0491 2541149, Fax: 0491 2542681Website: http//:www.mercycollege.edu.in

Email: [email protected]@gmail.com

Statement of ownership and other particulars

Place of publication : Mercy College Palakkad

Periodicity of publication : Annual

Printers Name and Address : Dr. Sr. Lilly.P.V, Principal, Mercy College Mercy College, Palakkad

ScientiaVolume 13. No.1 Jan-Dec.2017 ISSN: 0976-8289

Editorial Board

Chairperson : Dr. Sr. Lilly P.V., Principal, Mercy College, Palakkad- 678006.

Chief Editor :Dr. Jayasree S.,Associate Professor,Department of Zoology, Mercy College, PalakkadEmail:[email protected], Mob: 9446143023

Editorial Board :

1. Dr.C.P. Biji, Department of Zoology

2. Dr.R. Girija, Department of Zoology

3. Dr. Lakshmi M., Department of Physics

Advisory Board :

Dr. M. Chandrasekaran, Emeritus Professor,Department of Biotechnology,Cochin university of Science and Technology,KOCHI – 682 022. Kerala, IndiaEmail: [email protected]

Dr. P.R. Varghese, Research Coordinator, Jubilee Centre for Medical ResearchJubilee Mission Medical College &Research Institute, Thrissur, 680005, Kerala, India.

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Editorial

“The saddest aspect of life right now is that science gathers knowledge faster than society gathers wisdom.” ― Isaac Asimov

Each day brings new science to my virtual desk, and each day I learn something new. Being an editor is a dynamic, high-energy job, and for me the most satisfying part of it is that I get to publish fantastic scientific stories and interact with many amazing people doing science worldwide. The year 2017 marks the 13th year of publication of Scientia (ISSN: 0976-8289), an annual Peer Reviewed National Science Journal from Mercy college, Palakkad. We are happy to bring out this issue of Scientia which features 14 articles from various areas of science. Volume 13 introduces properties of thin films using van der Pauw technique and explains Vitamin C supplementation effects on silkworm, Bombyx mori L.

Description on the properties of nano structured Li-doped NiO films using the sol gel spin coating method and Manifestations in the epididymis in response to treatment of ursolic acid, a phytotherapeutic: a study in Wistar rat fetches new findings. Impact of environmental pollution was portrayed in the study, Variation of chlorophyll content in five common plant species to environmental stress due to proximity of steel industries. Intervention of life style disease was attempted in An Insilico Study on PPAR γ2 Gene among Obesity related type-2 Diabetes mellitus (T2DM). Concern to the biodiversity was reflected in the studies of Preliminary observations on the behaviour of Hierodula ventralis Giglio-Tos (Mantodea: Mantidae), Kerala, India. Antibacterial and Blood Clotting Properties of Pholcidan Spider Webs was an attempt to prove traditional knowledge through a blend of scientific mixing. Scientia editorial team congratulates all our contributors for their support and achievement attained in the year 2017.

We invite your continued support and contribution of articles/news items of interest/research papers etc. on science.

With warm personal regards

Dr. Jayasree S.Chief Editor

Contents

REVIEW

1. Measurement of temperature dependent electrical properties of thin films using van der Pauw technique

Ambily Krishnan, V. Geetha and Pradeesh Kannan 9

2. A review on nutritive effect of supplementation of mulberry leaves with Vitamin C on silkworm, Bombyx mori L.

Aneesha U. and C.V.Sreeranjitkumar 15

FULL PAPER

3. Investigation of the properties of nano structured Li-doped NiO films using the sol gel spin coating method

Anlin Lazar K., Aleena Thomas 20

4. Preliminary observations on the behaviour of Hierodula ventralis Giglio-Tos (Mantodea: Mantidae), Kerala, India

Binoy C.F. and Greeshma C.S. 27

5. Comparative study of In vitro antioxidant properties of Jatropha curcas Linn and Jatropha multifida Linn

Deepa M.K. and Jelly Louis 32

6. Bactericidal activity of nutmeg (Myristica fragrans) pericarp Flower Varghese, Teji K.T. and Philo T.J. 36

7. Manifestations in the epididymis in response to treatment of ursolic acid, a phytotherapeutic : a study in Wistar rat

Girija R., Kadalmani B. and Akbarsha M.A. 40

8. A Preliminary Investigation on the Antibacterial and Blood Clotting Properties of Pholcidan Spider Webs

Joyce Jose, Aiswarya Babu, Akhila Raju, Alwin Thankachan, and Sruthy M. Balan 53

9. Investigations on SnS & ZnO thin films for photovoltaic applications Melda Francis, Lakshmi M, Rosmy Joseph 59

10. Environmental Variability, Human Interventions and Ichthyodiversity of a fresh water stream of Kerala, India.

Radhika R 65

11. Variation of chlorophyll content in five common plant species to environmental stress due to proximity of steel industries

Rakshitha R. and Jelly Louis 72

12. Prevalence of pulmonary impairment symptoms among dry cement exposed group

Saji K.G. and Varghese P.R. 79

13. An Insilico Study on PPAR γ2 Gene among Obesity related type-2 Diabetes mellitus (T2DM)

Santhi.S and Jayasree.S 82

14. Biochemical alterations in Silkworm Bombyx mori on exposure to a new generation Pesticide – λ Cyhalothrin

Shahila Ismail K.I. and Sreeranjit Kumar C.V. 87

Instruction to authors 92 Subscription form 95

* Corresponding author, E-mail: [email protected]

Scientia ISSN: 0976-8289Vol. 13, No.1, Jan.-Dec., 2017, pp.9-14

Measurement of temperature dependent electrical properties of

thin films using van der Pauw techniqueAmbily Krishnan, V. Geetha and Pradeesh Kannan

Department of Physics, Govt. Victoria College, Palakkad- 678001, Kerala, India

AbstractA proper knowledge of the electrical properties of materials like electrical resistivity, type and concen-tration of charge carriers and their mobility is essential in understanding the transport mechanisms in them. This knowledge is crucial while preparing devices out of them. Here, the principles of electrical characterization involving a combination of van der Pauw and Hall techniques is described in detail. The fundamentals for setting up a measurement apparatus capable of measuring sheet resistances varying from a few ohms to terraohms and a very wide range of mobilities and carrier concentrations at different temperatures and magnetic fields are also explained.

Keywords: Resistivity, Mobility, Carrier Density, Van der Pauw method, Hall Coefficient

IntroductionDuring the early nineteenth century,

electrical resistance R or conductance G was considered as the basic parameter obtain-able from two terminal I-V measurements. But since samples of different shapes gave different values of resistance, it was under-stood that resistance could not be consid-ered as a standard parameter characteriz-ing different materials. Later, an intrinsic material property like resistivity ρ which does not depend on the geometry of the sample was chosen. This allowed scientists to quantify the current carrying capability of the material and compare with those of other materials. By the early 1900s, how-ever, it was realized that resistivity itself is not a fundamental material parameter because different materials could have the same resistivity and the same material synthesized by different techniques could exhibit different values of resistivity. This is particularly true for semiconductors. Their electrical conduction mechanisms could be understood only by applying quantum mechanics. This led to the definitions of

carrier density n and mobility µ. The values of conductivity σ (or ρ) together with n and µ and their variation with temperature can completely tell us about the complex electri-cal behavior of most materials.

The measurement of electrical resistiv-ity and Hall coefficient1 therefore play an important role in any research concerned with the electronic transport properties of materials. From the sign and magnitude of Hall coefficient, the type and density of carriers can be directly known. By simulta-neous determination of resistivity through van der Pauw technique, carrier mobility can be calculated. The temperature depen-dence of carrier mobility gives us an idea about the scattering mechanisms and the dependence of conductivity on temperature tells us about the mechanism of carrier transport.

Van der Pauw (VDP) methodThe van der Pauw (VDP) method1,2 pro-

vides a very convenient technique to mea-sure the resistivity of lamellar samples of any shape and dimension. The only dimension

Scientia Jan-Dec,2017

10

to be known accurately is the thickness of the film. The technique requires thin sam-ples of uniform thickness that are simply connected with no holes, non-conducting islands or inclusions with four very small contacts placed on the periphery. Electric current is applied very close to the ends and voltage is measured between two points where the current is uniform. The resis-tance of the voltage probe being infinitely high, virtually no current flows through the voltage path. So the error due to contact resistance is zero, in this four probe tech-nique. The four probes could be arbitrarily located around the edge of the sample and the specific probe location is less critical. By switching the probes for currents and volt-ages, it is possible to very accurately deter-mine the sheet resistance and resistivity if the thickness is accurately known.

In VDP method, the two characteristic resistances RA and RB are determined by measuring 8 voltages by switching the cur-rents in 8 different ways.

For example, one applies a dc current I12

into contact 1 and out of 2 and measures the voltage V43 from contact 4 to contact 3. By switching the probes for voltages and cur-rents, eight resistances are measured. RA is taken as the average of

(1)

Similarly RB is the average of the four measured resistances

(2)

Generally RA is different from RB. RA and

RB satisfy the van der Pauw equation,

(3)

Here ρ is the sample resistivity and d is the sample thickness. This relation can be solved analytically only when RA and RB are equal. Then

, (4)

and sheet resistance is given by (5)

This requires that the sample geometry is square and the probes are symmetrically placed. Otherwise the VDP equation may be solved numerically by Newton Raphson or binary search methods.

Hall MethodThe Hall coefficient (RH)1,2 in an isotro-

pic material is a measure of the transverse induced voltage produced by Lorentz forces on charge carriers perpendicular to both an applied electric current and magnetic field. After applying a magnetic field perpendicu-lar to the sample surface, a current is send

t ln 2rdR , where R = 2

RA + RB

Fig. 1. Connections to find the VDP resistances

Fig. 2.Connections to find the VDP resistances

Rs = dt

Fig. 3. Connections to measure the Hall voltages

Ambily Krishnan: Measurement of electrical properties of thin films

11

across it (diagonally) and the induced trans-verse voltage (the Hall voltage) is measured. The ratio R= V/I gives the Hall resistance.

The Hall field developed is proportional to the applied electric current density J and the perpen-dicular magnetic field B.

EH = RH ( J X B ) = RH JB (6)

If w is the width of the sample perpendicular to the applied current and d is the sample thickness (in the direction of B),

EH = VH/w and J = I/wd (7)

Substituting (7) in (6) and using Hall resistance R = VH/I,The Hall coefficient is measured as RH = VHd/IB = Rd/B. (8)

In steady state, the force exerted by the magnetic field on the charge carriers is exactly balanced by the Hall field force. eEH = evB where e is the electric charge and v is the drift velocity of the charge carriers. This gives EH = vBIf n is the density of charge carriers, the current density J = nev. Hence, v = J/ne. This gives EH = JB/ne (9)Comparing (9) with (6), RH = 1/ne (10)

Here the ohmic voltages should be avoided while measuring the Hall voltages. The VDP geometry provides an easy setup for Hall mea-surements. Four identical measurements are made with the current in both directions along both diagonals. This gives V24, V42, V13, V31 on apply-ing currents I13, I31, I42 and I24. Then the magnetic field is reversed and four more measurements are taken:V’24,V’42,V’13and V’31. Four Hall voltages are calculated by the following equations

(11)

This subtracts off the ohmic voltages.The carrier type is determined from the polarity of the voltage sum Vc + Vd + Ve + Vf. If this sum is positive, the sample is p-type and if the sum is negative, it is n-type.The Hall coefficient directly gives the carrier concen-tration becauseThe Hall coefficient is calculated as

(12)

The charge carrier density (13)

Conductivity is given by σ = neµ. (14)Carrier mobility is therefore Thus the combination of van der Pauw and Hall techniques provides the mobility too. By study-ing the temperature dependence of conductivity, which comes through both the temperature depen-dence of carrier concentration and mobility, it is possible to get an idea about the conduction (car-rier transport) mechanisms6 in the material.

Materials and MethodsThe basic components of the measurement sys-

tem3,4,5 are:

1. A precision current source with a picoammeter (either along with it or separate) capable of supplying currents from pA to 100-500 mA, depending on the resistance of the sample being used. Keithley 6220 current source was used in this study.

2. A high impedance nano voltmeter, Keithley 2182A was used for measuring the voltages

3. A switch system, Keithley 7001 employ-ing a Hall effect scanner card. Keithley 7065 was used to switch the currents and voltages between the four probe points.

4. A PC with labVIEWwas programmed for data acquisition and processing to get values of ρ, n and µ directly.

5. The magnet. We used an electromagnet providing a space between the pole pieces with a perpendicular electromagnetic field. For a higher magnetic field, the poles should be close, but this restricts the space for the sample holder, heater and vacuum enclosure. With an electro magnet, the direction of the magnetic field can be easily reversed.

6. The sample holder. To hold the sample, we used an enclosed flat container with embedded heaters beneath the sample fixing area. It was made from an electrically

Vc = 2V24 - V'24 ,Vd = 2

V42 - V'42 ,Ve = 2V13 - V13

l

,C = 2V31 - V'31

RH = 1/ne = IBd

8Vc + Vd + Ve + Vfa k

n =ed Vc + Vd + Ve + Vf^ h

8IB

µ = net1


12

insulating material with high thermal conductivity to reduce temperature gradi-ents across the sample holder, alumina.

The sample was held in position with the help of the probe tips which may be moved to and fro according to the sample size and held tightly in position with precise spring loaded screws. Heating coils and heat sens-ing thermo couples were mounted in the holder directly beneath the sample zone and can be controlled by external tempera-ture controller.

The metal for contacts should be inert

and should maintain good mechanical and electrical properties over a wide range of temperatures and should have a low See-beck coefficient (low thermo power). The usual methods of electrical wiring, insula-tion and shielding were used to minimize noise and stray voltages.

While making temperature dependent measurements, the temperature was var-ied in steps and care was taken to see that it remained almost constant during the measurements.

Measurement ExamplesGiven below are the results of measure-

ments made on sputtered films of cupric oxide (CuO) and cuprous oxide(Cu2O) at dif-ferent temperatures and magnetic fields.Cu2O is a p-type semiconductor and has a band gap of 2.1 eV. CuO is an intrinsi-cally p-type semiconductor with band gap

of 1.21-1.51 eV. Cu2O is a brown yellowish material and CuO is black in color. CuO is a more thermally stable material and oxida-tion of Cu2O produces CuO.

1. Cupric oxide thin filmCurrent applied = 10-6 A Sample thickness = 153 nm (Measured using Dektak 6M stylus profiler )

From the variation of conductivity with temperature, activation energy (Ea) of the material is calculated.

(6)

For the cupric oxide sample, Ea = 0.56 eV,

Rs = 2.64168 x 106 Ω and ρ = 52. 8336 Ω-cm

Hall Effect measurements: B= 5500 G, Vc = 0.009765 V, Vd = -0.00819863 V, Ve = 0.017022 V and Vf= -0.0118584 V

Average voltage, Vh = 0.000841332 V

From the variation of sheet resistance

v = e2KT-Ea

qr lnv = 2KT-Ea

Fig. 4. The sample holder

Table 1. Temperature dependence of conductivity of CuO

Temperature T (0C)

Resistivity ρ (Ωm)

Conductivity σ ( Ωm)-1 x10-3K-1 ln σ

27 0.4290 2.0325 3.333 0.7092650 0.3628 2.7563 3.095 1.013870 0.2422 4.1288 2.915 1.417990 0.14428 6.9309 2.754 1.9359

110 0.0853 11.7233 2.610 2.4615130 0.0473 21.1461 2.481 3.051150 0.03298 30.3214 2.364 3.411170 0.01436 69.637 2.257 4.2432

T1

Fig. 5. Variation of conductivity of CuO with temperature

Ambily Krishnan: Measurement of electrical properties of thin films

13

with magnetic field, magneto resistance of

the material is calculatedMagneto Resistance = 2.835 MΩ/T

2. Cuprous oxide thin film sampleCurrent applied = 10-6 A, sample thickness = 150 nmEa = 0.29 eV, Rs = 6.56 x 106 Ω, and ρ = 9.8 Ω-cm

Hall effect measurements were done at magnetic field B = 5400 G

Sample Type Mobility (cm2/Vs)

Carrier density (cm-3)

P-type 3.57 5.74 x 10 15

On repeating the Hall voltage measure-ments under different magnetic fields, the magneto resistance can be obtained.

ConclusionThe electrical conductivity of a mate-

rial is determined by the concentration of free carriers available to conduct current and their mobility (or freedom to move).

Both the carrier concentration and mobility are temperature sensitive for semiconductors. Thus the study of tempera-ture dependence of conductiv-ity and mobility through the combined Van der Pauw and Hall techniques provides valu-able information about the charge transport mechanisms and proves to be an indispens-able tool to both scientists and engineers working with mate-rials for electronic, optoelec-tronic and nano scale devices.

This article delivers the fundamentals of the design and principles involved in setting up such a useful experimental set up in any research laboratory.

References1. Dieter K. Schroder, 2006. Semiconductor Material and

Device Characterization, third ed. John Wiley & Sons, Inc.2. Robert Green, 2011. Hall Effect Measurements in Material

Characterization, Keithley Instruments, Inc., USA.

Sample Type Mobility (cm2/Vs) Carrier density (cm-3)P-type 5.79061 4.75715 x 10 15

Table 2: Variation with magnetic field:B in gauss Sheet Resistance in MΩ Resistivity in Ω-cm

5400 1.991 31.855500 2.023 32.365700 2.042 32.665900 2.092 33.476000 2.168 34.706100 2.197 35.16

Fig. 6. Variation of Sheet Resistance with magnetic field

Table 3. Temperature dependence of conductivity of Cu2O

Temperature T (0C)

Resistivity ρ (Ωm)

Conductivity σ ( Ωm)-1 x10-3K-1 ln σ

28 0.098512 10.1510 3.322 2.317550 0.072253 13.8402 3.095 2.627570 0.060165 16.6209 2.915 2.810690 0.04589 21.7912 2.754 3.0815

110 0.035181 28.4244 2.610 3.3472130 0.027475 36.396 2.481 3.5944150 0.021175 47.2255 2.364 3.8549170 0.016059 62.2703 2.257 4.1314

T1

Fig. 7. Variation of conductivity of Cu2O with temperature


14

3. Hall effect measurements-NIST’s website of National Metrology Lab.

4. Keith Neiman, 2011. Electrical Measurements on Nano Scale Measurements, Keithley Instruments Inc., USA.

5. Kasper A Borup, Eric S. Toberer, Leslie D. Zoltan, George Nakatsukasa, Michael Errico, Jean Pierre Fleurial, Bo B.

Iversoen and G. Jeffrey Synder, 2012. Measurement of the electrical resistivity and Hall coefficient at high tempera-tures, Rev. Sci. Instrum. 83, 123902.

6. C. W. J. Beenakker and H. van Houten ,1991. Quantum Transport in Semiconductor Nanostructures in: Solid State Physics, Philips Research Laboratories, Netherlands, 44, pp. 1-228.

∗ Corresponding author, E-mail: [email protected]


A review on nutritive effect of supplementation of mulberry leaves

with Vitamin C on silkworm, Bombyx mori L.

Aneesha U. and C.V. SreeranjitkumarDepartment of Zoology, Govt Victoria College, Palakkad, Kerala - 678001, India.

Abstract Sericulture is the science of rearing silkworms by using mulberry leaves; for this reason, silk pro-duction is directly depends on the larval growth and nutritive value of mulberry leaves. The quality and quantity of mulberry leaves may change due to environmental conditions. One of the alternative methods to improve the larval feeding is enrichment of mulberry leaves with the supplementation of various nutrients such as vitamins. Vitamins are a group of unrelated organic compounds needed only in minute quantities in the diet that are essential for specific metabolic reactions within the cell and necessary for normal growth and maintenance of health. Vitamin C plays a major role in the growth of silkworm. Many works are accomplished the effect of supplementation of vitamin C on cocoon charac-ters and various physiological and biochemical parameters. This review is an attempt to compile all the available literature on the effect of Vitamin C supplementation and its importance in the growth and development of silkworm

Keywords: silkworm; mulberry leaves; vitamin C

IntroductionSericulture is both an art and science of

raising silkworms, which produces luxu-riant silk thread in the form of cocoon. In India four types of silkworms were reared. Among the four types Bombyx mori. is the primary silk producing insect with great commercial value. Sericulture is an agro –based industry promoted as a secondary occupation and it is one of the important domesticated industries which provide financial security to rural people especially women. In order to make the silkworm rear-ing a profitable enterprise and owing to the monophagous feeding habit of the silkworm, scientists have tried different vitamins as food supplements.

Nutrition plays an important role in improving the growth and development of the silkworm, Bombyx mori L. and the

silk production is dependent on the larval nutrition and nutritive value of mulberry leaves and finally in producing good quality cocoons1.Feeding of nutritionally enriched leaves showed better growth and develop-ment of silkworms as well as improve the economic value of cocoons2. The influence of micronutrients on larval development and cocoon characters of silkworm were also studied3.Fortification of mulberry leaves with supplementary compounds to increase the larval growth and development has been carried out. These include vitamins4,5,6,7,8; amino acid9, 10, 11, 12; and minerals13, 14, 15. Stud-ies of determined that generally vitamins present in the mulberry leaves satisfy min-imum needs of silkworm but the amount of vitamins present in mulberry leaves varies on the basis of environmental conditions , usage of fertilizers in field and mulberry varieties and other field practices. Nh7n.


16

Ascorbic acid (Vitamin C-C6H6O8)Vitamin C is chemically the simplest of

the vitamins and perhaps for these reasons it was first isolated, characterized and its structure determined. It is a water soluble derivative of sugar molecule which includes

all the compounds having antiscorbutic activity. The name “ascorbic acid” comes from Greek root- ‘a” (no or anti) and “scor-butus” (Scurvy) or simple “anti-scurvy” acid; because it was known to dramatically cure this disease.

L-ascorbic acid is a white, odorless, crystalline compound, soluble in water but insoluble in fat solvents. It is readily oxi-dized to dehydro ascorbic acid, a biologi-cally less potent form. It is heat labile and prone to atmospheric oxidation, especially in the presence of metallic catalysts like copper, iron,etc. L-ascorbic acid acts as a reducing agent in the transport hydrogen. It is involved in many enzyme systems for hydroxylation; i.e., hydroxylation of tryp-tophan, tyrosine, or proline. Ascorbic acid plays a synergistic role along with vitamin E as intracellular antioxidants and free rad-ical traps. It is a powerful antioxidant, pro-tecting against oxidative damage to DNA, membrane lipids and also proteins

Vanderzant and Richardson 16 reported

that all the insects known to need Vitamin C are plant feeders.Vitamin C is known to serve as phagostimulant for phytophagous insects17. On the other hand, Vitamin C was shown to elicit no significant feeding response for several sugar-cane infeasting scarabaeids Antitrogus parvulus and Lep-idiota negatoria 18. Dadd andVanderzant et al, Chippedale and Beck and Levinson and Navon19,20,21,22 regarded dietary Vitamin C essential for the growth and development of several plant-feeding insects and its omis-sion from diet caused retarded growth and heaviest mortality at the moult from the fourth to the fifth instar.

Vitamin C and insects Felton and Duffey23 provided evidence of

an important role for ascorbate free radical (AFR) reductase, dehydroascorbate(DHA) reductase, glutathione and glutathione reductase as components of an oxidant- scavenging system in the midgut of larval Helicoverpa zea. Mathews et al.24 reported that ascorbate peroxidase (APOX) activity, which catalyzes the oxidation of VC with the concurrent reduction of hydrogen perox-ide (H2O2), was important in removing lipid peroxides in fat body tissues of Helicoverpa zea larvae.

Importance of vitamin C in silkworm

Vitamin C is the more effective antiox-idant and has got many protective effects, but the silkworm B.mori has been classified among the insects which are unable to syn-thesize Vitamin C in their body and depend on exogenous supply to fulfill the require-ment. The animals which cannot synthesize Vitamin C, lack an enzyme, L-gulono-1, 4-lactone oxidase, which is essential for

Aneesha and Sreeranjitkumar: Nutritive effect of Vitamin C on silkworm

17

synthesis of the Vitamin C intermediate precursor 2-keto-1-gulonolactone25. Anti-oxidant activity of ascorbic acid decreases reactive oxygen species and oxidative pres-sure, and as a result, the absorption of nutritious substances in the midgut would increase26. Ascorbic acid shows a particular behavior as it is very susceptible to degra-dation, especially when in solution, and/or exposed to light, oxygen and free radicals. Ito27 recorded relationship of ascorbic acid supplementation and growth of silkworm. The absence of ascorbic acid in the diet of first and second instar larvae postponed growth and development of silkworm. There is enough vitamin C in mulberry leaves and ascorbic acid content of growing larvae is dependent on amount of this vitamin in diet. The availability of artificial diet from the 1960s, permitted researchers to confirm the importance of Vitamin C for the silk-worm physiology on the basis of classical experiments carried out by Ito, though it is difficult to precisely estimate the amount of Vitamin C ingested by silkworm larvae fed fresh mulberry leaves. Vitamin C usually has been added to silkworm food in a quan-tity generally varying from 1-2% of the dry weight of the artificial diet, which is consid-ered as optimum content of this Vitamin28.

Vitamin C has always been regarded as indispensable for the growth and develop-ment of the silkworm, B.mori and its depri-vation in the diet affected larval growth and cocoon production. Cui reported that Vitamin C (0-.2%-0..3%) was necessary for growth and development of silkworm, while the amount beyond a certain concentration, growth and survival rate is decreased29. Vitamin C is present in all plants particu-larly in fruits and vegetables representing about 10% of total carbohydrate pool in favourable conditions 30,31,32. Vitamin C also present in mulberry leaves33. It was VC con-tent of 1.3 to1.8 mg/g34. There is very close relationship reported between the addition of Vitamin C in diet and silkworms growth. The growth of 1 gram fresh weight of larva needs about 0.668mg (0.067%) Vitamin C. Although the function Vitamin C to silk-worm physiological activities has not been clear, it is known to promote silkworm food

ingestion35. But, the content of Vitamin C in mulberry leaves is largely dependent on cli-matic and seasonal conditions (with a max-imum content in late spring time, or at the beginning of summer under temperate cli-matic conditions) and on the position of the leaf on the shoot (with the maximum con-tent in the apex and median leaves and min-imum content near the branch insertion). Furthermore, the choice of the mulberry variety affects the content of Vitamin C in the mulberry leaves. Vitamin C has been found to be reduced in the mulberry leaves grown under shade. Leaf preservation, even if it is protected from direct sunlight, is responsible for reducing Vitamin C content at a rate of at least 20% in 24 hours36. Dry-ing of mulberry leaves causes a complete degradation of Vitamin C, so mulberry leaves cannot be considered as a source of Vitamin C for larvae fed on artificial diet containing even a high percentage of dried, pulverized mulberry leaves37 measured the Vitamin C content in M5 mulberry leaf of different ages, fresh and preserved leaves and found that it varied according to the age and preservation of leaf. Gamo showed that the content of the VC in fresh mulberry leaves changed in relation to conditions influencing the food value of leaves and that the amount of Vitamin C in the larval body is dependent on the leaves.

In silkworm, fortification of mulberry leaves fed to silkworm has been reported to increase the food consumption, coefficient of utilization, larval weight ,silk filament length and weight38 and fecundity39,40. Fur-ther, it has been reported that excessive amounts of Vitamin C supplementation to silkworm diet have negative impact causing decrease in food intake and cocoon charac-teristics due to hypervitaminosis. Dietary supplementation with synthetic and plant based Vitamin C has been found to enhance the economic parameters of the mulberry silkworm significantly.

ConclusionThis review summarizes data show-

ing that supplementation of mulberry leaves with ascorbic acid have various


18

characteristic properties and functions. Ascorbic acid is the master nutrient because of their importance, occurrence and abun-dances. The effectiveness of ascorbic acid at lower concentrations indicates the need for elaborating comprehensive studies on the subject.

References 1. Etebari, K., Ebadi,R.and Matindoost,

L.2004. Effect of feeding mulberry’s enriched leaves with ascorbic acid on some biological, biochemical and economical characteristics of silkworm Bombyx mori.L.Int.J.Indus.Entomol.8:81-87.

2. Krishnaswami,S., Kurnararaj,S.S., Ija-yaragliavan, K. and Kastviswanathan, K.1971.silkworm feeding traits for evaluat-ing the quality of mulberry as influenced by variety, spacing and nitrogen fertilization.Indian.J.Seric.10(1):79-90.

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6. Nirwani, R.B., Hugar,I.I. and Kaliwal, B.B.1998. Supplementation of riboflavin on economic parameters and biochemi-cal changes of the silkworm, Bombyx mori L.Bull.Seric. Res.9:37-41.

7. Rahmathulla,V.K., Das, P.,Ramesh, M. and Rajan , R.K.2007.Growth rate pattern and economic traits of silkworm, Bombyx mori .L.under the influence of folic acid admin-istration.J.Appl.Sci.Environ. Manage. 11(4):81-84.

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Amino acid contents in posterior silk gland of Bombyx mori. Influenced by quality of mulberry leaves. Bull. Seric.Res.5,63-68.

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11. Shaha, B.N. and khan, A.R.1996.Effect of dietary supplementation of vitamins and minerals on the growth and development of Bombyx mori L. Bangladesh.J.Zool.24 (2),125-131.

12. Magadum, S.B., Hooli M.A. and Magadum, V.B.1992. Effect of application of juvenile hormone analogue in IV instar followed by thyroxine in the pure Mysore breed of Bom-byx mori. Sericologia.32(3),385-390.

13. Islam Z. and Khan A.R.1993. Growth and development of the mulberry silkworm, Bombyx mori of feed supplemented with manganese sulphate. J.bio-sci.1,21-30.

14. Ito, T. 1978. Silkworm nutrition In: The silk-worm –An important laboratory tool(eds Y. Tazima) Kodansha Ltd., Tokyo. 307.

15. Vanderzant, E.S. Richardson, C.D.1963. Ascorbic acid in the nutrition of plant feed-ing insects. Science.140,989-991.

16. Cohen A.C.2004. Insect diets: Science and techonology. CRC Press,29.

17. Allsop, P.G.1992. Sugars,amino acids and ascorbic acid as phagostimulants of larvae of Antitrogus parvulus and Lepidiota nega-toria.J.Eco.Entomol.85,106-111.

18. Dadd, R.H.1957. Ascorbic acid and carotene in the nutrition of the desert locust, Schisto-cerca gregaria. Nature, London.179,427-428.

19. Dadd, R.H.1960. Some effects of dietary ascorbic acid on locusts. Proceedings of the royal society of London. Series B,Biological Sciences 153(950):128-143.

20. Vanderzant, E.S.,PoolM.C.,Richardson C.D.1962. Role of ascorbic acid in the nutri-tion of three cotton insects.J.Insect Physiol., 8,287-297.

21. Chippendale, G.M.,Beck S.D. 1964. Nutri-tion of the European corn borer,Ostrinia nubilalis; Ascorbic acid as the corn leaf fac-tor. Entomol.Exp.Appi.7(3):241-248.

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22. Levinson, H.Z.,Navon A. 1969. Ascorbic acid and unsaturated fatty acids in the nutrition of the Egyptian cotton leaf worm, Prodenia litura.J.Insect Physiol.,15,591-595.

23. Felton, G.W. Duffey, S.S.1992. Ascorbate oxidation reduction in Helicoverpa zea as a scavenging system against dietary oxidants. Arch.Insect.Biochem.Physiol.,19(1),27-37.

24. Mathews, M.C.,Summers, C.B.,Felton, G.W.1997. Ascorbate peroxidase: A novel antioxidant enzyme in insects. Arch.Insect Biochem.Physiol.,34(1),57-68.

25. Ito T., Arai, N.1965. Nutrition of silkworm Bombyx mori L. IX. Futher studies on the nutritive effects of ascorbic acid. Bull.Seric.Expt.Statn.Japan.20,1-9.

26. Ito, T.1961. Nutrition on the silkworm Bom-byx mori L. IV. Effects of ascorbic acid. Bull.Seric.Expt.Statn.,17,134-137.

27. Ito, T. 1978. Silkworm nutrition In: The silk-worm –An important laboratory tool(eds Y. Tazima) Kodansha Ltd., Tokyo. 307.

28. Cui, W.Z.,Shang J.Y. Wang, B, Cheng A.W. 2003. Effects of vitamin C on feeding habit and growth and development of the silk-worm Bombyx mori L. Sericologia, 43,73-80.

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30. Noctor, G., Foyer, C.H.1998. Ascorbate and glutathione:keeping active oxygen under control. Ann. Rev. Plant Mol. Biol., 49,249-279.

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32. Smirnoff, N.,Conklin, P.L. Loewus, F.A.(2001). Biosynthesis of ascorbic acid

in plants: a renaissance. Ann. Rev.Plant Physiol. Plant Mol. Biol., 52, 437-467.

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Investigation of the properties of nano structured Li-doped NiO films

using the sol gel spin coating method Anlin Lazar K 1,2 ∗ Aleena Thomas1

1Department of Physics, Mercy College, Palakkad, Kerala, India2Department of Physics, Govt. Victoria College, Palakkad, Kerala, India

AbstractMetal oxide semiconductors have attracted a wide interest owing to their unique properties and mas-sive potential applications in different opto-electronic devices. The Li doped NiO thin films were pre-pared using sol gel spin coating method. Structural and optical properties of NiO-based nanoparticles can be modified by doping with lithium (Li). Lithium is doped in NiO nanoparticles with varying con-centrations (2, 4, 6, 8 and 10 wt %) of lithium. The effect of Li concentration and variation of annealing temperature on the structural, morphological, optical and electrical properties of Li: NiO thin films were studied. Characterization of prepared films was done using X-ray diffraction (XRD), Scanning electron microscopy (SEM), UV-visible and Hall measurements. The SEM image displays the surface morphology of Li-doped nickel oxide thin films. The UV studies revealed that the films have a band gap ranged between 3.49 eV to 3.65 eV. The maximum absorption for the films occurred within the UV region. The average transmittance and band gap energy of the Li: NiO films in the visible region increases with the increase of Li concentration and decreases with an increase in the annealing tem-perature. Hall Effect measurements provided positive Hall voltages substantiating p-type conduction in Li: NiO films. Electrical properties like resistivity, carrier mobility, and carrier concentration of Li: NiO film with varying Li concentration. The results suggest that Li: NiO thin films prepared by spin coating technique can turn out to be a promising transparent electrode for opto electronic devices and gas sensors.

Key Words: Sol-gel, Li doped NiO thin films, Spin coating, Structural properties, Opto electronic properties.

IntroductionTransparent conductive oxides (TCO)

are doped metal oxides used in optoelec-tronic devices such as flat panel displays and photo voltaic which include inorganic devices, organic devices, and dye-sensitized solar cell1. Most of these films are fabricated with polycrystalline or amorphous micro-structures. Typically, these applications use electrode materials that have greater than 80% transmittance of incident light as well as electrical conductivities higher than 103

S/cm for efficient carrier transport2. In gen-eral, TCOs for use as thin-film electrodes in solar cells should have a minimum carrier concentration on the order of 10-20 cm−3 for

low resistivity and a band gap greater than 3.2 eV to avoid absorption of light over most of the solar spectra. Mobility in these films is typically limited by ionized impurity scat-tering due to the large amount of ionized donors and is of the order of 40 cm2/(V·s) for the best performing TCOs3. Current trans-parent conducting oxides used in industry are primarily n-type conductors, ie, their primary conduction is as donors of electrons. This is because electron mobilities are typ-ically higher than hole mobilities, and the difficulty of finding shallow acceptors in wide band gap oxides to create a large hole population4. Suitable p-type transparent conducting oxides are still being researched, though the best of them are still orders of

Anlin et.al: Li-doped NiO filmsusing the sol gel spin coating method

21

magnitude behind n type TCOs. The lower carrier concentration of TCOs with respect to metals shifts their plasma resonance in the NIR and SWIR range. Different tech-niques have been adopted for the prepa-ration of lithium doped nickel oxide thin films. It includes sol gel5,6 ,spray pyrolysis7,8, chemical bath deposition9,10,electrochemical deposition11, SILAR method 12,13 ,sputter-ing14-16, thermal evaporation 17etc. Of the above deposition techniques, sol-gel process is a cost effective technique that can be used for large area film deposition.

Materials and methodsMicroscopic glass slides (2.5 cm x 2.5

cm x1.45 mm) were used as substrates for thin film deposition. Substrates were first cleaned using laboratory detergent followed by distilled water. Then they were cleaned ultrasonically with acetone and methanol each for 10 min using an ultrasonic cleaner. Finally the slides were dried in oven.

Precursors used in preparation process:• Nickel acetate tetra hydrate

(C4H6NiO4·4H2O) (starting materials)• Lithium chloride (LiCl) (dopant

precursor)• 2-Methoxyethanol (C3H8O2) (Solvent)• Di ethanolamine (MEA)(Stabilizer)

The precursor solutions for Li-doped NiO were prepared by the sol–gel method using nickel acetate tetra hydrate and lith-ium chloride as the starting materials. The nickel acetate was initially added into a mixture of 2-methoxyethanol and di-etha-nol amine. The molar ratio of MEA to nickel acetate was maintained to 1.0, and the con-centration of nickel acetate was 0.5 M. The resultant solution is stirred at 600C for two hours using magnetic stirrer, then aged for 24 h at room temperature and filtered to obtain green coloured homogeneous and clear solution. The lithium chloride was

Li:NiO Thin Films(2%. 4%. 6%. 8%. 10%)

Nickel Acetate Tetra hydrate+

2-Methothoxy ethanol+

Diethanol amine

Lithium Chloride+

2-Methothoxy ethanol

Filtered and stirred at 60°c for 1 h Stirred at 80°c for 1 h

Li doped NiO Solution(Doping% of Li 2%, 4%,

6%, 8%, 10%)

Stirred for 5 min

Spin Coating Technique

3000 rpm for 30 secRepeated five times

Preheated at 300°c for 15 min

Annealed at 400uC, 450uC,soo0 c. 55,:Pc or 1 h

Fig.1. Flow chart of experimental details of Li doped NiO thin films.

Scientia Jan-Dec, 2017

22

dissolved in 2-methoxyethanol under air atmosphere and the concentration of lith-ium chloride was 0.5 M. The resultant solu-tion was stirred using magnetic stirrer at 80°C for 1 h and then filtered to yield a clear and homogeneous solution. Finally, both stock solutions were mixed, with different volume fractions, to prepare the stoichiom-etry, transparent and stable nickel acetate solutions with Li contents ranging from 2 to 10%. The percentage of lithium in the solu-tion was defined as the ratio between the concentration of lithium ions (Li+) and the total concentration of cation (Li+ and Ni2+) in the solution. Li doped NiO thin films were deposited on glass substrate using static dispense spin coating method that was performed at room temperature.

Results and DiscussionsThin film fabrication technologies are

in high demand and have given the wide-spread use of coatings in all engineering and science fields. Mechanical, functional and geometrical properties of thin films can vary dramatically and this fact makes it difficult to find a general purpose characterization technique. After the production of thin films, they were characterized using different tech-niques concerning their electrical, optical, structural and morphological properties.

In order to study the effect of Li concentration on the micro structural properties of Li-doped NiO (NiO:Li) thin films, different values of doping concentration (2, 4, 6, 8 and 10%) were used.

Structural studyThe XRD pattern shows the effect of

Li on the structural properties of NiO

thin films prepared at different Li ratio. The films were annealed at 4500C, 5000C and 5500C respectively. The XRD shows three characteristic peaks, indexed as the reflec-tions from (111), (200) and (220) planes (JCPDS card No.75-0197)18 representing a typical face-centred cubic (fcc) structure of Nickel oxide nanoparticles. The crystal-line structure of NiO does not appear to be modified by the increase of the Li ratio. The films were polycrystalline, consisting of NiO cubic phase. It is observed that the intensity of the peaks decreases with increase in lith-ium concentration. This reduction in inten-sity can be attributed to the local lattice disorders resulting from doping. The broad peaks reveal the polycrystalline nature of the sample. The peak becomes wider (full width at half maximum), suggesting that the doping process of the investigated sys-tem brought about a progressive decrease in the degree of crystallinity of NiO phases. The increase in lithium concentration results in the substitution of Ni2+ sites by Li ions whose radius is 5% smaller than Ni2+.

Table 1. Effect of annealing on micro structural parameters of NiO:Li thin films.

Annealing temper-

ature (oC)FWHM

Crystal size (D)

(nm)

DislocationDensity (δ) (1019)

Interplanar distance(d) theoretical

(A0)

Interplanar distance(d)

practical(A0)

Lattice parameter(a)

theoretical(A0)

Lattice parameter(a)practical(A0)

450 0.842 1.857 2.897 2.041 2.041 4.083 4.082500 0.832 1.879 2.292 2.038 2.038 4.077 4.076550 0.654 2.391 1.747 2.039 2.037 4.078 4.074

Fig.2. XRD pattern of NiO films with lithuim doping at 5000C.


23

This led to a progressive decrease in their grain size with increase in the Li concen-tration. The crystallite size (D) is calculated using the Scherrer formula19, D = 0.9 λ/β cos θ. Where β is the full width half maximum of diffraction peak measured in radians, D is the grain size , λ is the wave length of x-ray, θ is the angle of diffraction. From the table 1 it is clear that the observed Inter planar distance (d) and Lattice parameter (a) val-ues are in good agreement with the stan-dard values.

Surface MorphologyIt can be seen that the film have a finer

microstructure with the nano sized grains

spreading on the substrate surface uni-formly. The grain size dependence of the film on the concentration of lithium may be due to different rate controlling steps involved in the formation of lithium doped nickel oxide thin films.

Optical studiesTransmittance

The Figure 4 depicts the optical trans-mittance spectra of NiO: Li films at anneal-ing temperature 5000C. All films were highly transparent in the visible region. The transmittance increases with Li con-centration. The highest average transmit-tance value was 86.5%, which was obtained in the 10% NiO:Li thin film. The low trans-mittance is due to the NiO secondary phase and Li dopants being aggregate.

Absorption studiesThe optical absorption spectra can be

calculated by using the following equation20

Where, A is a constant, Egis the semicon-ductor band gap and n is a number equal to ½ for direct gap and 2 for indirect gap compound. Since NiO is a direct band gap compound, n=½. The above equation can be employed to plot against to deter-mine the energy band gap (Eg). The linear portion of the curve when extrapolated intersects the x-axis and gives the value of Eg.

Fig.3.SEM of NiO thin film annealed at 5000C

Fig.4. Plot of optical transmittance spectra of NiO:Li films at annealing temperature 5000C

Fig.5. Plot of (αhν) 2 versus (hν) of NiO:Li thin film


24

Table 2.Band gap of NiO:Li film at annealing temperature 5000C.

Concentration of Li Band gap (eV)

2% 3.5064% 3.5706%8%

10%

3.5913.6133.643

From table 2 it is clear that the band gap energy increases with the increase of the Li concentration. This is because the dopant Li ions act as scattering centres and affect the carrier mobility21,22.

Electrical studies:A measurement of Hall voltage is often

used to determine the type of semicon-ductor (n-type or p-type, the free carrier density and the carrier mobility. The Hall Effect describes the behavior of the free carriers in a semiconductor when applying an electric as well as a magnetic field. Hall Effect measurements provided positive Hall

voltages substantiating p-type conduction in NiO:Li films. Fig. 6 is the graph of resis-tivity vs. Li concentration. It shows that the film resistivity is varying with Li con-centration. Two stages of resistivity change were found as Li content increased in the NiO films. The value reaches a maximum of 3.35x106 Ωcm at Li concentration of 4%

and then the resistivity slightly decreased to 1.09x106 Ωcm when the Li concentration reaches 10%. It is very difficult to know the ratio of holes to total cations since the nickel vacancy may be partially singly charged and partially doubly charged23. It is generally believed that charged nickel vacancies are the predominating point defects for electri-cal properties.

When doped with Li the Nickel vacancies are get occupied by Li, which can be repre-sented by Eq. (1)24.

---(1)

Initially the incorporated Li ions occupy the native Ni vacancies, consuming the electric hole concentration and lowing the non-stoichiometry of the NiO film. There-fore, the initial increase in resistivity and the decrease in excess oxygen resulted from Li ions filling nickel vacancies.

With increased Li doped into the film, some Li ions may substitute Ni2+ in the nor-mal crystal sites and create new holes .This leads to a slight decrease in resistivity.

ConclusionsNanocrystalline lithium doped nickel

oxide thin films were fabricated by low-cost sol gel spin coating technique with LiCl as the doping source. Films obtained by this method are adherent and have an uniform surface. Film structure, morphol-ogy, transparency, optical band gap energy and resistivity were examined at different Li concentration (2, 4, 6, 8 and 10 wt%) and at different annealing temperature (4500C to 5500C). The XRD result revels that the intensity and crystallinity of the peak decreases with Li concentration and increases with annealing temperature. In both case the observed inter planar dis-tance (d) values are in good agreement with the standard results. The XRD pattern also shows polycrystalline nature. The SEM results depicted a uniform surface mor-phology. The optical studies shows that the

Fig. 6. The graph of resistivity vs. Li concentration


25

transmittance increases with Li concentra-tion and decreases with annealing tempera-ture. The optical band gap energy is found to be increased with Li concentration and decreased with annealing temperature. The p-type electrical conductivity is confirmed from Hall Effect measurement. The electri-cal resistivity is found to be increased with Li doping up to certain Li concentration and then it gets decreased with Li doping. The resistivity also gets decreased with increas-ing annealing temperature. NiO:Li films fabricated by spin coating should be use-ful as a less expensive p-type transparent electrode for solar cells and displays. These results suggest that NiO:Li thin films pre-pared by spin coating technique can turn out to be a promising transparent electrode for opto electronic devices. The films also have significant role in hydrogen sensing detector and electro-chromic devices.

AcknowledgementThe authors wish to acknowledge STIC,

CUSAT for their help and support during the characterization of the samples. The authors would like to gratefully acknowl-edge the University Grant commission and DST-FIST, for providing the financial assis-tance and infrastructure to carry out this work.

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S.J. Yaghmoura, F.M. Al-Marzoukia ., 2009. Structure and optical properties of nano-crystalline NiO thin film synthesized by sol–gel spin-coating method, Journal of Alloys and Compounds, 486, 9–13.

2. Sta, M. Jlassi, M. Hajji, H. Ezzaouia., 2014. Structural, optical and electrical properties of undoped and Li-doped NiO thin films pre-pared by sol–gel spin coating method, Thin Solid Films. 555,131–137.

3. Foaad S. Hashim, Khansaa H. Mohsin ., 2015. Effect of Li Doping on Structure and Optical Energy Gap of NiO Films Prepared by Sol–Gel Technique ,Journal of KUFA – PHYSICS ,Vol.7 No.1.

4. S. Irudaya Sahaya Lancy, M. Priya Dharsh-ini, V. Shally, Sr. Gerardin Jayam ,.2016.

Effect of Lithium Doping Concentration on the Structural, Morphological and Optical Properties of NiO Nanoparticles, Interna-tional Research Journal of Advanced Engi-neering and Science, Vol1, No 2, 55-58.

5. S. Anandh Jesuraj, M.Haris, P.Immanuel ., 2013. Structural and Optical Properties of Pure NiO and Li-Doped Nickel Oxide Thin Films by Sol-Gel Spin Coating Method, International Journal of Science and Research (IJSR) 2319-7064, UGC Sponsored National Conference on Advanced Technol-ogy Oriented Materials (ATOM-2014), 8-9th Dec-2014.85-87.

6. Guo Wen, K.S. Hui and K.N. Hui,. 2012. High conductivity nickel oxide thin films by a fac-ile sol-gel method, Materials Letters,10.109.

7. D. Paul Joseph, M. Saravanan, B. Muthu-raaman, P. Renugambal, S. Sambasivam, S. Philip Raja, P. Maruthamuthu and C. Ven-kateswaran ., 2008. Spray deposition and characterization of nanostructured Li doped NiO thin films for application in dye-sensi-tized solar cells, Nanotechnology. 19, 485707 (10pp).

8. Wu Chia-Ching and Yang Cheng-Fu., 2013. Investigation of the properties of nano struc-tured Li-doped NiO films using the modified spray pyrolysis method, Nanoscale Research Letters,8,33.

9. J. C. Osuwa, G. I. Onyejiuwa., 2013 Struc-tural And Electrical Properties Of Annealed Nickel Oxide (NiO) Thin Films Prepared By Chemical Bath Deposition. Journal of Ovonic Research Vol. 9, No. 1, P. 9 – 15.

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11. Li Luo, Deniz Bozyigit, Vanessa Wood, and Markus Niederberger., 2013. High-Qual-ity Transparent Electrodes Spin-Cast from Preformed Antimony-Doped Tin Oxide Nanocrystals for Thin Film Optoelectronics, Chem. Mater. 25, 4901−4907.

12. K.K. Purushothaman, S. Joseph Antony, G. Muralidharan., (2011) Optical, Structural and Electrochromic Properties of Nickel


26

Oxide Films Produced By Sol–Gel Tech-nique. Solar Energy. 85 ,978–984.

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17. R.K.Gupta, Z. Serbetci, F. Yakuphanoglu., 2012. Band gap variation in size controlled nanostructured Li–Ni co-doped CdO thin films, Journal of Alloys and Compounds ,515, 96– 100.

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Preliminary observations on the behaviour of

Hierodula ventralis Giglio-Tos (Mantodea: Mantidae), Kerala, India

Binoy C.F.* and Greeshma C.S.Research and PG Department of Zoology. St.Thomas College (Autonomous),

Thrissur-680001, Kerala, India

AbstractHierodula ventralis Giglio-Tos (1912) belongs to family Mantidae and subfamily Mantinae, is a natu-ral predator, generally green in colour and commonly seen among green vegetation. Their ethological characterization revealed mainly seven characteristics such as locomotion, antennal shaking, focusing, swinging, striking, grooming and resting. Their activities could be divided into three phases such as prefeeding, feeding and post feeding behaviour. From the observations it was evident that resting was the major behavioural state shown by H.ventralis (61%), followed by swinging (18%), focusing (8%) ,antennal shaking (6%) and locomotion (4%). Antennal shaking was the only character shown by them in all the three phases. The transition in behaviour occurred by the presence of prey and during feeding. Striking is the unique behaviour shown by them which was purely based on prey capture and devoted 2% time for this. The least observed character was grooming (1%). Based on these behaviour categories an ethogram of H.ventralis was prepared.

Key words: Hierodula ventralis, Mantidae, Mantinae, Ethogram, Kerala, India

IntroductionAbout 168 species of mantids were iden-

tified from India1 and out of this 40 species were recorded from Kerala2. As per records five species of Hierodula were identified in Kerala, they are H. membranacea (Bur-meister), H. bipapilla (Audinet-Serville), H. keralensis Vyjayandi & Narendran, H. tenuidentata Saussure, and H. ventralis Giglio-Tos3. H.ventalis is green in colour and seen among green vegetation. In India they are mainly distributed in Kerala, Chandi-garh, Madhya Pradesh and Maharashtra and in Kerala they are seen in Pathana-mthitta, Thrissur, Ernakulum, Idukki, Wayanad and Palakkad districts2. They have an average of 7cm length with wide tri-angular head and globular compound eyes. Fore legs are modified as raptorial legs with internal and external spines. Abdomen is

broad and generally narrow towards poste-rior region. Wings are longer than the abdo-men while the forewing opaque with broad coastal area.

Mantids are peculiar group of insects showing interesting behaviours. They remain motionless for hours which makes them, the sit and wait predators4. They have the ability to rotate their head about 180 degree to observe the movements of prey for successful capture and consumption. Usu-ally they proceed slowly towards the prey till they come within their raptorial legs in a comfortable range5. The mantis was able to attack prey throughout a large three-di-mensional capture zone by changing body orientation relative to its perch6.

Behavior is the link between organisms and environment and is one of the most


28

important properties of animal life. It plays a critical role in biological adaptations. The conservation of organism requires knowl-edge about natural behavior in order to develop effective reserves and effective pro-tection measures. Forty species of mantids were found in Kerala with only very few tax-onomic works2. But there is little informa-tion regarding their behavioral characters. The present study deals with the behav-ioral characterization of Hierodula ventra-lis Giglio-Tos which is a pioneering attempt in Kerala.

Materials and methodsStudy area

The work was carried out in Pamboor a small locality in Kuttur village, Thris-sur Taluk, Thrissur District, Kerala. The area comes under Kolazhy Panchayath and belongs to Central Kerala Division. The study was conducted from June 2016 – Jan-uary 2017.

Nine H .ventralis adults were housed in cylindrical glass jars having 10 cm perime-ter and 25 cm length. Instantaneous time sampling method was applied and deter-mined the vivid type behaviour of mantids. Every day one hour was used for observing the activities of each individual. During this one hour pre feeding, feeding and post feeding behaviour were observed. Food were provided to them after 30 minutes and observed the changes in their behaviour. The time taken to complete each type of behaviour was recorded. The observation fields were divided into three phases such as pre feeding, feeding and post feeding period. The data were documented and based on the observation an ethogram was prepared and represented graphically.

Results and discussion H. ventralis showed seven types of

behaviour such as locomotion, anten-nal shaking, focusing, swinging, striking, grooming and resting. The different catego-ries and the time allocated for each during the study are given in Table 1. According to the observations The maximum time

was allocated for resting (61%) followed by swinging (18%), focusing (8%), antennal shaking (6%) and locomotion (4%). Groom-ing (1 %) happened the least number of times during the study period.

During the pre feeding period, H.ventra-lis showed three different behaviors such as locomotion, antennal shaking and rest-ing activities (Fig. 1). Maximum time spent (94%) was for resting, 1% time for anten-nal shaking and 5% time for locomotion. Resting behaviour was observed almost all the time and the transition in behaviour occurred mainly during the feeding period.

During the feeding period, H.ventralis showed four activities such as swinging (57%), antennal shaking (15%), striking (5%) and focusing (23%). As the swinging activity increased the capturing success also increased. Feeding period was the most active period and they spend maxi-mum time for swinging activity followed by focusing and antennal shaking. Swing-ing behaviour was a preparatory phase for capturing the prey. This will be preceded by focusing activity. Focusing helps the man-tids to efficiently catch the prey by striking. The least time was required for striking, a unique activity of praying mantids that helps them to quickly grasp the prey. The striking activity was almost equal on the basis of time for each attempt to capture the prey (Fig. 2).

Grooming, antennal shaking, locomotion and resting were the post feeding behaviours shown by H. ventralis (Fig.3).. Similar to pre feeding period, resting was the major char-acter shown by the species (91%) followed by locomotion (6%) and grooming (2%). The grooming of fore legs (Fig. 4) and hind legs (Fig. 5) after the feeding activities were observed only during this phase. The anten-nal shaking (1%) was the least observed activity during this period.

Comparing the three phases of behaviour it was observed that antennal shaking was the only activity shown by H.ventralis in all the three phases. However, antennal shak-ing was more during the feeding period than pre and post feeding phases. Resting and

Binoy and Greeshma: Behaviour of Hierodula ventralis in Kerala

29

locomotion activities were seen in both pre and post feeding periods and the time dura-tion for these activities were almost equal in both phases. Swinging, head focusing and striking were the interesting behaviours observed during the feeding phase. These activities helped the species in successful capture of the prey.

H.ventralis generally indulged in a period of visual search, a slow approach to the prey, and capture by an extremely rapid grasping movement of the raptorial protho-racic legs and the ingestion of the captured food. The present study determined that the striking activities of the species were mainly based on the relative closeness of prey along with head focusing. The size and

Table 1. Different behavioral categories and their time allocation by H. ventralis during the study

Behaviour Definition Time allocated (%)

Locomotion Movement around the habitat (jar) 4

Antennal shaking Vibrating motion of antenna 6

Resting

Hanging down from the upper surface or clinging to twigs in jar without any movement

61

Swinging Side to side movement of body while seeing a prey 18

Focusing Movement of head related with the movement of prey 8

Grooming Cleaning of forelegs, hind legs and antennae with mouth after feeding. 1

Striking Quick stretching of forelegs for grasping the prey 2

Pre Feeding Period Feeding Period Post Feeding PeriodL 5% 1% A

R94%

Fig. 1.Ttime allocated for locomotion, antennal shak-ing and resting

Note: R-Resting, L-Locomo-tion, A- Antennal shaking

Fig. 2. Time allocated for swinging, antennal shak-ing, focusing and striking Note: S-Swinging, F –Fo-cusing, A-Antennal shak-ing, ST-Striking

Fig. 3. Time allocated for lo-comotion, antennal shak-ing, resting and grooming Note: R-Resting, G-Groom-ing, L- Locomotion, A- An-tennal shaking

F5% T

S

GL A

R

6%

91%

1%2%

S

23%

15% 57%

A


30

weight of the prey and the space inside the captive area also played a major role in recognizing and capturing the prey7, 8. As the number of prey increased, H. ventralis showed less movement and they adopted a still posture and whenever the prey were at close range of foreleg they quickly captured and consumed it. The size of the prey and predator was important because as the size of the prey decreased it was too small to fit in the raptorial leg of the mantid and hence avoided such prey.

The study revealed that behavior of H.ventralis was related with their feeding strategies. They were activated by the pres-ence of prey and a single movement of the prey attracted their attention and created responses in them. Therefore vision9, 10 and audition may be the more prominent fac-tors that help the mantids to capture their prey and ultimately lead to their successful survival.

AcknowledgementWe express our sincere gratitude to Dr.

M.C. Vyjayandi, Retd. Professor, Providence College, Kozhikode, Kerala, for identifying the Mantid species. Sincere thanks are also to the Principal and management, St. Thomas College (Autonomous), Thrissur, for providing facilities and support.

References1. Mukherjee T.K., Asish Kumar Ghosh, Asish

Kumar Hazra, 1995. The mantid fauna of India (Insecta: Mantodea). Oriental Insects (29): 185-358.

2. Vyjayandi M.C. 2007. Mantid fauna of Ker-ala. Zoological Survey of India, 267 : 1-169.

3. Giglio- Tos, E. 1912.* Mantidi Esotici V Man-tes Tenodera, Hierodulae, Rholllboderae. Boll. Soc. Entomolital., 43: 3-167. * Cross reference.

4. Sureshan .P.M., Samanta .T., & Radhakrish-nan .C., 2007. Mantid (Insecta: Mantodea) fauna of Orissa with some new records for the state. Zoo print journal 22(1):2539-2543.

5. Gelperin A, 1968. Feeding Behaviour of the Praying Mantis: A Learned Modification. Nature 219:399 – 400.

6. Corrette B. J., 1990. Prey capture in the praying mantis Tenodera aridifolia sinensis: coordination of the capture sequence and strike movements. Journal of experimental biology 148:147-180.

7. Iwasaki Taku, 1991. Predatory behavior of the praying mantis, Tenodera aridifolia II. Combined effect of prey size and predator size on the prey.Journal of Ethology(9):77–81.

8. Yamawaki Yoshifumi, 2011.Defence behaviours of the praying mantis Tenod-era aridifolia in response to looming objects. Journal of Insect Physiology (57): 1510–1517.

9. Prete Frederick R., Robert Theis, Justin L. Komito, Jessica Dominguez, Salina Domin-guez, Gavin Svenson,& Frank Wieland,2012. Visual stimuli that elicit visual tracking, approaching and striking behavior from an unusual praying mantis, Euchomenella mac-rops (Insecta: Mantodea). Journal of Insect Physiology (58):648–659.

10. Schirmer Aaron E., Frederick R. Prete, Edgar S. Mantes, Andrew F. Urdiales, WilBogue, 2014. Circadian rhythms affect electroretino-gram, compound eye color, striking behavior and locomotion of the praying mantis Hiero-dula patellifera. Journal of Experimental Biology 217: 3853-3861.

Fig. 4. Grooming of fore leg Fig. 5. Grooming of hind leg



Comparative study of In vitro antioxidant properties of

Jatropha curcas Linn and Jatropha multifida Linn

Deepa .M.K.1 and Jelly Louis1

Department of Botany, Mercy College, Palakkad - 678 006, Kerala, India

AbstractJatropha plants gained considerable significance in recent years due to its property of biodiesel produc-tion. Another reason is the medicinal properties of some species of Jatropha. In the present study in vitro antioxidant activities of fresh leaves of Jatropha curcas and Jatropha multifida were analyzed. Antioxidant properties were determined using DPPH radical scavenging activity and reducing power assay. The highest DPPH radical scavenging activity is seen in J.curcas and lowest in J.multifida. IC50 value of J.curcas is 76µg/ml and that of J.multifida is 88µg/ml. The reducing powers of all samples were increased with increasing concentration, and it is highest in J.curcas.

Keywords: Antioxidant, Radical scavenging activity, DPPH, Reducing power.

IntroductionPlants are a rich source of useful drugs

since ancient times. In India, plants have been used for many therapeutic applica-tions by our traditional system of medicines such as Ayurveda, Unani, Homeopathy, and Siddha. Traditional medicine systems gave birth to many active compounds in early years of natural chemistry, but reports of natural products from different sources declined rapidly in last few decades and is looking for new active compounds.

All living organisms contain an antiox-idant defensive mechanism to counter the free radicals, reactive oxygen species (ROS), reactive nitrogen species (RNS) and other oxidants produced as byproducts of the metabolism. Most of the diseases caused by overproduction and reactive mechanism of free radicals1 2.

Many medicinal plants, spices, aromatic plants using as food supplements and medi-cines are natural antioxidants3. It has been an upsurge of interest to look for potential antioxidants from plants sources.

Euphorbiaceae, the spurge family, is a large family of flowering plants, with about 300 genera and around 7500 species. Among these, Jatropha L is the one of the main genus, belonging to the sub family Crotonoideae, which has about 170 species.

The name Jatropha is derived from the Greek word ‘jatros ‘(doctor) and ‘trophe’ (food).The genus Jatropha has a widest distribution, with species found in Africa, India, South America, West Indies, Central America and the Caribbean. Two species of Jatropha is selected for present study. They are Jatropha curcas and J. multifida.

J. curcas is a semi-evergreen shrub or small tree, reaching to a height of 6 m. Leaves are 10-15 cm long, broadly ovate, cordate, acute usually palmate, 3-5 lobed. Flowers 7.5 mm across, yellowish green, in loose, axillary, cymose panicle .Fruit 2.5 cm long and ovoid. It is resistant to a high degree of aridity, allowing it to be grown in deserts.

The seeds contain 27-40% oil that can be processed to produce high quality biodiesel


32

fuel, usable in a standard diesel engine. The twigs are used for tooth brushing when the gums are swollen. The sap of the plant and leaves are applied to wounds and refractory ulcers; very effective in treatment of sca-bles, eczema, ringworm and toothache.

J. multifida is also known as coral plant is a shrub or small tree with a sin-gle trunk, a loose, spreading crown and

a typical height in cultivation of 6-10 ft. The latex of J. multifida is applied for the treatment of wounds, scabies and ulcers. The ether extract of the roots showed anti-biotic activity against Escherichia coli. Seed oil is sometimes used as cathartic, although it may cause strong irritation and poison-ing. Seeds are used fresh as purgative and emetic. The leaves contains saponins, they are used as purgative, and in the treatment of dysentery.

Materials and methodsFresh leaves of Jatropha curcas, J.multi-

fida were collected from different localities of Palakkad district in March 2017. Plant leaves were washed thoroughly with tap water .The leaves were dried under shade at room temperature for 3-5 days. Then the dried leaves were powdered using mechan-ical grinder, and stored in air tight con-tainers for further use .The crude extract was prepared by cold maceration method. The powdered leaves were extracted with ethanol for 3-4 hours using magnetic stir-rer. Then extract was filtered by Whatman No: 1 filter paper and the pooled extracts

were then evaporated to dryness .Then it is stored in refrigerator for further use.

Antioxidant activities DPPH Radical Scavenging Activity4

The free radical scavenging activity of the ethanolic leaf extract of Jatropha spe-cies was measured by DPPH4. According to

this method, 0.1mM solution of DPPH in ethanol was prepared and 2 ml of the solu-tion was added to 3 ml of sample extract in distilled water at different concentration (20-100µg/ml). The mixture was shaken vigorously and allowed to stand at room temperature in dark for 30 minutes .Then the absorbance was measured at 517 nm.

Reducing Power Assay5 The reductive abilities of sample extract

were estimated using the method5. In this method, different concentration of sample extract was mixed with 2.5ml of Phosphate buffer (0.2 M, pH 6.6) ,and 2.5 ml of Potas-sium Ferricyanide (1% w/v) are added to it. The resulting mixture was incubated at 500c for 20 minutes, followed by addition of 2.5ml Trichloroacetic acid (10% w/v). The mixture is centrifuged at 3000 rpm for 10 minutes .Then collect the upper layer of the solution (2.5 ml), mixed with distilled water (2.5 ml) and 2.5 ml Ferric chloride solution (0.1% w/v). The absorbance is measured at 700 nm. All the tests were done in triplicate and results were expressed as mean ± stan-dard deviation.

Fig.1a. Jatropha curcas Fig.1b. Jatropha multifida

Deepa and Jelly Louis:Study of in vitro antioxidant properties of Jatropha

33

ResultsDPPH Radical Scavenging Activity

Lower absorbance of reaction mixture indicated higher free radical scavenging activity. Percentage of inhibition of DPPH radical is calculated by using formula

% inhibition of DPPH free radical = [(A0

- A1)/A0]*100Where A0 is the absorbance of control

reaction and A1 is the absorbance of the sample.

The antioxidant activity of each sample was expressed in terms of IC50 (micro molar concentration required to inhibit DPPH radical formation by 50%), calculated from the graph after plotting inhibition percent-age against extract concentration.

Results shown in table indicate that per-centage of DPPH radical scavenging activ-ity is increasing with increasing concen-tration. J. curcas has highest antioxidant activity with IC50 value 76 µg/ml than the J. multifida having IC 50 value 88µg/ml.

Reducing Power AssayThe reducing power of ethanolic leaf

extract of Jatropha species were increasing with increasing concentration. In this study J.curcas showed highest reducing power as shown in table 2.

DiscussionReactive oxygen species (ROS) are oxy-

gen centered free radicals such as hydrogen peroxide, superoxide, hydroxyl and per-oxyl ROO-.Over production of ROS results in oxidative stress, leads to the cell mem-brane, protein denaturation, lipid perox-idation and oxidative DNA damage that has been considered generally linked with many chronic diseases in human beings, including diabetes, cancer, cardiovascular diseases and inflammations. Antioxidants have been defined as a substance when present at lower concentration compared with oxidizable compounds (eg: DNA, pro-tein, lipid and carbohydrate), which delay or prevent oxidative damage5

.

For in vitro antioxidant screening, dif-ferent methods such as DPPH free radical

Table 1. DPPH Radical Scavenging Activity of ethanol leaf extract of Jatropha species.

Sl. No:

Sample concen-tration (µg/ml)

Inhibition of DPPH (%)

J.curcas J.multifida

1 20 31.25± 0.003 27.5± 0.002

2 40 37.5 ± 0.0005 36.25± 0.001

3 60 46.25± 0.001 41.25± 0.0014 80 51.25± 0.002 47.5± 0.0025 100 71.25± 0.001 53.75± 0.002

IC50 values 76 88Values are expressed as Mean ±SD.

Fig.2. Percentage inhibition of DPPH of leaf eth-anol extract of Jatropha curcas and Jatropha multifida.

Table 2. Reducing power activity of Ethanolic leaf extracts of Jatropha species.

Sl. No:

Sample concent-ration(µg/ml)

Absorbance at 700nm

J.curcas J.multifida

1 20 0.179 ±0.001 0.122 ± 0.0022 40 0.193 ± 0.0011 0.140 ± 0.00053 60 0.212 ± 0.0011 0.142 ± 0.00174 80 0.221 ± 0.003 0.180 ± 0.00115 100 0.276 ± 0.001 0.207 ± 0.001

Values are expressed as mean ±SD

Fig.3. Reducing power assay of Jatropha curcas and Jatropha multifida leaf extracts


34

scavenging, metal ion chelating, hydrogen peroxide scavenging, reducing power assay are commonly used. However, the total anti-oxidant activity of an antioxidant cannot be evaluated using one single method, due to oxidative process. Therefore, at least two methods should be employed in order to evaluate the total antioxidant activity.

The model of scavenging the stable DPPH radical is a widely used method to evaluate antioxidant activities in a relatively short time compared to other methods. The effect of antioxidants on DPPH radical scaveng-ing was thought to be due to their hydro-gen donating ability. DPPH is a stable free radical and accepts an electron on hydrogen radical to become a stable diamagnetic mol-ecule. The decrease in absorbance of DPPH radical is caused by antioxidants because of the reaction between antioxidant mol-ecule and radical progresses those results in the scavenging of the radical by hydro-gen donation. It is visually noticeable as a discolouration from purple to yellow. In this study, absorbance of reaction mixture decreases with increasing concentration of the solution but percentage of free radical scavenging activity increases with increas-ing concentration. IC50 value was calculated from the graph after plotting inhibition per-centage against extract concentration. J. curcas has the highest antioxidant activity with IC 50 value 76 µg/ml than the J. multifida with IC50 value 88 µg/ml.

The reducing power assay is often used to evaluate the ability of an antioxidant to donate an electron. In this assay, the abil-ity of extracts to reduce Fe3+ to Fe2+ was determined. The presence of antioxidants in the extracts resulted in reduction of the Ferric Cyanide complex (Fe3+) to the Fer-rous Cyanide form (Fe 2+), thereby changing the solution to various shades from green to blue, depending on the reducing power of the compounds. In this study, absorbance of reaction mixture increases with increasing concentration, i.e., higher the absorbance of reaction mixture, higher would be the reducing power. Reducing power is highest in J. curcas and lowest in J. multifida6.

ConclusionJ.curcas and J. multifida are the promis-

ing plants in the traditional medicines used for the various therapeutic applications. In the present study these two plants showed significant in vitro antioxidant activity. But highest antioxidant activity was noted in the J.curcas when compared to J.mul-tifida. This may be due to the presence of phytochemicals such as terpenoids, tan-nins, flavonoids and phenolic compounds. A further study of isolation and characteri-zation of bioactive molecules from the plant is required for therapeutic and multiple applications.

AcknowledgmentWe are thankful to the Head of the

Department of Botany at Mercy College Palakkad for providing us all facilities and support for our work. We also acknowledge DST-FIST for the financial aid for setting up our research lab.

Reference1. Alam M. N., Bristi N. J., Rafiquzzaman M.,

Alam N., and Bristi N. J., 2013. Review on in vivo and in vitro methods evaluation ofan-tioxidant activity. Saudi Pharm. J., 21(2): 143–152.

2. Valko M., Leibfritz D., Moncol J., Cronin M. T., Mazur M., and Telser J., 2007. Free radi-cals and antioxidants in normal physiological functions and human disease. Int.J Biochem Cell Biol., 39( 1) :44–84.

3. Veeru P., Kishor M. P., and Meenakshi M., 2009. Screening of medicinal plant extracts for antioxidant activity. J. Med. Plants Res. 3: 608–612.

4. Shimada K., Fujikawa K., Yahara K., and Nakamura T., 1992. Antioxidant properties of xanthone on the auto oxidation of soybean in cylcodextrin emulsion. J Agric Food Chem. 40: 945-948.

5. Oyaizu M., 1986. Studies on products of browning reaction: Antioxidant activities of products of browning reaction prepared from Glucosamine. Jpn. J. Nutr.; 44: 307-315.

6. Uppal G. and Nigam V., 2012. In vitro antiox-idant activity of Ethanolic extract of Euphor-bia hirta Linn. Int.J. of Current Research. 4 (10): 155-159.


Bactericidal activity of nutmeg (Myristica fragrans) pericarp

Flower Varghese1 , Teji K.T.1 and Philo T.J. 2

1Dept. of Zoology, Morning Star Home Science College, Angamaly-6835732Dept. of Botany, Morning Star Home Science College, Angamaly-683573

AbstractAntibiotics are substances which are used against an infection caused by bacteria, fungi etc..Bacteria are unicellular organisms which show resistance or sensitivity to certain antibiotics present in the nature. In this study antibacterial activity of aqueous, diethyl ether, ethyl acetate and methanol extract of pericarp of Mystrica fragrance against Escherichia coli were analysed. Methanol extract showed highest sensitivity and 2.2 cm clear zone was observed. Diethyl ether extract of Nutmeg pericarp was found to be resistant to E. coli where as the others were sensitive.

Keywords : Antibiotic sensitivity, Nutmeg pericarp, Methanol extract, Diethyl extract

IntroductionThe worldwide distribution of multidrug

resistance in both community and health care associated bacterial infections has impaired the current antimicrobial ther-apy, warranting the search for other alter-natives1. Herbal medicine are the finished, labeled medicinal products that contain active ingredients from aerial or under-ground parts or other plant materials or combination thereof, whether in crude state or as plant preparations. As per WHO reports about 4 billion people of the world population presently use herbal medicines for their primary health care as alternative system of medicine i.e., ayurvedic, homeo-pathic, naturopathic, oriental and native American Indian medicine2. Extracts from all parts of the plant show pharmacological properties, recognized by popular use and corroborated by the scientific community.

Antibiotics are a special kind of chemo-therapeutic agent usually obtained from living organisms. The word antibiotic has to refer to a metabolic product of one micro-organism that in very small amounts is detrimental or inhibitory to other micro-organisms. It has been known for many

years that antagonisms can exist between microorganisms growing in a common envi-ronment. Several thousand antibiotic sub-stances have been isolated and identified since 1940.

Antibiotics can be classified in several ways. For example, some are bactericidal and others are bacteriostatic. They may be grouped on the basis of chemical struc-ture. A third way of classifying antibiotics is on the basis of their mode of action that is the manner in which they manifest their damage upon microbial cells. Inhibition of cell wall synthesis, damage to the cytoplas-mic membrane, inhibition of nucleic acids and protein synthesis, inhibition of spe-cific enzyme systems etc are the mode of actions3. The antimicrobial activity of plant extracts and phytochemicals was evalu-ated with antibiotic susceptible and resis-tant microorganisms.4,5,6 ,7 . Antibiotics are substances which used against an infection caused by bacteria, virus, fungi etc… Bac-teria are unicellular organisms which show resistance or sensitivity to certain antibiot-ics present in nature. In this study antibac-terial activity of Nutmeg pericarp extract of aqueous, ethyl acetate, diethyl ether and

∗ Corresponding author: [email protected]


36

methanol against Escherichia coli were analysed.

Nutmeg (Myristica fragrans) (Fig.1) belongs to the family Myristicaceae. It is a tropical evergreen tree. It is native to Indo-nesia. It contains essential oil myristic acid,

geraniol, camphor, oleic acid and eugenol. Nutmeg is a bitter spicy herb. It stimulates digestion and improves appetite. It also used for digestive tract infection, colic flatu-lence, nausea etc…

Material and methods Collection of test material

Healthy fresh fruits of M. fragrans were collected, washed, sliced and shade dried in oven. Dried pericarp was powdered using an electrical grinder. Fine powder was obtained by sieving.

Preparation of extracts2gm of pericarp powder was weighed in

an electronic balance. Pericarp powder was mixed in 10ml of distilled water for aqueous extract preparation. For all other extracts 10ml of solvent was added in each 2gm pow-der. The mixtures were stirred for 24 h in an electrical shaker at120rpm and left to stand for 48 h at room temperature. The super-natants were transferred into three watch glasses and used in this study.

Preparation of antibiotic discAutoclaved Whatmann No. 1 filter

paper discs, 6mm in diameter, were put in watch glass containing the extracts for 1hour and dried under sunlight. They were used for antimi-crobial sensitivity test.

Microorganism

Escherichia coli.Preparation of nutrient medium

500ml nutrient agar medium was pre-pared by dissolving 2.5g peptone, 1.5g beef extract, 10g agar agar and 4g sodium chloride in distilled water in a conical flask. It was then sterilized.

ProcedureThe method used for antibacterial sensi-

tivity is Kirby-bauer disc diffusion method8. The nutritive media after sterilization was cooled down to ear bearing heat and was poured into petri plates that were already sterilized .The nutrient medium was allowed to solidify. When it solidified 300µl of bacterial strain was taken and poured into the solidified medium. Using L-road the culture was spread evenly in aseptic conditions.

Preparation of pericarp powderAfter which the prepared discs were

placed on the medium using sterilized forceps . They were incubated over night and the zone of inhibition was noted. The clear zones around the disc was mea-sured using a scale.

Results The antimicrobial activity of Nutmeg

pericarp extracts of aqueous, ethyl acetate, diethyl ether and methanol against Esche-richia coli is presented in Table 1. Diethyl ether extract did not show sensitivity to bac-teria so no clear zone was observed. Meth-anol extract had highest sensitivity and 2.2 cm clear zone was observed. All other extracts were also sensitive to bacteria (Fig. 2-5).

Table 1. Antibacterial activity of .fragrans pericarp ex-tracts against E. Coli

Extracts Bacteria usedMeasurement of zone of inhibition

Interpretation

Aqueous Escherichia coli 1.6cm SensitiveEthyl acetate Escherichia coli 1.1cm SensitiveDiethyl ether Escherichia coli 0 ResistantMethanol Escherichia coli 2.2cm Sensitive

Fig. 1 Pericarp of Myristica fragrans

Flower Varghese et al : Bactericidal activity of nutmeg pericarp

37

DiscussionThe ability of extracts to inhibit the

growth of bacterial strains is an indication of its antibacterial potential that might be utilized in the management of bacterial infections in future, so the plant or quintes-sence may be employed to develop a poten-tial antimicrobial drug. The antimicrobial activity shown by the extract might be due to some antimicrobial substances present in these plants. Here diethyl ether extract of Nutmeg pericarp was found to be resistant to E. coli where as the others were sensi-tive. The antibacterial activities observed in the present study could be attributed to compounds such as alkaloids, glycerides and flavonoids, steroids, terpenoids, sapo-nins, tannins and anthraquinone present in the plant parts.9, 10 It also reported that M. fragrans had antimicrobial, antibacterial, larvicidal and molluscicidal properties.

ConclusionPlant extracts have great potential as

antimicrobial compounds against micro-organisms. Thus they can be used in the treatment of infectious diseases caused by resistant microbes. With the rise in disease causing that have become resistant to syn-thetic drugs, phyto-medicine is now gain-ing importance. The results of the present study confirm the importance of laboratory testing of medicinal plants used in indige-nous medicine in search of new substances capable of inhibiting E. coli.

AcknowledgementWe express our sincere thanks to Ker-

ala State Council for Science Technology and Environment for the financial sup-port given under SPYTiS- II 2016(125/SPYTiS-II/2016/KSCSTE).

Fig.2 Aqueous Fig.3 Ethyl acetate Fig.4 Diethyl ether Fig.5 Methanol


38

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2. Kumaran A. M., D’souza P., Agarwal A., Bokkolla R. M., Balasubramaniam M., 2003. Geraniol, the putative anti-helmintic principle of Cymbopogon martinii. Phytother Res. 17 (8): 957.

3. Pelczar M.J., Chan E.C.S., Kring N. R., 1993. Microbiology. Tata Mc Graw-HillEducation Ltd. Delhi.

4. Almagboul A. Z.; Bashir A. K.; Farouk A.; Salih A. K. M., 1985. Antimicrobial activity of certain Sudanese plants used in folkloric medicine. Screening for anti-bacterial activ-ity. Fitoterapia. 56, 331-337.

5. Al-Sarraj S. M., Redha F. M. J., Mahmoud M. J., Hussein W. A., 1985. Modified extraction procedure for the active constituents of some Iraqi medicinal plants. Fitoterapia . LVI, 56:58.

6. Bouhnik Y., Flourie B., D’Agay-Abensour L., Pochart P., Gramet G., Durand M., Ram-baud J.C., 1997. Administration of trans-galacto-oligosaccharides increases faecal bifidobacteria and modifies colonic fermen-tation metabolism in healthy humans. J Nutrn. 127, 444–448.

7. Dabur R., Amita Gupta, Mandal T. K., Lavekar T. K., 2007. Antimicrobial activity of some Indian medicinal plants. Afr. J Tra-dit. Complement Altern Med., 4(3), 313-318.

8. Bauer A. W., Kirby W. M., Sherries J. C., Turck M., 1966. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol., 36(4): 493-496.

9. Sankar Narayan Singha, 2012. Phytochem-ical analysis and antibacterial potential of Moringa oleifera Lam. IJSID, 2(4): 401-407.

10. Minu jose, Sini jose, Lini Mohandas, Alphonsa Vijaya Joseph, 2014. Effects of Moringa oleifera Lam. on human patho-genic bacteria.in: Proc. Natl. Semin.Trends in Biodiversity studies. St.Terasa’s college Ernakulam, December 4-5, 201-207.



Manifestations in the epididymis in response to treatment of ursolic acid, a phytotherapeutic : a study in Wistar rat

Girija R1, Kadalmani B2 and Akbarsha MA3

1 Department of Zoology, Mercy College, Palakkad - 678 006, Kerala, India. 2Department of Animal Science, Bharathidasan University, Tiruchirappalli-620 024, India.3Visiting Professor, King Saud University, Saudia Arabia, Riyadh, Kingdom of Saudi Arabia.

AbstractUrsolic acid (UA), a triterepenoid lactone used widely in the cosmetic industry, is considered to be der-matologically innocuous. Preliminary studies from our laboratory indicated UA to produce testicular pathology. Therefore, in this study, an attempt was made to find the manifestations in the epididymis to ursolic acid treatment. Wistar strain male albino rats were administered UA at a daily dose of 10mg/kg body weight intraperitoneally for 55 days and the various segments of the ductus epididymidis were subjected to histopathological analysis adopting resin embedding, semithin sectioning and transmis-sion electron microscopy. In the initial segment and the intermediate zone the narrow cells underwent hyperplasia and their apical portions protruded deep into the lumen. At all the segments of the epidid-ymis, the Golgi cisternae of the principal cells appeared hypertrophied. In the proximal as well as distal cauda epididymides, clear cells underwent hypertrophy and hyperplasia. Their cytoplasm increased in the accumulation of dark lysosomal bodies and vacuoles. Further, in all the segments of the epididymis the intraepithelial lymphocytes and basal cells became abundant, and particularly the former had their distribution at all levels along the height of the epithelium, several of them being located along the apical border. Several of basal cells appeared plumby. The basal cells were found to be ladenend with lipofuscin inclusions. The responses of the epididymal epithelium to UA treatment suggest them to be mechanisms to combat the arrival of Sertoli cell fragments and dead / defective sperm in the epi-didymal lumen, consequent upon the toxic effect of UA on Sertoli cell in the seminiferous epithelium.

IntroductionUrsolic acid (UA) is a pentacyclic triter-

penoid compound (C30H48O3 ; Mol. Wt 456.68) isolated from the leaves of Ocimum sanctum ( Fam: Lamiaceae), etc., which may occur in their free acid form as glycones of triter-penoid saponin linked to one or more sugar moieties 1. Ursolic acid naturally occurs in a large number of vegetarian foods and medicinal herbs 2,3 . UA was considered to be pharmacologically inactive 4, it`s alka-line salts as potassium/sodium ursolates were in use as emulsifying agents in phar-maceutical, cosmetic and food preparations 4,5. However, upon closer examinations, UA was found to be medicinally active topi-cally and internally 2. Novel UA derivatives,

including ursane-type triterpenoid sapo-nins, are naturally produced as secondary metabolites through complex metabolic pro-cesses in different parts of the plant 6,7,8,9,10. Contemporary scientific research, which led to the isolation and identification of ursolic acid, revealed and confirmed that several pharmacological effects, such as anti-tu-mor, hepato-protective, anti-inflammatory, anti-ulcer, anti-microbial, anti-hyperlip-idemic and anti-viral, can be attributed to UA2. However, its anti-inflammatory, anti-tumor and anti-microbial properties are relevant to the cosmetic industry.

Ursolic acid has also been recommended as an ingredient in burn ointments, since it inhibits the activity of inflammatory


40

enzymes such as human leukocyte elas-tase (HLE), lipoxygenase and cyclooxygen-ase 5,11,12. Ursolic acid also encourages hair growth by stimulating the peripheral blood flow and activating the hair mother cells. Alopecia-preventing and dandruff-prevent-ing effects of UA have also been discovered 13. Ursolic acid also exhibits an antimicro-bial effect as it inhibits the growth of sev-eral strains of Staphylococci, Microsporium lenosum and Candida albiicans 14,15. Ursolic acid has been used to treat photo-aged skin, as it inhibits the appearance of wrinkles and age-spots by restoring skin collagen bundle structures and elasticity 16. Notwithstand-ing its numerous pharmacological prop-erties, UA is an ideal cosmetic ingredient because its overall toxicity has been consid-ered low. Infact, it has been termed derma-tologically innocuous. Ursolic acid is known to inhibit 12-O-tetradecanoylphorbol-13-ac-etate (TPA)-induced tumor initiation and promotion, inflammation and ornithine car-boxylase activity in mouse skin 17, 18,19. Theo-retically, inhibition of tumorigenesis means arrest of cell proliferation. Spermatogenesis is a dynamic process of germ cell prolifer-ation and differentiation from stem sper-matogonia to spermatozoa 20. Therefore, a preliminary attempt using light microscopy made by us to find the effect of this phyto-chemical on the male reproductive system suggested it to be a potent disruptor of sper-matogenesis, and there were indications of pathological changes in the epididymal epi-thelium 21. The latter prompted us to take up a focused study to find the manifesta-tions in the epididymis to UA treatment.

Materials and MethodsAdult Wistar strain male albino rats, 90

day old, weighing 150-200g, raised from a stock procured from Fredrick Institute for Plant Protection and Toxicology (FIPPAT), Padappai, Chennai, India, were placed in groups of five per cage. The animals were maintained with standard pellet feed (Sai Durga Feeds and Foods, Bangalore, India) and water ad libitum. The UA (Sigma Chemical Co., MO, USA) was dissolved in minimum quantity of ethanol and diluted quantitatively in physiological saline and

administered to 10 rats in the experimental group through intraperitoneal (i.p.,) route at a daily dose of 10 mg/kg body weight for 55 days, according to Akbarsha et al. 21, the duration of one complete spermatogenic cycle 22. Rats in the control group, in equal number, received the vehicle alone. At the end of the experimental period, the repro-ductive system of five rats in each group was perfused with Karnovsky`s fluid under mild MS222 anesthesia and dissected to remove the testis and epididymis. After removing the connective tissue around, thin slices of testis and different segments of epididymis (initial segment, intermediate zone, caput, corpus and cauda) were fixed in 2.5% glutar-aldehyde in cacodylate buffer, post-fixed in 1% osmium tetroxide and embedded in thin viscosity resin (Spurr mix, Sigma Chem-ical Co., MO, USA). Semi-thin sections (1μm thickness), cut in an ultra-microtome (Reichert Jung, Austria), were stained with toluidine blue-O (TBO) and observed in a Carl Zeiss (Germany) Axio 2 Plus research microscope. Images were captured through a CCD camera in a computer and processed using Carl Zeiss Axiovision software. Areas in TBO-stained sections were chosen for transmission electron microscopic analysis. Ultrathin sections cut in a Leica (Germany) ultra-microtome were stained with uranyl acetate and lead citrate and observed in a Phillips (Holland) 201C transmission elec-tron microscope (TEM). The seminiferous tubules and the cell types lining the epi-didymal epithelium, namely principal cell (PC), basal cell (BC), clear cell (CC), apical cell (AC), narrow cells (NC) and intraepi-thelial leukcocytes (IELs, i.e., lymphocytes as well as macrophages), and also the lumi-nal content were critically observed.

The remaining five rats in each group were dissected under MS222 anesthesia to expose the epididymis. The connective tissue capsule around the epididymis was incised and it was transferred to an embryo cup. After a thorough rinse in physiological saline, the cauda epididymidis was macer-ated to release the semen. Using a micropi-pette, 0.05µl of the semen was transferred to an embryo cup and diluted 200 times. From this preparation, hanging drops were

Girija et al: Toxic manifestations of Ursolic Acid in Epididymis

41

made for observation in the microscope to analyse the nature, duration and grade of motility and the vitality of the sperm according to the methods prescribed 23. Sperm counts were made using Neubauer`s counting chamber.Sperm smears, stained with Giemsa`s / Papanicoloau`s stain, were used to record the sperm abnormalities.The investigation was approved by the Institu-tional Animal Ethics Committee (Registra-tion No. 418/01/a/CPCSEA/dt. 4.6.2001).

ResultsUrsolic acid treatment resulted in pro-

duction of spermatozoa possessing different kinds of abnormalities, as observed in the

epididymal spermatozoa (Fig.1). One major abnormality was the failure to shed the CD by a large number of the cauda epididymidal spermatozoa (Fig.2). The most characteristic structural organization of CD relates to the presence of numerous less dense irregular inclusions and moderately dense irregular structures around the abnormal axonemes (Fig.3). Cauda epididymidal sperm counts, motility and vitality decreased (Table 1). In several spermatozoa the plasma membrane was detached from the nucleus (Fig. 4). Ser-toli cell fragments containing germ cells were present in large numbers in the lumen of the epididymis. They contained late-step spermatids and cell debris, several of which were undergoing disintegration (Fig. 5). Germ cells, prematurely released from the seminiferous epithelium, resembling round spermatids, were also present in the ductus epididymidal lumen (Fig. 6). Macrophages were found in large numbers in the lumen of the epididymis (Fig.7). The luminal

macrophages contained spermatozoa and cell debris, in cytoplasmic vacuoles, and also large electron-dense vesicles/vacuoles, which resembled primary lysosomes and/or multivesicular bodies (MVBs) (Fig.8).

Since many of these changes reflected testicular pathology, sections of testis were subjected to light microscopic analysis. In the UA-treated rats spherical masses, separated out from the rest of the epithe-lium and Sertoli cell, were observed in the seminiferous epithelium. Such masses con-tained several nuclei in common cytoplasm and appeared syncitial (Fig.9). In some of the seminiferous tubules spacious cavities appeared in the epithelium and multinu-

cleate masses containing intensely stain-ing nuclei were found to lie loose in these cavities. In certain of the extreme cases, the seminiferous epithelium decreased in height, but towards the lumen it appeared densely fibroid with some of the tubules containing uni-/multinucleate cells entan-gled in the fibroid extensions (Fig.10). Apart from this fibrosis of the adluminal compartment of SC`s due to the exfoliation of masses of germ cells, large lumina were formed around the early stages spermato-cytes (Fig.11). The Leydig cells were intact, but appeared hypertrophied (Fig.12).

The most characteristic change observed in the initial segment of the epididymis was occurrence of abundant NCs which had their apical portions protruding deep into the lumen. The apical cytoplasm of NCs was rich in dark dense bodies which may be pri-mary/secondary lysosomes (Fig.13). Basal to the nucleus, the cytoplasm contained

Table.1 Data on sperm parameters of male rats treated with ursolic acid

GroupSperm motility

Sperm vitality

Sperm counts (x 106 /ml)

Cauda epi-didymidal sperm retaining CD (%)

GradeDuration

(min)

ControlRapid, progressive

43.2 ± 1.2 +++29.87 ±

3.956.2 ± 0.7

UA-treatedSluggish, non-progressive

12.3 ± 0.9 *

+18.38 ± 1.52 *

47.3 ± 1.0 *

Values are Mean ± SD* p <0.01


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lipofuscin-like inclusions. The protruding apical ends of the NCs had their cytoplasm profusely vacuolated (Fig.14), and piched off into the lumen. In the intermediate zone of the epididymis, the Golgi zone of the PCs was densely vacuolated (Fig.15). The PCs of the caput epididymidis contained vacuoles in the apical cytoplasm and such vacuoles enclosed particles comparable to sperm heads (Fig.16,17). At all the segments of the epididymis, the Golgi cisternae of the PC`s appeared hypertrophied and the sacs opened up (Fig.18). Further, IELs and BCs were more abundant than in the controls and, particularly, the IEM`s had their dis-tribution at all levels along the height of the epithelium, several of them located along the apical border (Fig.19, 20, 21,22). In the

proximal as well as distal cauda epididymi-des, the CC`s underwent hypertrophy and hyperplasia, while the PC`s appeared com-pressed (Fig.23). Consequently, the mem-brane between adjacent PC`s became indis-tinct resulting in apparently bi- and tri-nu-cleate PC`s (Fig.24). The apical cytoplasm of the CC`s was densely vacuolated, and the rest of the cytoplasm was packed with large lysosomal bodies (Fig.23, 25). The highly heterogenous nature of the content of the lysosomal bodies was evident in the TEM pictures (Fig.25, 26). The CC`s were only very sparsely microvillated (25, 27). Another significant trend noticed was, irre-spective of the segments of the epididymis, the BC`s increased in abundance. Several of these cells even appeared plumby (Fig.15,

Fig.1.Papanicolaou-stained sperm smear of UA –treated rat. A) fused sperm. B & C) curved sperm. D) agglutinated sperm. E) coiled sperm. F) double-headed sperm. G) detached tails. H) bro-ken heads

Fig.2. Low power TEM of the cau-da epididymis of UA-treated rat showing the lumen (LU). Several sperm in the lumen retain the cy-toplasmic droplet (arrowhead).EP, epithelium.

Fig.3. TEM of a TS of the cauda epi-didymal sperm through the cyto-plasmic droplet of UA-treated rat showing abnormal axoneme (A), less dense inclusions (asterisks) and irregular moderately stained structures (arrows).

Fig.4. TEM of LS of a cauda epidid-ymal sperm showing plasma membrane (PM) detached from nucleus (NU) (arrows).A sperm re-taining the cytoplasmic droplet is also shown (arrowhead)


43

Fig.5. TEM of a Sertoli cell fragment (SF) in the lumen of epididymis undergoing disin-tegration.SM,sperm;LY,lysosomes.Fig.6. TBO-stained semithin section of the cauda epididymidal lumen of UA-treated rat showing the presence of immature germ cells (GS).EP, epithelium, SM, sperm.

Fig.7. TBO-stained semithin section in the canda epididymial lumen showing the presence of luminal macrophages (LM).SM, sperm

Fig.8. TEM picture of epididymal luminal macrophage containing phagocytosed sperm (SM), and also in the process of phagocytosing a sperm (arrowhead). DB, electron dense bodies; MVB, multivesicular body; VA, digestion vacuoles.

Fig.9. H&E-stained paraffin section of testis of UA-treated rat, showing the symplasts in the seminiferous epithelium(SE)(arrows).

Fig.10. H&E-stained paraffin section of seminiferous tubules of UA-treated rat, show-ing depletion of germ cells, and fibroid nature of the Sertoli cells towards the lu-men (F)


44

Fig. 11. TEM of the basal aspect of seminiferous epithelium showing a lumen between a germ cell (GC) and Sertoli cell (asterisks).

Fig. 12. Light micrograph of the interstitial compartment showing hypertrophy of Leydig cells. LC, Leydig cells; ST, seminiferous tubules.

Fig.13. TEM of the apical cytoplasm of a narrow cell (NC) showing dense accumulation of lysosomal granules (LY) apical to the nucleus, and a lipofuscin inclusion basal to the nucleus (arrowhead).

Fig.14. TEM of a narrow cell (NC) showing the apical ends protruding (PE) into the lumen (LU).The protruding end has a basal constriction (arrowhead) indicating its pinching off.

Fig.15. TBO-stained semithin section of the epithelium of intermediate zone of the epididy-mis of UA- treated rat showing vacuolated Golgi zone. In the PC`s the apical vacuoles contain sperm heads (arrowheads), BCs are increased (arrows).


45

Fig.16.TEM of PC`s of the caput epididymidis of UA- treated rat show-ing apical vacuoles containing phagocytosed sperm heads ( ar-rowheads).

Fig.17. One of the vacuoles (VA) of figure.17 magnified. SM, sperm head.

Fig.18. TEM showing hypertrophied Golgi cisternae of PCs.Fig.19. TBO-stained semithin section of the epithelium of caput epi-

didymidis of UA-treated rat showing the abundance of intraepi-thelial macrophages (IEM) at different heights of the epithelium. BC, basal cell.

Fig.20. TBO-stained semithin section showing the epithelium of cor-pus epididymidis of UA-treated rat showing the abundance of intraepithelial macrophages (IEM) at different heights of the epi-thelium. Note basal cells (BC)

Fig.21.TEM of the intraepithelial macrophages (IEM) located at the basal aspect of the epididymal epithelium.

Fig.22. TEM of an intraepithelial macrophage (IEM) located closer to the lumen.


46

Fig.23. TBO-stained section of the epithelium of cauda epididymidis of UA –treated rat showing hypertrophy and hyperplasia of clear cells (CC). Basal cells (arrows) are also increased. The principal cells (PCs) are com-pressed.

Fig.24. TEM of the epithelium of the cauda epididymidis of UA- treated rat. The cross wall between principal cells (PC) has disappeared thus making the principal cells syncitial. BC,basal cell; CC, clear cell.

Fig.25. TEM of the epithelium of the cauda epididymidis of UA- treated rat showing clear cell (CC) with lysosomal granules (LY) and lacking stereo-cilia (arrowhead).Lumen contains sperm retaining cytoplasmic droplet (arrows). The principal cell (PC) is compressed.

Fig.26. TEM cauda epididymidis showing the highly heterogenous nature of the lysosomal bodies (LY) in the clear cells (arrowheads).

Fig.27. TBO-stained semithin section of corpus epididymidis showing a clear cell with apical end protruding into the lumen vacuolated and lacking stereocilia.

Fig.28. TEM of a basal cell of UA- treated rats showing the presence of heter-ogenous lipofuscin (LF) granules. Less dense LF is seen in the indentations of the nucleus (NU).


47

20,23). In the BC`s lipofuscin inclusions were increased, in which case each inclu-sion had a homogenous content, but differ-ent inclusions differed among themselves in electron density (Fig. 28).

DiscussionThe results obtained in this study indi-

cate that UA is a potent disruptor of sper-matogenesis. From the disruption of the seminiferous epithelium caused by UA, resulting in formation of multinucleate giant cells, we conceive that the treatment has caused opening up of the intercellu-lar bridges between germ cells which has already been reported 21. This result is sim-ilar to that of Weber et al. 24 , wherein treat-ment with cytochalasin D resulted in pro-duction of multinucleate giant cells termed symplasts. In a series of study on male reproductive effect of carbendazim (MBC), Nakai and his associates have shown that MBC causes disruption of microtubule of the cytoskeleton of the body of SCs. This results in the apical portions of the SCs breaking away and such fragments carry with them the associated germ cells, which reach the lumen of the seminiferous tubules and arrive at the ductus epididymidis 25, 26. Therefore, the SC fragments noticed in the lumen of the ductus epididymidis, in this study might have been produced due to the disruption of the cytoskeletal framework of the SCs by UA, resulting in breaking away of the apical portion of the SCs, thus deplet-ing the germ cells in the seminiferous epi-thelium. The spermatotoxic effect of UA, indicated by decrease in the sperm counts, motility and vitality of spermatozoa and an increase in the percentage of abnormal sperm, might be due to the direct effect of UA on testis affecting spermatogenesis 21.

One of the most prominent ultrastruc-tural changes occurring in the epithelium of the ductus epididymidis on treatment with UA is hyalination of the Golgi of PC`s resulting in large empty spaces in the supranuclear cytoplasm. The vacuolation of Golgi apparatus leading to hyalination of Golgi area in of UA-treated rats implies to us either a toxic manifestation of UA on the

Golgi or the heightenend activity of Golgi towards formation of secretory granules to fulfill a physiological requirement. It is an established fact that under normal cir-cumstances sperm leaving the testis are not physiologically mature, and the matu-ration takes place only during epididymal transit 27. The changes occurring during sperm maturation involve addition of new proteins, removal of the existing proteins and modification and/or translocation of the existing proteins. These changes occur due to roles by proteins that are secretory products of the PCs 28,29 . Therefore, hyalina-tion and vacuolation of the Golgi, from this point of view, is due to UA toxicity. However, the heightenend endocytic uptake of cell debris/sperm heads by the apical portion of PC`s, their processing in membrane-bound vesicles and the abundance of MVB`s are reflections of heightenend activity of PC`s towards processing of the endocytosed dead sperm/cell fragments, in which process the Golgi apparatus should play the role of pro-viding the lysosomal enzymes. This implies hypertrophy of the Golgi so as to provide the required lysosomal enzymes.

Studies indicate a role for BCs in pro-tecting the epithelium and lumen from the attack of electrophiles.The intense reac-tivity of BC`s to the various subunits of GST`s indicated a role to BC`s in protecting the spermatozoa in the lumen from harm-ful electrophiles entering via the circula-tion 30,31. Thus, in the present study, the increased abundance of BC`s in the pseu-dostratified epithelium in UA-treated rats may point to a similar role. The appearance of plumby BC`s in UA-treated rats is yet another interesting observation. The only earlier report of existence of such plumby BC`s was by Seiler et al. 32 in mice aged 12 days. According to these authors, such plumby BC`s become more flat in aged mice to remain so thereafter. During the process of their establishment, they are not pro-duced from already existing epithelial cells but recruited from elsewhere. During the course of their establishment they become plumby and, subsequently, take the flat shape. Thus, the plumby BC`s in the epithe-lium of UA-treated rats, considered along


48

with the increase in the BC`s, suggest fresh recruitment of BC`s. Accumulation of LF material is considered to be due to a com-bination of oxidative damage and decline in the degradation pathways. Liposomes filled with LF material have been observed in the BC, suggesting a role for it in scavenging reactive oxygen species (ROS) 33. Basal cells are reactive for several glutathione-S-trans-ferases (GST) and Cu-Zn-SOD. Therefore, it has been proposed that BCs have a protec-tive role in detoxifying electrophilic chem-ical substances 34. The results in the pres-ent study in respect of BCs, thus, reflect augmentation of the defense mechanisms to combat electrophilic attack as caused by UA.

According to Seiler et al.32 and Yeung et al.33 BC has origin from circulating /inter-stitial monocytes and express macrophage antigens. The leaky blood- epididymal bar-rier provides for leakage of sperm antigens from the lumen into the intercellular spaces between the epithelial cells. The BC`s play a critical role in the surveillance of sperm antigens so as to prevent the generation of antisperm antibodies 33. It has been pro-posed that BC`s increase in abundance and accumulation of LF material in the aging rat consequent upon leaky blood-epididy-mis barrier 35. Therefore, it is conceivable that UA treatment might have caused dis-ruption of the blood-epididymis barrier as comparable to that caused during aging. This would render access of sperm antigens through the intercellular spaces, and there is response of recruitment of fresh BCs as well as accumulation of LF material in the existing BCs.

Narrow cells are confined to the initial segment and intermediate zone30,36. The presence of invariably all the detoxifica-tion enzymes has indicated a role for NCs in detoxification mechanisms37. The prod-uct of detoxification would accumulate in MVBs for lysosomal degradation. The NCs in, order to cast off such contents, would responded with an autotomy where-upon the apical portion is autotomized. This interpretation corroborates the observation of Martan and Allen38 and Martan et al39

that NC`s would be extruded into the lumen of the epididymal duct, where they complete their degeneration.

Abe and Takano40, in the mouse, and Goyal and Williams41, in the goat, were the first to make a clear cytological distinction between intraepithelial lymphocytes and intraepithelial macrophages. According to these authors, epididymal tissue lympho-cytes are a resident population, and macro-phages arrive from the circulation. Lympho-cytes do not enter the lumen of the epidid-ymis whereas macrophages can access into the lumen. The lymphocytes are considered to be concerned with surveillance of sperm antigens 35, 42, 43, 44, whereas macrophages are concerned with phagocytosis of dead sperm from the lumen as well as the intercellular spaces between the epithelial cells 45. The ultrastructural evidences obtained in the present study suggest most of the migra-tory leukocytes to be macrophages. There-fore, the increase in the abundance of mac-rophages is considered to be concerned with phagocytosis of dead sperm from the lumen as well as the intercellular spaces between the epithelial cells.

The response of increased vacuolation and lysosomal bodies in the CC`s may be interpreted from the points of view of phys-iological as well as pathological responses. Generally, CC`s are attributed with roles of endocytosis and disposal of the cytoplas-mic droplet shed by the maturing sperma-tozoa46. In the present study, the hypertro-phy and hyperplasia of the CC`s and the increase in the lysosomal bodies reflect their heightenend endocytic activity. The arrival of immature germ cells from the testis into the epididymal duct, after their processing in the epididymal lumen, would contribute to accumulation of cell debris in the lumen of the distal segment of the epididymis. The corpus and cauda epididymides alone pos-sess the CC`s, and there is no other mech-anism by which the epididymis can cleanse the debris.Such mechanisms can be sought in their endocytic removal by the CCs. The hypertrophy and hyperplasia of CCs results


49

in compression of the PCs and the apparent disappearance of the crosswalls between them.

Thus, the present study indicates that UA, a phytotherapeutic, can produce toxic manifestations of disruption of spermato-genesis, and also epididymal sperm matu-ration. The epididymis can also be a target to the toxic effect of UA. However, epididy-mis is a versatile organ capable of manag-ing such adverse conditions through alter-ations in structure, and/or augmentation of the inherent physiological roles of the epi-thelial cells.

Acknowledgments The ultracut and TEM facility of Well-

come Trust Laboratory, Christian Medi-cal College and Hospital, Vellore, India, is gratefully acknowledged. The authors are grateful to the Department of Science and Technology (DST), Govt. of India, New Delhi, for the grant No.(SP/SO/C-31/98) and the assistance under FIST scheme to the Department.

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26. Nakai M., Hess R.A., Netsu J., Nasu T., 1995. Deformation of the rat Sertoli cell by orl administration of carbendazim (methyl 2-benzimidazole carbamate). J Androl 16, 410-416.

27. Eddy and O`Brien. 1994. The Spermatozoon , in: Knobil E., Neill J.D., (Eds.) The Physiol-ogy of Reproduction. Raven Press Ltd., New York, 1117- 1190.

28. Dacheux J.L., Druart X., Fouchecourt S., Syntin P., Gatti J.L., Okamura N., Dacheux F., 1998. Role of epidymal secretory proteins in sperm maturation with particular refer-ence to the boar. J Reprod Fertil 53, 99-107.

29. Cooper, T.G., Interactions between epidid-ymal secretions and spermatozoa. 1998. J Reprod Fertil 2, 119- 136.

30. Veri J.P., Hermo L., Robaire B., 1993. Immu-nocytochemical localization of the Yf subunit of glutathione S-transferase P shows regional variation in the staining of epithelial cells of the testis, efferent ducts and epididyimis of the male rat. J Androl 14, 23-44.

31. Papp S., Robaire B., Hermo L. 1995. Immu-nocytochemical localization of the Ya,Yc,Yb1 and Yb2 subunits of glutathione S-transfer-ases in the testis and epididymis of adult rats. Microsc Res Tech 30,1- 23.

32. Seiler P., Wenzel I., Wagenfeld A., Yeung C.H., Nieschlag E., Cooper T.G., 1998. The appearance of basal cell in the develop-ing murine epididymis and their temporal expression of macrophage antigens. Int J Androl 20, 217- 26.

33. Yeung C.H., Nashan D., Cooper T.G., Sorg C., Oberpenning F., Schulze H., Nieschlag E., 1994. Basal cells of the human epididy-mis-antigenic and ultrastructural similari-ties to tissue-fixed macrophages. Biol Reprod . 50, 917-926.

34. Hoffer A.P., Hinton B.T., 1984. Morpholog-ical evidence for a blood-epididymis barrier and the effects of gossypol on its integrity. Biol Reprod 30, 991- 1004.

35. Levy S., Robaire B., 1999. Segment-specific changes with age in the expression of junc-tional proteins and the permeability of the blood-epididymis barrier in rats. Biol Reprod 60, 1392- 1401.

36. Seiler P., Cooper T.G., Yeung C.H., Nieschlag E., 1999. Regional variation in macrophage antigen expression by murine epididymal basal cells and their regulation by testicular factors. J Androl 20, 738- 46 .

37. Adamali H.I., Somani I.H., Huang J-Q., Mahuran D., Gravel R.A., Trasler J.M., Hermo L., 1999. Characterization and devel-opment of the regional and cellular specific abnormalities in the epididymis of mice with


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β-hexosaminidase A deficiency. J Androl 20,803 -824.

38. Martan J., Allen J.M., 1964. Morphological and cytochemical properties of the holocrine cells in the epididymis of the mouse. J Histo-chem Cytochem 12, 628- 639.

39. Martan J., Risley P.L., Hurban Z., 1964. Holocrine cells of the human epididymis. Fertil Steril 15, 180-187.

40. Abe J., Takano H., 1989 . Early degeneration of the epithelial cells in the initial segment of the epididymal duct in mice after efferent duct cutting. Arch Histol Cytol 52, 299-310.

41. Goyal H.O., Williams C.S., 1991. Regional differences in the morphology of the goat epi-didymis: a light microscopic and ultrastruc-tural study. Am J Anat. 190, 349- 69.

42. Serre P., Robaire B., 1998. Segment-specific morphological changes in aging brown Nor-way rat epididymis. Biol Reprod 58, 497 -513.

43. Seiler P., Cooper T.G., Nieschlag E., 2000. Sperm numbers and condition affect the number of basal cells and their expression of macrophages in the murine epididymis. Int J Androl 23, 65-76.

44. Serre V., Robaire B., 2002. Interactions of the immune system and the epididymis, in: Robaire B., Hinton B.T., (Eds.) The Epidid-ymis: From Molecules to Clinical Practice. Plenum Press., New York, 219-33.

45. Pollanen P., Cooper T.G., 1994. Immunol-ogy of testicular excurrent ducts. J Reprod Immunol 26, 167-216.

46. Hermo L., Dworkin J., Oko R., 1988. Role of epithelial clear cells of the rat epididymis in the disposal of the contents of cytoplasmic droplets detached from spermatozoa. Am J Anat 183, 107-124.



A Preliminary Investigation on the Antibacterial and Blood Clotting

Properties of Pholcidan Spider WebsJoyce Jose, Aiswarya Babu, Akhila Raju, Alwin Thankachan, and Sruthy M Balan

Research and PG Department of Zoology. St.Thomas College (Autonomous), Thrissur-680001, Kerala, India

Abstract

Folklore on the wound healing properties of spider web, especially those coated by kitchen soot was investigated by examining the antibacterial activity and blood clotting property of two species of com-monly occurring Pholcidan species’ web. It was found that addition of spider web into 2 ml blood sample significantly reduced the clotting time when compared to control. Antibacterial properties were not displayed against either E.coli or Staphylococcus. The efficacy of old spider webs coated with soot as a natural bandage seemed doubtful as the investigators were not able clean debris and dirt from the web.

Key words – pholcidae, spider web, antibacterial effect, blood clotting,

IntroductionSpiders are cosmopolitan in distribu-

tion and occupy a super predatory position in ecosystems. The family Pholcidae (cel-lar spiders, daddy long legs spiders) with about 1000 tropical species has adapted well to human habitats and is commonly found in corners and dark spaces in and around buildings, especially in basements. Characteristic features include a cephalo-thorax typically about as long as it is wide, extremely long and thin legs with flexible

tarsi held in a curved position. Pholcidan movement is by rapidly flexing their legs so that the body gyrates in a circular motion. Web structure varies considerably within the family1. The common pholcids which have been reported from Kerala are Sme-ringopus pallidus and Crossopriza lyoni 2 (Fig. 1 and 2).

Spiders, produce cobweb made out of proteineous spider silk from its spinner-ets3. Spider web has been traditionally used for wound healing because it contains high amounts of vitamin K and is also able to

Fig.1.Smeringopus pallidus Blackwall,1858 Fig.2. Crossopriza lyoni Blackwall,1867

Joyce Jose et al:Antibacterial and Blood Clotting Properties of Pholcidan Spider Webs

53

stimulate the immune system4. In addi-tion, phospholipids hydrate and potassium nitrate present in the spider silk prevent the growth of fungi and bacteria on the silk5. Studies6 have shown that some of bisphos-phonate (Phosphonates) peptides in spider silk have antibacterial activity as reported from spider silk of Pholcus phalangioides and Tegenaria domestica7, 8. Wound healing properties of spider web and improved blood clotting has also been studied9

Spider webs especially, the black webs were believed to have antibacterial and anticoagulant properties from time imme-morial. Sudanese folklore9, traditional European wound medicines3, Greeks’ and Romans’ treatment for wounded soldiers10 mention wound healing and blood clotting properties of spider web. There have been previous studies 5,6,7,8,9,11,12 documenting blood clotting and wound healing using dif-ferent spider web and web preparations

In Kerala too cowebs have been associ-ated with treatment of wounds and stop-ping of bleedings. Webs associated with the cooking range, blackened by the carbon from constant deposition of soot have been especially valued in Malayalam folklore. In this context the objectives of the study were to determine the blood clotting properties of pholcidan spider webs and to investigate the antibacterial activity of pholcidan spi-der webs and thus evaluate a traditional knowledge of wound healing in a scientific manner to prove or disprove it.

Materials and MethodsMaterials

1 ml blood/ experiment, spider web made into thin square sheets of 1 cm², test tubes ,test tube stand ,forceps ,needle, stop watch, grey (non sooty) and black (sooty) web cap-sules, sputum, Mac Conkey plate streaked with sheep blood agar ,inoculation wire loop ,nutrient broth, cotton swab ,forceps, Muel-ler -Hinton agar plate, tape, spirit lamp., etc.

Collection and treatment of webWebs were collected using plastic drink-

ing straws from different regions of houses. The cobwebs collected from the kitchen chimney regions of house had high accumu-lation of soot from the cooking range and were named as black webs. The cobwebs collected from the regions other than the kitchen chimneys of the house were called grey web. The cobwebs were cleaned man-ually by removing dead insects and other large debris. The soot and dust deposited on the cobwebs could not be removed even by pressure washing because the microscopic particles tend to adhere very strongly to the webs. The web was shaped into thin square sheets of 1 cm² dimension. Ten test tubes were arranged in a stand and five were labeled as control. No spider web was added to the control. In the remaining five test tubes, one spider web square of 1 cm2 was added using forceps and needle carefully. One ml of blood was added to each test tube immediately after taking it from the donor. The blood in the control as well as test sam-ples was observed for clot formation every 1 minute. The experiment was repeated three times.

The cobwebs of pholicidan spider were investigated for their antibacterial activity using gram positive strains of Staphylococ-cus and gram negative strains of Escherichia coli bacteria. The sputum sample collected from the infectious patient, was streaked into the Mac Conkey plate containing sheep blood agar using inoculation loop of diam-eter 4 mm in order to get isolation colo-nies .Pure isolated colonies were obtained from the Mac Conkey plate by continuous streaking. The desired colonies were taken from the nutrient broth and streaking was done with the cotton swab. The plate used for this technique was Mueller-Hinton agar plate. Fourteen MH plate were prepared out of which seven were streaked with E. coli and the remaining with Staphylococcus .One plate each of E .coli and Staphylococ-cus were taken as control The black web capsules collected were administered into 3 plates streaked with E. coli and plates and 3 plates streaked with Staphylococcus. Grey


54

webs capsules were similarly administered using forceps. The MH plates administered with black and grey web were piled and tied together with a tape .It was incubated for 24 hours.

Data was consolidated, tabulated and entered in MS Excel The difference between clotting time of blood with and without cob-web gauze treatment was tested using a two tailed paired student’s t test. Significance level fixed at 5%.

Results and DiscussionNo clear zone was formed around the spi-

der web pellets of either grey or black colour. Rather in all the bacterial culture plates the bacteria spread out enormously in the

whole plate. Thus there is no antibacterial property for the web (Fig.3, 4, 5 and 6).

In all the three repetitions it was observed that without spider web, 1 ml of blood took around five minutes to show visible and large clots but the samples with spider web started clotting before the third minute and had completed clotted by the end of it (Fig.7). The calculated t value (for mean time taken for clotting in control and test) was 4 which is greater than the tabular value of 2.14 at 5% significance and degrees of freedom 14. Therefore it was concluded that there is a significant difference in the clotting time of the control (blood without web gauze) and the test (blood with web gauze).

A previous study7 compared the growth of a Gram positive and a Gram negative

Fig.3.E.coli test and control plates-grey web Fig.4.E.coli test and control plates-black web

Fig.6.Staphylococcus test and control plates-black web

Fig.5.Staphylococcus test and control plates-grey web


55

bacterium in the presence and absence of silk produced by the common house spider Tegenaria domestica. They demonstrated that fresh native web silk of Tegenaria domestica can inhibit the growth of the Gram positive bacterium, Bacillus subtilis

but had no significant inhibition of growth against Gram negative bacterium, Esche-richia coli. They stated that antimicrobial effect against B. subtilis appeared to be short lived thus the active agent poten-tially acts in a bacteriostatic rather than

Fig.7. Clotting experiment with spider webs


56

bactericidal manner. Our study showed similar results as the cobwebs which were more than 24 hours old showed no effect on bacterial growth at all

Roozbahani8 demonstrated the antimi-crobial properties of spider silk Pholcus phalangioides produced in sterile condi-tions against two bacteria, Listeria mono-cytogenes and Esherichia coli using well diffusion method and Macro Broth Dilution method. The results showed that the anti-microbial compounds present in the solu-tion spider silk greater inhibitory effect on gram-positive bacteria L. monocytogenes than Gram-negative bacteria E. coli.

A study from Sudan9 collected and ana-lyzed webs of members of the family Phol-cidae (Order:Aranea) to detect potential substances. High concentrations of glycine and alanine indicated presence of fibroin and sericin. FTIR analyzer (Fourier Trans-form Infra Red) detected pyrrolidine, potas-sium hydrogen phosphate and potassium nitrate. Dressing of laboratory mice (Albino wister) skin wounds with these webs kept the wounds moist, leading to a 7 days faster recovery in comparison with other mice which were dressed with Panderm® cream.

We were unable to compare the effects of spider web on wound healing but we were able to reproduce the blood clotting results mentioned in this study. They stated that9

there was a significant reduction in blood clotting time which are consistent with our findings.

Another significant study12 on spider wound healing developed a spider web based ointment which showed better heal-ing, lower wound epithelization period and heals wounds with less scabs and other abnormalities.

A lot of information regarding spider web wound healing properties is available on the internet but most of them are not trust wor-thy or seem to be contradictory in nature. Only three studies 7, 9, 12 seemed to be credi-ble and repeatable. While the effectiveness of spider web in speeding up blood clotting was proven in many previous studies the

antibacterial effect of spider webs gave con-tradictory answers.

Local folk lore insists on the usage of soot rich cobwebs but our study found no significant difference in the anti microbial activities of soot rich or normal cobwebs. In addition our inability to clean the cobwebs satisfactorily raised a doubt of the cobweb gauze itself being a carrier of infection.

We conclude that the spider (pholcidan) web gauze was effective to speed blood clot-ting time but did not have any significant antimicrobial activities. Local and global folklore insists on the wound healing prop-erties of spider web but we feel that further studies are necessary both in insitu and exsitu medium to get a consensus. The onus should be not only on developing new prod-ucts but also on examining how spider web as such may improve or worsen a wound con-dition. This would be of use in emergency situations where conventional bandages to staunch bleeding may not be available. Our study underlines the fact that while there is some truth in the wound healing folklores, spider web cannot be used at present as an effective bandage and techniques to clean and process it must be developed.

AcknowledgmentWe are grateful to the owner and staff of

Mother Lab, Thrissur who helped us with col-lecting voluntary blood samples. We are grate-ful to the staff at PolyClinic Thrissur who facil-itated antibacterial studies by permitting us to use their resources and premises. We thank the Principal of the college Dr. Jenson P.O. and Dr. Francy K Kakkassery Head of the Department of Zoology for moral support. Except for the first author who has given maximum contribution to this article the order of authorship is purely alphabetical and does not reflect the quantum of individual contributions which was same.

References1. Bradley R A. 2013. Common Spiders of

North America. UC Press. 2. URL: http://www.southindianspiders.org/.

Accessed on 21/03/2017


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3. URL:https://en.wikipedia.org/wiki/Spider_web. Accessed on 21/03/2017

4. *Heimer,S.1988.WunderbareWeltderSpinnin.URANIA.

5. Chakraborty, D.S.2009. Antibacterial activities of cobweb protein. European Congress of Clinical Microbiology and Infectious Diseases.

6. *Gellynck, K., Kiekens, P., Mertens, J. 2006. Coconzijde en spin rag in weefs elengineer-ing Silk and spider silk in tissue engineer-ing. Academiejaar.

7. Wright, S, Goodacre, S L. 2012. Evidence for anti-microbial activity associated with common house spider silk. BMC Research Notes, 5:326. http://www.biomedcentral.com/1756-0500/5/326

8. Roozbahani, H , Mahdi A, Naser G, Khosro I. 2014. Evaluation of antimicrobial activity of spider silk Pholcus phalangioides against two bacterial pathogens in food borne. Int. J. Adv. Biol. Biom. Res, 2014; 2 (7), 2197-2199

9. Manal, E. M. Siyamand Hind, M. Abushama (undated) Study on the useful effects of spi-der webs (family Pholcidae) in epidermal recovery. Department of Zoology , Faculty of Science, University of Khartoum, Sudan

10. URL:http://www.healthydietbase.com/usingspiderwebstohealwounds/. Accessed on 21/03/2017

11. Gosline,J.M.;Guerette,P.A. Ortlepp,C.S. and Savage,K.N.1999.The mechanical Design of spider silks: from fibroin sequence to mechanical function. The Journal of Experi-mental Biology.

12. Preeti Kumari, Chahar MK, Veerapur VP, Spandana G, Thippeswamy B S and Badami S. 2012. Spider web ointment: A traditional based apprach in cutaneous wound healing. Indian Journal of Traditional Knowledge. Vol 12 (4) 2013 65* Originals not seen



Investigations on SnS & ZnO thin films for photovoltaic applications

Melda Francis1, Lakshmi M2, Rosmy Joseph2

1Department of Physics, Christ College, Irinjalakkuda-680125, Kerala,India.2Department of Physics, Mercy College, Palakkad - 678 006, Kerala, India

AbstractIn this work, SnS and ZnO thin films were prepared by chemical bath deposition and sputtering tech-niques respectively. The morphological studies of both the films were done with scanning electron microscopy. The absorption spectrum of the films was recorded using UV-visible spectrophotometer. From the Tauc plot, the optical band gap of the films was calculated. Annealing increased the crystal-linity and decreased the band gap of both the materials. The structural characterization was done by X-ray diffractometer. On comparison with the standard JCPDS data, the crystal structure of SnS was found to be orthorhombic and that of ZnO was found to be hexagonal wurtzite structure. Optimisation of various deposition parameters was carried out for the successful deposition of SnS and ZnO. A pho-tovoltaic junction of the configuration ITO\SnS\ZnO\Ag was also tried. The formation of photovoltaic junction was confirmed from I-V characteristics measured using Keithley 2401 source measuring unit. The efficiency of these cells was not appreciable and it may be attributed to high resistance of ZnO layer and defects in SnS layer.

Keywords: SnS, ZnO, CBD, SnS/ZnO heterojunction

IntroductionTin monosulphide (SnS) is a layered

semiconductor whose energy band gap is in the range of visible light of the solar spectrum. Due to low cost, low toxicity and earth abundant nature, it is a suitable can-didate for future multifunctional devices, particularly in light conversion applica-tions. This material finds applications in a number of solid state devices such as photo-voltaic cell1–5, photoconductive cells6, inter-calation battery systems7 etc. Although the reported efficiencies of SnS based solar cells are low, the cost-per-watt of the fabri-cated cells is found to be competitive. SnS films usually exhibit a p-type conductivity and its direct and indirect band gap ranges from 1.1eV -2.35eV 8 and 0.9eV -1.3eV9 respectively. There are mainly three types of crystal structures for SnS: orthorhombic, zinc blende and NaCl structure. SnS films prepared by methods involving vacuum

evaporation, spray pyrolysis, chemical bath deposition and electrochemical deposi-tion usually have a layered orthorhombic structure.

ZnO has attracted much attention within the scientific community as a “future material”. Zinc oxide (ZnO; II-VI compound semiconductor) is one of the best candidate materials among attractive metal oxides for optoelectronic devices due to its wide direct band-gap, high exciton binding energy and good efficiency which will result in high gain. The band gap of ZnO is 3.44 eV at low temperature and 3.37 eV at room tempera-ture10. This enhances applications in opto-electronics in the blue/UV region, includ-ing the light emitting diodes, laser diodes and photo detectors. The other applications of ZnO are in solar cells, gas sensors, etc. There are several deposition techniques for ZnO thin films which include chemical vapour deposition, spray pyrolysis, pulsed laser deposition, sputtering etc.

Melda Francis et al: SnS and ZnO thin films.

59

In the present work, Chemical Bath Deposition technique was chosen for the synthesis of SnS and RF magnetron sput-tering for ZnO. The prepared thin films were characterized structurally, morpholog-ically and optically using XRD, SEM, UV - Vis Spectrophotometer, etc. The formation of the SnS/ZnO heterojunction was fabri-cated and I-V characterisation was carried out using Keithley 2401 source measuring unit.

Materials and MethodsSynthesis of SnS Thin Films - Chemical Bath Deposition

30 ml of 0.15M SnCl2.2H2O is taken in a 100 ml beaker and 30ml of 0.7M NH4F is added to it and stirred well. 5ml of 2M Na2S2O3.5H2O and 6 ml of 0.25% NH4OH is added sequentially to this solution. NH4F acts as the buffer solution and NH4OH reg-ulates pH. The deposition time is about 18 hour and it resulted in dark brown SnS thin films.

The reaction of SnS formation on the glass substrates is as follows:

Trials were made for optimizing vari-ous parameters like pH, deposition tem-perature, etc. for the successful deposition of thin films. The pH was optimized to be

5.6 and deposition temperature at 25oC. The effect of pre-cooling was also studied. Pre-cooling all the precursor solutions for 25oC prior to deposition was needed for the slow and steady initialisation of nucleation. Preparation of ZnO Thin Films by RF Magnetron Sputtering

The target used is Zinc oxide (99.999% pure, 2.00” diameter × 0.125” thick). The

RF power for the target was optimized to be at 80W and the working pressure during the deposition process was 5.5×10-3 mbar. The coating time was about 20 minutes. The distance between target and substrate holder was set at 8cm. The ZnO films thus obtained were almost transparent but had very high resistance (in MΩ range) which was undesirable.

Fabrication of the SnS/ZnO heterojunction

SnS thin films were chemically deposited on an ITO/ ZnO layer. Silver paste was used as the top electrode. The fabricated hetero-junction has ITO/SnS/ZnO/Ag configuration and the structure is schematically shown in figure 1.

Results and DiscussionsThe prepared thin films were character-

ised structurally, morphologically, optically using XRD, SEM and UV - Vis Spectros-copy respectively. The X - Ray Diffractom-eter used for the structural analysis was Bruker Kappa Apex II model and X - Ray source was Cu (Wavelength 1.5406Ao). The SEM imaging was carried out using JEOL JSM-6390LV and absorption studies using INTEK UV Professional spectrophotome-ter in the wave length range 190-1100 nm. The I-V characteristic was plotted using Keithley 2401 source measuring unit.

SnCl2+2NH4OH [SN(NH3)2Cl2+2H2O,

S2O32-+H+ S+HSO3

-,

2S2O32- S4O6

2-+2e-,

S+2e- S2-

Sn(NH3)2Cl2+S2- SnS+2NH3+2Cl-

Glass

ITO

p-SnS

n-Zno

Ag electrode

Fig 1: Fabricate cell structure


60

Structural Analysis - X-Ray Diffraction Studies

SnS Thin Films The XRD spectrum of the as prepared

sample, shown in fig 2.1 exhibits prominent peaks corresponding to 2θ values 27.40o and 30.45o. These peaks are found to be the characteristic peaks of orthorhombic SnS of the standard JCPDS datasheet. The SnS films prepared were annealed at 100OC for 30 minutes. The XRD spectrum of the annealed sample is given in fig 2.2. The

spectrum showed well resolved peaks which can be correlated with the standard JCPDS data card. The crystal structure is found to remain orthorhombic even after annealing.

The crystallite size of the SnS film was evaluated using Scherrer’s formula.

where K is shape factor (0.94), λ is the wavelength of X-ray (0.1541nm), β the FWHM and θ is the Bragg’s angle. The crys-tallite size of the as prepared film is around 7nm and that of annealed film is around 21nm. The increase in the crystallite size may be attributed to the effect of annealing.

ZnO Thin FilmsThe as grown ZnO thin films showed

amorphous nature which is clear as shown

in figure 2.3. However, crystallinity was found to increase on annealing. The XRD spectrum of the annealed sample is shown in figure 2.4. The peaks were compared with the standard JCPDS data card. The prom-inent peak at 2θ = 34.35o, corresponding to 002 plane, matched well with the standard value and the structure is confirmed to be

Fig 2.1: XRD spectrum of the as prepared SnS thin film.

Fig 2.2: XRD spectrum of the annealed SnS thin film.

Fig 2.3: XRD spectrum of the as prepared ZnO thin film.

Fig 2.4: XRD spectrum of the annealed ZnO thin film.


61

hexagonal wurtzite. The grain size was cal-culated using Scherrer’s formula and it was found to be around 8nm.

3.2 Morphological Studies - SEM ImagingSnS Thin Films

The SEM image of the as prepared sam-ples was not so uniform and was discontin-uous. The morphology was of granular form and the grain size was found to be in nano-meter regime. On annealing at 100oC for 30 minutes, the film becomes more uniform, dense and continuous. The granular shape is lost which may be due to the diffusion of sulphur on annealing. The SEM images of the as prepared and annealed samples are given in fig. 3.1 and fig.3.2 respectively.

ZnO Thin Films The SEM images of as grown and

annealed ZnO thin films are shown in fig-ures 3.3 and 3.4 respectively. The images

didn’t show any particular shapes, struc-tures or clusters. But, a sheet like formation is observed in both the samples.

Optical Studies - UV - Vis Spectroscopy

The absorption spectra of the as prepared and annealed SnS thin films were taken using UV - Vis Spectrophotometer. The band gap can be easily found out from the Tauc plot i.e. the graph is drawn between

and the photon energy for as- prepared and annealed thin films. Here the band gap is direct so that m can be taken as 2. The energy gap ( ) value is calculated by extrapolating the straight portion of the plot.

In general, the band gap of the thin films basically depends on different factors like crystallinity, composition, grain size, strain, lattice parameters etc. The value of

Fig 3.1: SEM image of the as prepared SnS thin film. Fig 3.2: SEM image of the annealed SnS thin film.

Fig 3.3: SEM image of the as prepared ZnO thin film. Fig 3.4: SEM image of the annealed ZnO thin film.


62

Fig 4.1: Tauc plot of the as prepared SnS thin film Fig 4.2: Tauc plot of the anealed SnS thin film.

Fig 4.3: Tauc plot of the as prepared ZnO thin film. Fig 4.4: Tauc plot of the annealed ZnO thin film.

Fig 5.1: I-V Characteristics

SnS as prepared(a

hv)2

hv


63

band gap is found to decrease from 2.01eV to 1.66eV as result of annealing. Here, this decrease can be attributed to the increase in crystallite size and thereby increase in cell volume as an effect of annealing. The Tauc plots of the as prepared and annealed samples are given in figure 4.1 and 4.2 respectively.

The Tauc plots of the as prepared and annealed samples of ZnO thin films are shown in fig 4.3 and 4.4 respectively. The band gap of the ZnO films slightly decreased from 3.27 eV to 3.22 eV on annealing. Here also the decrease of the band gap on anneal-ing can be associated with the increase in the crystallinity.

V - I CharacteristicsThe V - I characteristics of the fabricated

heterojunction is shown in figure 5.1. The relevant cell parameters were determined. The efficiency of the fabricated solar cell is low which may be due to the inherent defects in the chemically deposited SnS layer and the high resistivity of ZnO layer. The lattice mismatch between the p region and n region of the sample cell may also contribute to the poor efficiency of cell. A change in conductiv-ity of ZnO can be achieved with appropriate metal dopants, such as Al, Ga, In etc. Hence controlled doping of the window layer may help to improve the efficiency of the hetero-junction. Further, work in this area should lead to a better understanding of this pho-tovoltaic junction and thereby improve the cell output. The fill factor obtained is about 0.38.

Conclusion In the present work, fabrication of a het-

erojunction was tried with ITO/SnS/ZnO/Ag configuration. The methods adopted for the preparation of SnS and ZnO films were CBD and RF sputtering respectively. The struc-tural and morphological characterization was carried out using XRD and SEM. The absorption and band gap studies were done by UV - Vis Spectroscopy. The efficiency of the fabricated cell was low and needs to be improved by suitable doping methods.

AcknowledgementWe acknowledge STIC,CUSAT Cochin

for providing open facilities like XRD and SEM for the completion of this work. We also acknowledge DST-FIST for the finan-cial aid for setting up our research lab.

References 1. Albers W., Haas C., Vink H.J., Wasscher

J.D., 1961. Investigations on SnS. Journal of Applied Physics 32(10), 2220–2225.

2. Nikolic P.M., Todorovic D.M., 1987. Pho-to-acoustic and thermo-acoustic properties of single-crystal SnS compared with its near-in-frared optical and transport measurements. Journal of Physics C: Solid State Physics 20(1), 39–44.

3. Ghosh B., Roy R., Chowdhury S., Banerjee P., Das S., 2010. Synthesis of SnS thin films via galvanostatic electrodeposition and fabri-cation of CdS/SnS heterostructure for photo-voltaic applications. Applied Surface Science 256(13), 4328–4333.

4. Sharon M., Basavawaran K., 1987. Photo-electrochemical studies on p-SnSe electrodes. Solar Cells 20(4), 323–332.

5. Blaesser G., Rossi E., 1988. Extrapolation of outdoor measurements of PV array I–V char-acteristics to standard test conditions. Solar Cells 25(2), 91–96.

6. Kawano K., Nakata R., Sumita M., 1989. Effects of substrate temperature on absorp-tion edge and photocurrent in evaporated amorphous SnS2 films, Journal of Physics D: Applied Physics 22(1), 136–140.

7. Whittingham M.S., Jacobsen A.J., 1982. Intercalation Chemistry, Academic Press, New York.

8. F.Gode, E.Guneri, O.Baglayan, 2014. Effect of tri-sodium citrate concentration on struc-tural,optical and electrical properties of chemically deposited tin sulphide films. Applied Surface science (2014).

9. E Turan, M Kul, A S Aybek and M Zor. 2009. Structural and optical properties of SnS semiconductor films produced by chem-ical bath deposition, J.Phys, D:Appl.Phys, 4, 2454, 08.

10. Mang, K. Reimann and St. Rubenacke, Solid State Commun. 1995. 94, 251.

* Corresponding author, E-mail: [email protected]


Environmental Variability, Human Interventions and Ichthyodiversity of a

fresh water stream of Kerala, India.Radhika . R*

P G and Research Department of Zoology, N.S.S Hindu College, Changanassery - 686102, Kerala, India.

AbstractKerala is blessed with many rivers and fresh water streams. But most of them are severely under threat by human interference and anthropogenic interventions. The water bodies are harnessed by developmental activities such as reclamation, solid and liquid waste disposal after domestic and indus-trial purposes. During the last decades, scientific evidences has presented that the fresh water streams and low lying areas are experiencing the adverse consequences of hazards related to anthropogenic stress and climate change was analysed. The present work focused on primary production, seasonal and annual fluctuations, characteristics, relationship of environmental factors with fishery potential and annual variation of ichthyofaunal diversity from a lotic ecosystem in Vakathanam village, Kotta-yam district, Kerala. The data gathered from three different spots and the samples were collected by two days in every week for two consecutive years.

Keywords: ichthyofauna, diversity, fish potential, lotic, ecosystem

IntroductionRivers and streams in Kerala form the

backbone of capture fisheries and producing livelihood to fishermen. However, day by day increasing anthropogenic pressures on the rivers have adversely affected the fish pro-duction potentialities and them no longer to support rich biota. Loss of Marine bio diver-sity are largely the result of the conflicting usage of the coastal habitat and hence lead to degradation. The best way to conserve biodiversity is to protect habitat diversity1. The studies on prevailing quality of envi-ronment form the basis for understanding impacts of a particular anthropogenic stress 2. Studies on the various aspects of the phys-ico chemical parameters of this water body is essential for assessing the water quality and various biogeochemical processes and also improving future guidelines on the estuarine management 3 . The environmen-tal threats coupled with overfishing and irrational exploitation of natural resources have led to the degradation and deteriora-tion habitat decline and disappearance of many valuable fish species. Fresh water

biodiversity is associated with physical, temporal, chemical and biological charac-teristics of their immediate environment and the changes in these factors affect bio-logical life. Temperature, Light, Dissolved oxygen, CO2, Dissolved nutrients, pH etc., are the main factors affecting the produc-tivity of fresh water ecosystems. The study of fish fauna is useful to assess the relative fertility and health of water body. The stud-ies on prevailing quality of environment form the basis for understanding impacts of anthropogenic stress on ecosystem.

MethodologyStudy area

The present investigation was carried out by selecting three sites (S1, S2, S3) which are 2 km. apart of a stream which is situated in Kottayam-Puthuppally-Then-gana road in Vakathanam and belongs to Vakathanam grama panchayath. This stream passes through the places such as Pandanchira(Fig.1), Pathayapallikadavu (Fig.2), Vettikalungu (Fig.3)and ends to Kodoorar in Puthuppally (Table 1).

Radhika :Ichthyodiversity of a fresh water stream of Kerala

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Fig.1 Pandanchira Fig.2 Pathayapallikadavu Fig.3 Vettikalungu Fig.4 Kodoorar Fig.5 Vallisneria Fig.6 Polluted water into the stream

1 2

43

5 6


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Table 1. Details of sampling sites

Site No.

Site of the sample

Characteristics of site

S1 Malikakadavu Water is crystal clear, deeper than S2

S2 PandanchiraWater is clear, shallow, and rich with phytoplankton, water plants.

S3 Vettikalungu

Turbid water which is polluted, domestic wastes and garbage dumping in this region.

Sampling and Analytical Methodology

The data gathered from three different spots and the samples were collected by two days in every week during two successive years from March to June. The sampling was carried out from sample sites of the stream at a distance of 2 km between sampling sites. Fauna and Flora were assessed. Hand held scoop nets, Hooks and line, Cast Net were used for collecting fishes Tempo-rary bunds were constructed to catch the trapped fish and others were collected by the net. The collected specimens were dipped in 10% forma-lin after collection from water in a container that allows proper spreading of their fins. Preparation of 10% dilution was adopted during the sampling time. By the end of each sampling the specimens were examined and classified into families. Each container was labeled properly against the physi-cal data sheet of the sampling station and brought to the further taxonomic exercise and counting of fishes obtained each 3 sites. Morpho-taxonomic counts and identification have been done 4. Water samples were kept in polythene bottles of one litre capacity. Samples were brought to the wet labora-tory at Post Graduate and Research Department of Zoology, N.S.S.Hindu College, Changanacherry. The samples were kept cool in darkness until the analysis was finished. The samples were analysed for temperature, pH, CO2, Nutrients, Primary Pro-ductivity, Transparency, flora and fauna 5,6 .

Statistical AnalysisStandard deviation and mean were also

calculated using Microsoft Excel, 2007.

Results Water samples were collected from

the selected sites of the water body under study and were analysed for various phys-ico-chemical parameters. General hydro-graphic variables, nutrients such as nitrate, phosphate were determined in the water samples taken from the study area. Aver-age concentration of various hydrographic parameters and their variations were also determined.

Primary Production The floristic composition and vegeta-

tion of this freshwater ecosystem are typ-ically low. Few numbers of Casia fistula (kanikonna), Mangifera indica (mango), Spondia spinata (ambazham), Macaranga indica(Vatta), Acasia, Albizea lebeck (vaka), Artocarpus hirsutus (angili) etc are situated on the banks of this stream which helps to enhance organic matter. Water plants like Pistia, Hydrilla, Vallisnaria, Nymphea (Fig.5) etc were found in the study are were sparse. Phytoplankton such as Spirogyra and some algae were also found. (Fig.4 ,5). Primary productivity in non rainy season is minimum (0.025±0.48) at site 2 and max-imum at site 1(0.032±0.38). While during rainy season PP showed peak (0.025±0.005). In second year of study, in non rainy season, Primary productivity is minimum at site 2 (0.02±0.01) and maximum at site 1and 3(0.03±0.01). During rainy season, mini-mum at site 3(0.02±0.01) and maximum at site 1(0.03±0.02).

Environmental FactorsThe variations of general hydrographic

parameters estimated in the water column are furnished below.

TemperatureDuring the first year of investigation,

Temperature was minimum during rainy season at the site2 (25.5±0.5)and maximum temperature was at site 1(27±0.81). During second year, Temperature was minimum during rainy season at the site2(26±0.81)and maximum temperature was at site 3(27±26.25).


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pH pH was slightly acidic or slightly alka-

line during both seasons and showed fluc-tuations from 6.32 to 7.05±0.5 in first year. While during second year, pH was slightly acidic in both seasons (6.65 to 6.68±0.31).

Nitrate Among nutrients nitrate showed a varia-

tion from (0.3±0.08)at site 1 and( 0.52±0.05) at the site 3, during non rainy season. But in non rainy season it varied from (0.225±0.09)at site 1 to (0.4±0.12)at site 3. In this study, nitrate showed a variation from (0.225±0.09) at site 1 to( 0.52±0.05) at the site 3,during non rainy season. But in non rainy season it varied from (0.3±0.08)at site 1 to (0.4±0.18)at site 3.

Phosphate Phosphate was minimum (8.4±0.84) at

site 1 but maximum at site 1(9.07±0.4).Which in rainy season minimum (7.9±1.15)at site 3 and maximum is (8.1±1.28)at site 1.

Total Organic Carbon (TOC)

TOC was minimum (0.47±0.09) at site 1 but maximum at site 2(0.51±0.13) during non rainy season. In rainy season it is min-imum (0.46±0.03) at site 2 but maximum( 0.5±0.11) at the site 1.In 2014, TOC was minimum (0.2±0.08) at site 2 but maximum at site 1(0.27±0.17 during non rainy season. In rainy season it is maximum (0.2±0.08) at site 1 and 2 but minimum( 0.2±0.11) at the site 3.

Dissolved Oxygen In 2013, dissolved oxygen in non rainy

season was minimum (6.25±1.5) at site 3 and maximum (7.62±1.6)at site 1.But in rainy season it varied the minimum DO is (5.5±0.57) at site 3 and maximum (7.6±1.6). During 2014, Dissolved oxygen in non rainy season was minimum (7.25±0.95) at site 3 and maximum (7.62±1.6)at site 1. But in rainy season there is no variation of DO in all sites (7.62±1.6). Dissolved oxygen was found to be higher during rainy season due to water turbulence.

CO2 In 2013, Carbon dioxide in non rainy sea-

son the minimum (8.2±0.374) at site 2 and maximum (8.42±0.45) at site1.It is varied in rainy season minimum (7.5±0.42) at site 1 and maximum (8.2±0.37) at site 2. In 2014, Carbon dioxide in non rainy season CO2 was (6.67±0.53) at site 1,2 and 3. In rainy season (7.12±0.85).

TransparencyIn first year of study, in non rainy sea-

son transparency was minimum (1.07±0.25)and maximum (1.62±0.47) at site2,but in rainy season it is minimum(0.78)and max-imum(1.2). In the consecutive year, trans-parency was in non rainy season minimum (0.09±0.12 at site2 and maximum at site 3. But in rainy season it is minimum (0.07±0.1) at site1 and maximum (1.01±0.22) at site 2.

IchthyofaunaThe occurrence of fishes was showed in

the tables and graphs. The following fish species were identified and quantify from the study area. They are Anabas testu-dineus, Horabagrus brachysoma, Channa orientalis, Heteropneustes fossilis, Puntius sarana, Danio malabaricus, Ompok mal-abaricus, mystis gulio, Puntius fasciatus, Rasbora daniconius, etc., In this study the Anabas is obtained large quantity in two years at the site 1. But in pre monsoon sea-son a sharp decline is seen. The ichthyofau-nal composition is depicted in Fig. 7-17 and Table 2. There is a sharp decline in the fish during second year.

DiscussionBeing in the tropical climatic region

the diurnal and seasonal fluctuations in the atmospheric temperature significantly affect the water temperature. Fluctuations in the water temperature experienced in the present study may be due to the timing of collection and influence of season. The physiographical and temporal variations of the rivers are reflected in water tempera-ture. The present studies are in good agree-ment with the earlier studies. The import-ant nutrients which affect the growth of phytoplankton are nitrates, phosphates


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Fig. 7 Anabas testudineus Fig. 8 Heteropneustes fossilis Fig. 9 Danio malabaricus Fig. 10 Puntius sarana( kuruva paral) Fig. 11 Clarius batrachus Fig. 12 Channa orientalis (vatton) Fig.13 Horabagus brachysoma(manjakoori) Fig.14 Ompok malabaricus Fig. 15 Mystus gulio (vellakkoori) Fig.16 Rasbora daniconius

7 8 9

13121110

14 15

16


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etc. Nitrate is an important factor for con-trolling the occurrence and abundance of phytoplankton7. The important source is the biological oxidation of organic nitroge-nous substances or produce indigenously in the water. Higher concentration during rainy season suggests that the rain fall was supposed to be responsible for increasing nitrates in water 8 . Phosphate is one of the major nutrients responsible for biological productivity. A rise in phosphate content is attributed to allochthonous input like gar-bage and agricultural runoff. In the pres-ent study there is no annual fluctuation. Organic production is favoured by several

factors such as vegetation, texture of sediment and degree of oxida-tion. Besides this rainwater runoff to the stream play a vital role in the accumulation in the organic mat-ter. Anthropogenic activities also contribute to the organic matter deposition.

The occurrence and abundance of fish species is related to the water quality, which is the physico-chem-ical and biological properties and water level in the season. During rainy season in 2013 Anabas is the more obtained species. But Rasbora daniconius were obtained in least amount. The rainy season the more collected fish species is the Anabas and the least amount is Rasbora daniconius. During non rainy sea-son in the second year of study Ana-bas obtained more. Anabas can be sustained in any condition. During this year, polluted water enter into the stream (Fig. 6 ) and resulted in the sharp decline of fishes during this year. The quantity of Horaba-grus and Heteropneustes was close to the quantity of Anabas. The small fishes like Rasbora, Puntius etc., were very least amount, as they cannot survive the polluted water. The decline in fishery in the second year of study is due to anthropogenic stress. Pollution from decay of garbage, house hold wastes, small industrial wastes etc causes serious damage to the ecosys-

tem and especially ichthyofauna. Decrease in the plankton affects the productivity of the water body. It will affect the health and wealth of the streams.

ConclusionPhysico-chemical variables such as dis-

solved oxygen, nutrients and major elements either individually or collectively influence the abundance and distribution of phyto-plankton production which in turn affect the secondary and tertiary production. Knowl-edge on the various aspects of the parameters of the water column is essential for assessing

Table 2. Occurrence of Fish during Monsoon and Pre mon-soon seasons for two consecutive years

SpeciesPre monsoon

season (fish in Kg.)

Monsoon season

(fish in Kg.)2013 2014 2013 2014

Anabas testudineus 74.5 45.25 298 71Heteropneustes fossilis 72.75 45.5 291 67Mystus gulio 49.75 16.5 199 21.4Horabagrus brachysoma 64 36.25 256 56.5Mystus vittatus 23.5 14.5 94 20.15Ompok malabaricus 12.5 2.5 50 4.41Puntius sarana 26.25 12.75 105 7.75Rasbora daniconius 8.25 2.5 33 3.12Channa orientalis 16.5 77.75 66 3.56Puntius fasciatus 14.5 2.75 58 3.75Total 362.5 256.25 1450 258.64

Fig. 17 showing the occurrence of fish during two consecutive years


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the water quality and various biogeochemi-cal processes controlling the distribution and abundance of various species of organisms. Scientific evidences has been presented during the last decades that fresh water streams and low lying areas are experienc-ing the adverse consequences of the hazards related to climate change and anthropo-genic stress. So the all relevant ecosystems are suffering degradation affecting seriously their sustainability and the services they provide. The present study helps to clarify and quantify the changes and impacts in fresh water bodies and the decline of ich-thyofauna recently. During the second year of study there was influx of polluted water from houses and dumping of garbage lead to the deterioration of water quality and decline in fish landings. Physicochemical changes likely to affect the biological com-ponents of all ecosystems will have different effect at species, population and ecosystem levels.

Pollution leads to vulnerability of small pelagic fish population. Oxygen depletion inevitability leads to biological impacts ranging from altered microbial activity like enhanced denitrification to whole commu-nity displacement like loss of fisheries, inva-sion of displaced species into a new habitat which are poorly understood. As well oxygen losses in the water bodies are accompanied by increased acidity as CO2 level rise, these two trends may have synergistic impacts on biota.

Streams and rivers play a significant role as they serve the purpose of various activities. But natural resources were bru-tally exploited and destroyed in the name of development.The loss in agriculture and gain in industrialization and population explosion have impaired natural resources. The present investigation showed annual differences in environmental factors which in turn affects the fishery potential of the study area. The aquatic communities and more specifically fish communities are very good indicators of disturbances, and since these communities play an important role in the aquatic food chain, their study can effec-tively depict the comprehensive account of

aquatic ecosystem structure and function. For ecosystem restoration, it is very essen-tial to collect information on the structure and functions of these water bodies.

Acknowledgments I express my gratitude to the authorities

of N.S.S.Hindu College, Changanacherry, Kottayam, Kerala for their valuable sup-port by providing facilities for the comple-tion of work.

References 1. Tiwari L.R., Vijayalakshmi R. Nair., 1998.

Ecology of the phytoplankton from Dhar-matar creek, West coast of India. Ind J Marine Sci. 27, 302-309.

2. Kulkarni V.A. , Velamala S.Naidu., Tanaji G. Jagtap., 2011. Marine ecological habitat: A case study on projected thermal power plant around Dharamtar creek. Ind J. Environ. Biol.32, 213-219.

3. Beenamma Jacob., 1993. Studies on the Sul-phur Chemistry of a Tropical Estuarine sys-tem. Ph. D Thesis.

4. Munro., 1982. Estimation of biological and fishery parameters in coral reef fisheries. in: Pauli D., and Murphy G.I., (Eds.)Theory and Management of tropical fisheries. ICLARM Conf. Proc., 9, 71-81.

5. Anderson L. G., Turner D.R., Wedborg M., Dyrssen D., 1999. Determination of alka-linity, in: Methods of Sea Water Analyses. Grasshoff K., Ehrhardt M.,and Kremling K., (Eds.), Verlag Chemie, Weinheim, 127-147.

6. APHA., 2005. Standard Methods for the Examination of water and waste water. In : 21st edition. Andrew. D. Eaton., Lenore S. Clesceri., Eugine W. Rice., Arnold . E. Green-berg., (Eds) Centennial edition.

7. Rice D.L., 1982. The detritus nitrogen prob-lem: New observations and perspectives from organic geochemistry. Mar. Ecol. Prog. Series. 9, 153-162.

8. Anna E.O. , Adedipe, N.O., 1996. Water qual-ity monitoring and environmental status in Nigeria. FEPA Monograph 6, FEPA, Abuja, Nigeria, 239.



Variation of chlorophyll content in five common plant species to

environmental stress due to proximity of steel industriesRakshitha R* and Jelly Louis

Department of Botany, Mercy College, Palakkad - 678 006, Kerala, India

AbstractThe present investigation have been done, to compare the effect of industrial pollution on the chlorophyll content of leaves of five different species collected from Kanjikode, Palakkad district and compared with Vallady (non-polluted site) 10 kms away from polluted site. Determination of chlorophyll a, chlorophyll b and total chlorophylls content were done by using UV-Visible spectrophotometric analysis. A high reduction in the concentration of chlorophyll content was recorded in the leaf samples of plant species collected from polluted sites when compared with non-polluted site.

Keywords: Leaves, chlorophyll, concentration, industrialization, pollutants, chl a, chl b, total chloro-phyll content.

IntroductionIndustrial pollution is one of the burning

serious environmental problems in Kerala. As the society develops it lead to more air pollution which is a global problem. The increase in the number of industries and automobile vehicles are continuously add-ing toxic gases and other substances to the environment. The photosynthetic activity and plant pigment formation are suffered due to traffic and industrial pollution. Dust emitted from different industries affect pho-tosynthesis, respiration, transpiration and allows the penetration of phytotoxic gaseous pollutants into plants. It causes their phys-iological and metabolic activities.

Plants play main role in maintaining and determining the ecological balance by tak-ing part in cycling of nutrients and gases like carbon dioxide and oxygen etc.¹, ², 3. Pho-tosynthesis process is affected by air pol-lution and gets inactivated because pollut-ants are accumulated and absorbed on the surface of leaf. It also affects concentration

of photosynthetic pigments in leaf because leaves are more exposed to these pollut-ants. Gaseous pollutants enter the plants through the stomata in leaves. It affects sto-matal response and damages them.

Chlorophyll is the principal photoreceptor in photosynthesis, the light-driven process in which carbon dioxide is “fixed” to yield carbohydrates and oxygen. Chlorophyll is found in high concentrations in chloroplasts of plant cells. Chlorophyll molecules are specifically arranged in and around photo-system that are embedded in the thylakoid membrane of chloroplast. Chlorophyll is an antioxidant compound which is present and stored in the chloroplast of green leaf plants and mainly it is present in the green area of leaves, stems, flowers and roots4,5.

Two types of chlorophyll exist in the pho-tosystem of green plants: chlorophyll a and b. The difference between these two chloro-phylls is a methyl moiety in chlorophyll a replaced by a formyl group in chlorophyll b. The ratio of chlorophyll a to chlorophyll b in


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higher plants is approximately 3:1.Chloro-phyll ‘a’ is being more severely affected than chlorophyll ‘b’. Chlorophyll ‘a’ is degraded to phaeophytin through replacement of Mg⁺² ions in chlorophyll molecules, while chloro-phyll ‘b’ forms chlorophyllide ‘b’ through the removal of phytol group of the molecule⁶.Out of various photosynthetic pigments, chlorophyll a appears to be more sensitive

than chlorophyll-b and carotenoids which is also been observed in other plants⁷ ⁸ ⁹.

Chlorophyll is the index of productivity¹⁰ and synthesis or degradation of chlorophyll play a leading role in the tolerance of the plants to air pollution¹¹. Hence higher the chlorophyll contents greater is the toler-ance of plants to pollution. Photosynthetic pigment can also be affected at even low concentration of mixture of SO₂ and NO₂¹². Decrease in chlorophyll contents have been reported by Pandey and Pandey and Kumari¹ᶟ ¹⁴. Higher content of chlorophyll is present in various plant species¹⁵.

The estimation of chlorophyll content in plant species, helps to identify the impact of air pollution and tolerance level of the plants growing in that area. The present study was undertaken to assess the compar-ison of chlorophyll content in leaves which is collected from polluted and non- polluted sites of Kanjikode area, Palakkad District, Kerala.

Fig. 2.Satellite view of study area – Kanjikode

Fig. 3. satellite view of polluted site (industrial area of Kanjikode) and non-polluted site (10 km far away from polluted site)

Fig.1. Structure of chlorophyll a and chlorophyll b

Rakshitha and Jelly Louis: Variation of chlorophyll content

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Materials and MethodsStudy Area

Present study was carried at Kanjikode located at Palakkad district, Kerala, during the month of April 2017. A major criterion for selecting this place to this study is Kan-jikode is one of the largest industrial towns in Kerala, situated in Puduserry Pancha-yath, Palakkad district. It is situated 12 km from Palakkad town and 33 km from Coim-batore which is located on NH 47. A total of ten different plant species were selected at random from different parts of the study area(Table.1). Sampling was done for once only. Same sample plants were collected from polluted and less polluted site and subjected to standard chemical procedures for determination of chlorophyll content. The plant species were collected from sur-rounding area of this industrial site (with in 1 km) i.e. polluted site. The same plant species were collected from 10 kms away from the polluted site i.e. less polluted site (Fig 2 and 3).

Extraction of chlorophyll content Fifty milligrams of finely cut fresh

leaves were taken and ground with 10-15 ml of 80% acetone (80 ml acetone + 20 ml distilled water) by using clean motor and pestle and homogenized for 1 min. It was then filtered and cen-trifuged at 2000-2500rpm for 5mins. The supernatant was transferred and again centrifuged. It was repeated for 3 times. Then the supernatant was transferred to 1 cm cuvette by using Pasteur pipette. The absorbance was measured at 645, 663 using UV-visi-

ble spectrophotometer at 645nm and 663nm against the solvent (acetone) blank.

Soil pH analysispH of the soil from the study area was

determined by pH meter.

Estimation of Chlorophyll contentThe concentrations of chlorophyll a, chlo-

rophyll b and total chlorophyll were calcu-lated using the following equation:Chlorophyll “a” (mg/g) = 12.7(A₆₆₃) – 2.69(A₆₄₅) / 1000 x W x V Chlorophyll “b” (mg/g) = 22.9(A₆₄₅) – 4.68(A₆₆₃) / 1000 x W x V Total Chlorophyll “a”+ “b” (mg/g) = 20.2(A₆₄₅) + 8.02(A₆₆₃) /1000 x W x V Where:A₆₆₃= absorbance at 663nmA₆₄₅= absorbance at 645nmW = fresh weight of leaf sample taken V = volume of leaf extract

Table.1.Plant species selected for the Study

Sl. No.

Name of plant species

Family Habit

1. Morinda tinctoria Rubiaceae Tree

2. Azadirachta indica Meliaceae Tree

3. Tectona grandis Verbenaceae Tree

4. Cassia fistula Fabaceae Tree

5. Calotropis gigantea Asclepiadiaceae Shrub

Table 3.Concentration of photosynthetic pigments (mg/g) in the leaves of 5 different plant species collected from polluted and non-polluted sites.

Sl. No.

Name of plant species Polluted Less-polluted

Chl a Chl b Chl a+b Chl a Chl b Chl a+b1. Morinda tinctora 0.186±0.001 0.098±0.002 0.284±0.001 0.237±0.003 0.145±0.001 0.382±0.0012. Azadirachta indica 0.195±0.002 0.123±0.004 0.318±0.001 0.237±0.002 0.174±0.002 0.411±0.0013. Tectona grandis 0.199±0.001 0.116±0.003 0.315±0.002 0.270±0.002 0.150±0.001 0.420±0.0014. Cassia fistula 0.207±0.006 0.105±0.003 0.312±0.001 0.242±0.003 0.157±0.003 0.399±0.0015. Calotropis gigantea 0.213±0.002 0.039±0.004 0.252±0.001 0.231±0.002 0.126±0.002 0.357±0.001

±means standard deviation


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Fig. 4. The graph showing concentration of chlorophyll content of different plant species at less-polluted sites.

Fig.5. The graph showing concentration of chlorophyll content of different plant species at polluted sites.

Fig.6.The graph showing comparison of concentration of chlorophyll a in different plant species at less-polluted and polluted site.

Fig.7. The graph showing comparison of concentration of chlorophyll b in different plant species at less-polluted and polluted site.

Fig. 8.The graph showing comparison of concentration of chlorophyll a+b in different plant species at less-polluted and polluted site.


75

Result and DiscussionspH of the soil samples collected from pol-

luted and non-polluted sites are presented the Table 2. The concentration of chloro-phyll a, chlorophyll b, and total chlorophyll of the selected plant species are presented in Table 3. Total chlorophyll was found to be higher in Azadirachta indica at polluted site and Tectona grandis at non-polluted site as compared to other plant species. In the polluted sites, soil samples show more acidity (pH 5.45) due to the presence of heavy metals and pollutants¹⁶. But in non-polluted sites, soil sample shows little alkalinity (pH – 7.66).Table 2. Soil pH analysis of collected samples from polluted and non-polluted sites

Polluted sites Less-polluted sites

Soil pH 7.66 5.45

The results obtained from the study in the polluted and less-polluted plant species were compared. Due to pollution, plants from polluted sites shows decrease in their photosynthetic pigments. Plants shows a significant reduction in total chlorophyll content, chlorophyll ‘a’ and chlorophyll ‘b’ at polluted sites as compared to less-polluted sites.

Morinda tinctoraThe concentration of chlorophyll ‘a’ in the

leaves of Morinda tinctora at polluted sites was recorded as 0.186 mg/g which was 0.237 mg/g at less-polluted site. The concentra-tion of chl ‘b’ at polluted site was recorded as 0.098 mg/g which was 0.145 mg/g at less-polluted site. The concentration of chl ‘a+b’ at polluted site was recorded as 0.284 mg/g which was 0.382 mg/g at non-polluted site. Thus a reduction of 12.05% in chloro-phyll ‘a’, 19.34% of chl ‘b’ and 14.54% in chl ‘a+b’ was recorded in samples from polluted site compared to less-polluted site.

Azadirachta indicaThe concentration of chlorophyll ‘a’ in

the leaves of Azadirachta indica at pol-luted sites was recorded as 0.195 mg/g

which was 0.237 mg/g at less-polluted sites. The concentration of chl ‘b’ at polluted site was recorded as 0.123 mg/g which was 0.174 mg/g at less-polluted sites. The con-centration of chl ‘a+b’ at polluted site was recorded as 0.318 mg/g which was 0.411 mg/g at non-polluted sites. Thus a reduc-tion of 9.72% in chl ‘a’, 17.17% in chl ‘b’ and 12.75% in chl ‘a+b’ was recorded in samples from polluted sites compared to less-pol-luted sites.

Tectona grandisThe concentration of chlorophyll ‘a’ in the

leaves of Tectona grandis at polluted sites was recorded as 0.199 mg/g which was 0.270 mg/g at less-polluted sites. The concentra-tion of chl ‘b’ at polluted site was recorded as 0.116 mg/g which was 0.150 mg/g at less-polluted sites. The concentration of chl ‘a+b’ at polluted site was recorded as 0.315 mg/g which was 0.420 mg/g at non-polluted sites. Thus a reduction of 15.13% in chlo-rophyll ‘a’, 12.78% in chl ‘b’ and 14.28% in chl ‘a+b’ was noted in samples from polluted sites compared to less-polluted sites.

Cassia fistulaThe concentration of chlorophyll ‘a’ in the

leaves of Cassia fistula at polluted sites was recorded as 0.207 mg/g which was 0.242 mg/g at less-polluted sites. The concentra-tion of chl ‘b’ at polluted site was recorded as 0.105 mg/g which was 0.157 mg/g at non-polluted sites. The concentration of chl ‘a+b’ at polluted site was recorded as 0.312 mg/g which was 0.399 mg/g at less-polluted sites. Thus a reduction of 7.79% in chloro-phyll ‘a’, 19.84% in chl ‘b’ and 12.23% in chl ‘a+b’ was recorded in samples from polluted sites compared to less-polluted sites.

Calotropis giganteaThe concentration of chlorophyll ‘a’ in

the leaves of Calotropis gigantea at pol-luted sites was recorded as 0.213 mg/g which was 0.231 mg/g at less-polluted sites. The concentration of chl ‘b’ at polluted site was recorded as 0.039 mg/g which was 0.126 mg/g at less-polluted site. The con-centration of chl ‘a+b’ at polluted site was


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recorded as 0.252 mg/g which was 0.357 mg/g at non-polluted site. Thus a reduc-tion of 4.05% in chlorophyll ‘a’, 52.72% in chl ‘b’ and 17.24% chl ‘a+b’ was recorded in samples from polluted sites compared to less-polluted site.

From the study it is indicated that a decline in chlorophyll content growing in industrial areaare most likely to be dam-aged by air pollution. The reduction in chlo-rophyll content is due to the degradation of chlorophyll into phaeophytin by the loss of magnesium ions. Chlorophyll content may differ in different period of time under dif-ferent conditions of pollution stress and dif-ferent meteorological conditions. These pig-ments also exist in highly organized state, and under stress they may undergo several photochemical reactions such as oxidation, reduction, pheophytinisation and reversible bleaching.Any alteration in chlorophyll con-centration may change the morphological, physiological and biochemical behaviour of the plant. The present result also correlated with the study of number of workers¹⁷ ¹⁸.

ConclusionThe results of this study indicated a

decline in chlorophyll content, acidity nature of the soil and morphological varia-tions in the leaves in the plants growing in industrial area shows that the air pollution caused by industries is an operative ecologi-cal factor causing deterioration in the qual-ity of our environment. This might be due to the presence of different toxic pollutants present in the industrial environment.

AcknowledgementI express my sincere thanks to Dr.Lilly

P.V., The Principal, Dr.Jelly Louis, Head of the department of Botany, Mercy College, Palakkad for supervision and providing me with the necessary facilities required for the work and encouragement. We also acknowledge DST-FIST for the financial aid for setting up our research lab.

References1. Steubing L., Fangmier A., and Both R.,

1989. Effects of NO₂, SO₂ and O₃ on popu-lation development on morphological and physiological parameters of native herb layer species in a beech forest. J. Environ. Pollut. Vol. 58:281-301.

2. Agbaire P.O., 2009. Air pollution toler-ance indices (APTI) of some plants around Erhoike-Kokori oil exploration site of Delta State, Nigeria, International. J. Physical Sci. Vol. 4:366-368.

3. Mahecha G. S., Bamniya B. R., Nair, Neelima, Saini D., 2013. Air pollution toler-ance index of certain plant species-A study of Madri Industrial Area, Udaipur (Raj.), India.Int. J. Innovative Res. in Sci. Engg.and Tech. Vol. 2(12):7927-7929.

4. Mirza H., Kamrun N., Md. Mahabub A., 2, Roychowdhury R., Fujita M. 2013. Physio-logical, Biochemical, and Molecular Mech-anisms of Heat Stress Tolerance in Plants. Int. J. Mol. Sci. Vol. 14: 9643-9684.

5. Srichaikul B., Bunsang R., Samappito S., Butkhup S., and Bakker G., 2011. Compar-ative study of chlorophyll content in leaves of Thai Morus alba Linn. J. Plant Sci. Res.Vol. 3: 17-20.

6. Rao D. N. and LeBlanc F. 1966. Effects of SO₂ on the lichen alga with special reference to chlorophyll. J. Bryologist Vol. 69: 69-75.

7. Jamrich V., 1968. In chlorophyll stability a factor in the power of resistant against fumes. J. VO Zvolene. Vol. 1: 7 – 14.

8. Dubey P.S., and Pawar K., 1985. Air pol-lution and plant responses Review of work done at Vikram Univ. Centre. Vol. 1: 101 – 117.

9. Tiwari S., 1991. Studies of Air Pollution Tolerance Indices of some planted trees in urban areas of Bhopal with reference to Eco-planning of Industrial areas. Ph .D. Thesis, Barkatullah University, Bhopal, India.

10. Bell J. N. B., 1980. Response of plants to sulphur dioxide. J. NatureLond.Vol. 284: 399-400.


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11. Dedio W., 1975. Water relations in wheat leaves as screening tests for drought resis-tance. Can. J. Plant Sci. Vol. 55: 369-378.

12. Strand M., 1993. Photosynthetic activity of Scot pine (Pinus sylvestris L.) needles during winter is affected by exposure to SO2 and NO2 during summer. J. New Phytol. Vol. 23: 133-141.

13. Pandey J. and Pandey U. 1994. Evolution of air pollution phytotoxicity in a seasonally dry tropical urban environment.J. Environ. Monit. Assess. Vol. 33 (3): 195-213.

14. Kumari S.I., Rani P.U. and Suresh C.H. 2005. Absorption of automobile pollutants by leaf surfaces of various road side plants and their effect on plant biochemical constit-uents. J. Poll Res. Vol. 24 (3): 509-512.

15. Karthiyayini R., Ponnammal N.R. and Joseph R., 2005. Air pollution tolerance

index of certain plants of Coimbatore- Ooty highways, near ITI area, Coimbatore, Tamil-nadu.J. Poll Res. Vol. 24(4):801-803.

16. Sajeesh K, Richard Scaria, Pankajakshan P & Akshara C., 2015. Impact of Industrializa-tion on Soil Resources, a Study on Kanjikode Industrial Region, Palakkad. BEST Jour-nals ISSN 2348-0521. Vol. 3(5): 7-14.

17. Bansal S., 1988. Studies on the effect of certain atmospheric pollutants on fruit diseases of Lycopersicon esculentum Mill. caused by Alternaria alternate. Bhopal University.

18. Sandelius A.S., Naslund K., Carlson A.S., Pleijel H., and Sellden G., 1995. Exposure ofspring wheat (Triticum aestivum) to ozone in open top chambers effects on acyl lipid composition and chlorophyll content of flag leaves. J. The New Phytologist. Vol. 131: 231–239.



Prevalence of pulmonary impairment symptoms among

dry cement exposed groupSaji K.G.1 and Varghese P.R2

1Department of Zoology, St.Aloysius College, Elthuruth, Thrissur, 680611, Kerala, India2Jubilee Centre for Medical Research, Thrissur, 680005, Kerala. India

AbstractCement is an inevitable material of construction industry. Workers associated with this industry have many occupational hazards. This study is to understand the health hazards in dry cement exposed group. Epidemiological study was conducted on health related features. Labourers were categorized in to five classes based on duration of exposure. Lung function impairment symptoms are presented here. It is observed that cement exposure reduces their lung function capacity. The presently practiced per-sonal protective measures are not effective to prevent the entry of cement dust in to their body during their labour.

Key words: cement dust, health hazard, lung function impairment symptoms

Introduction Portland cement is an extremely import-

ant construction material used for infra-structure development and a key to eco-nomic growth. Cement industry helps to improve human life standard around the world, creates direct and indirect employ-ment and benefits to associated industries.

Cement is the mixture of Calcium Oxide (CaO), Silicon oxide (SiO2), Aluminium tri-oxide (Al2O3), small amounts of magnesium, sodium, potassium and sulphur, heavy met-als like nickel (Ni), chromium (Cr), lead (Pb), ferric oxide, magnesium oxide (MgO), selenium, thallium and other impurities1. Cement dust is particularly alkaline, an irritant dust, and might therefore be con-sidered to pose a greater risk of respiratory tract damage than many other poorly solu-ble dusts generically regarded as “low toxic-ity dusts”2.

Cement exposure by skin, eye or inhala-tion results in many health effects. Severity of health effects depends on duration, level

of exposure and individual’s sensitivity. A cement plant can be a significant source of air pollutants and cement can affect respi-ratory symptoms and lung function. Cement mill workers are exposed to dust at various manufacturing and production process.

Most of the research works were con-centrated among cement mill workers due to huge cement exposure. Construction workers and loading and unloading work-ers were also the victim of cement expo-sure. Construction workers need to handle cement in different combination at different time interval. Whereas loading workers are handling only dry cement. Shifting cement bags from the wagons to the neighbouring depots or directly to the trucks waiting for the purpose are the tasks of the loading and unloading group. They are exposed chiefly to cement dust in dry form. The aim of the present study is to realize the health haz-ards among dry cement exposed group and effectiveness of preventive measure as a security gadget.

Saji and Varghese: Prevalence of pulmonary impairment symptoms

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Materials and methods The present study was conducted

between December 2011 and March 2013 in Thrissur district of Kerala state. The study group comprises loading and unloading workers in the cement good shed of railway station at Ollur and Thrissur.

A questionnaire was prepared focusing questions on all known and other probable health hazards to which the study group were exposed. All the members of the study group were visited personally and data col-lected individually.

The study comprises 266 samples. The study group is categorized into 5 classes based on their duration of exposure (5-9 years, 10-14 years, 15-19 years 20-24 years and 25+ years). Questionnaire helps to iden-tify their duration of exposure, personnel habits, ailments and mode of security mea-sures among these workers. Health hazards studied include persistent cough, breath-lessness, wheezing, sneezing, burns and rashes on skin, irritation of skin, eyes and throat, hair loss and colour change on body. Pulmonary function impairment symptoms are analysed and presented here.

ResultsThe study group comprises 266 cases

(Table 1). Sixty eight percent cases have more than ten years of exposure while twenty one percent have more than twenty years of exposure.

Table 1. Frequency of loading workers based on duration of exposure

Years of exposure Frequency5-9 84

10-14 5915-19 6620-24 35

25+ 22Total 266

Persistent cough, breathlessness, wheez-ing, irritation on throat and sneezing were considered as symptoms related to lung

function impairment. The percentage symp-toms of lung function impairment are rep-resented in Table 2.Table 2. Percentage of lung function impair-ment symptoms in loading workers

Symptoms Percentage

Persistent cough 54.9Breathlessness 47.4Wheezing 26.3Irritation on throat 13.2Sneezing 8.3

The analysis of personnel habits showed that bidi, cigarette and combined use of both items among smoking group of labourers. Table 3 represents comparison of smokers and non-smokers with lung function impair-ment symptoms. Among non-smokers per-sistent cough, breathlessness, wheezing and sneezing are reported in higher per-centage than smokers.

Table 3. Comparison of lung function impair-ment symptoms in percentage among smokers and non smokers of loading workers

Symptoms Smokers Non- smokers

Persistent cough 44.52 55.48

Breathlessness 42.06 57.94

Wheezing 41.43 58.57Irritation to throat 57.14 42.86

Sneezing 40.91 59.09

Loading workers use cotton clothes or low cost masks during their labour. The per-centage of workers using any kind of face mask during the work place is 36.1%

DiscussionLoading workers of two centres in Thris-

sur district were focussed in the present study. They were exposed to cement dust during their working hours relentlessly.


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Labourers were categorized in to five classes on the basis of duration of exposure. In which, highest frequency (37%) were observed in first class (5-9 years) and lowest (9.7%) observed in last class (25+ years). A slight fluctuation observed in second class (10-14 years) and from third class onwards, the frequency showed decreasing pattern. This may be due to the health distress among these labourers by chronic exposure.

The health condition of the study group was assessed by analysing signs and symp-toms presented by them. In the present study persistent cough (54.9%), breathlessness (47.4%) and wheezing (26.3%) are the symp-toms reported associated with respiratory tract impairment. Manjula3 have reported 45.3% of clinical parameters of upper respi-ratory tract infection among the cement dust exposed workers. Sana4 reported 89% of cement workers complained of wheez-ing problem and 90% suffered from cough. Sundarraj5 reported 21.3 % of chronic cough and 36.9 % of breathlessness in his study. Ahmed and Smith6 reported 70.6 % with respiratory symptoms in cement workers.

While comparing, respiratory ailments are more in non-smokers than smokers (Table-3). The major symptoms like per-sistent cough and breathlessness are the indicators of pulmonary impairment. These two were higher in non smoker group. This may be because the smokers are already exposed to factors which may be severe than cement dust. Even though non smoker group were not exposed to toxic material, cement dust accelerate lung function impairment and decline their lung capacity.

Nearly 36% percentages of loading work-ers were regularly using face mask during their labour hours. The face mask or the personal protective equipment used by

them is only a kind of cotton cloth used as bath towel or hand kerchief. Manjula3 also reported use of cotton clothes as a security measure among cement workers in their study. It is clearly evident that the present practice of using cotton cloths as face mask is not enough.

The diameter of cement dust is only 0.05 – 5 µm and the mesh size of cotton clothes/ handkerchief / bathroom towel are larger in mesh size, so it is not effective. Constant use of industrial mask which can protect mouth, nose and ear can prevent the entry of cement dust since its mesh size was lower than cement dust.

References1. EL-Sewefy, A. Z., Awad, S., Metwally, M.,

1970. Spirometric measurements in an EgyptianPortland cement factory. J. Egypt. Med. Asso. 53, 179-186.

2. Meo, S. A., 2004. Health hazards of cement dust. Saudi Medical Journal. 1153-1158.

3. Manjula, R., Praveena, R., Clevin, R., Ghat-targi, C., Dorle, A., Lalitha, D., 2013. Effects of occupational dust exposure on the health status of portland cement factory workers. International Journal of Medicine and Pub-lic Health. 3(3), 192.

4. Sana, S., Bhat, G. A., Balkhi, H. M., 2013. Health Risks Associated With Workers in Cement Factories. International Journal of Scientific and Research Publications. 3(1), 2250–3153.

5. Sundararaj, A., 2013. The prevalence of res-piratory morbidity and the risk factors asso-ciated, among the workers of cement indus-try in south India: A cross sectional study.

6. Ahmed, H.O., Newson-Smith, M.S., 2010. Knowledge and practices related to occu-pational hazards among cement workers in United arab emirates. J Egypt Public Health Assoc. 85(3–4), 149–167.



An Insilico Study on PPAR γ2 Gene among Obesity related type 2 Diabetes

mellitus (T2DM)Santhi.S1 and Jayasree.S

1Bioinformatics Centre, Mercy College, Palakkad - 678 006, Kerala, India.Department of Zoology, Mercy College, Palakkad - 678 006, Kerala, India.

AbstractObesity and type 2 diabetes mellitus (T2DM) are major worldwide public health problems . T2DM is characterized by chronic hyperglycemia associated with insulin resistance and relative insulin defi-ciency. T2DM is believed to be attributable to the combined effect of genetic and environmental fac-tors. Peroxisome proliferator-activated receptor gamma 2 (PPARγ2) is one of the main candidate genes that are implicated in T2DM. A common proline 12 alanine (Pro12Ala) polymorphism in PPARγ2 has been shown to be associated with T2DM. The peroxisome proliferator- activated receptor γ2 (PPAR γ2) is a nuclear receptor transcription factor involved in lipid metabolism, adipocytes differentiation and proliferation, and insulin sensitivity by regulating the expression of adipocyte-specific developmental genes. Previous studies have suggested that genetic variation in PPAR may constitute a predisposing factor for obesity, type 2 diabetes, or insulin resistance, and a Pro12Ala substitution in exon B of PPAR 2 has been associated with BMI and type 2 diabetes in several populations, but the results has been inconsistent. The aim of this work was to investigate the possible role of PPARγ2 gene polymorphism, as a genetic risk factor for T2DM. So in this study a functional single nucleotide polymorphism (SNP) that predicts a proline to alanine substitution (Pro12Ala) within the coding region of this gene was analyzed by some of the Insilco method, like SNP and Predict SNP that predict the association of Pro12Ala genetic variation that can alter the function of the gene products. The result obtained in the study shows allele change occurred as deleterious it proves that individuals carrying the Ala12 risk allele may be more prone to develop T2DM mediated obesity. These findings suggest that the PPAR-γ2 Pro12Ala variant may increases the risk factor for development of Type 2 Diabetes in Obese People.

Key Words: PPAR-γ2, Obesity, T2DM, PREDICT SNP, Deleterious

IntroductionDiabetes mellitus (DM) is a group of

common metabolic disorders that share the phenotype of hyperglycemia. Globally, about 5.4% of adult population has been estimated to have type 2 diabetes mellitus (T2DM)

1. Several types of diabetes mellitus exist and are caused by a complex interaction of genetics and environmental factors2. Type 2 diabetes has a strong genetic component. There is evidence that the Pro12Ala poly-morphism is linked to obesity and T2DM3. The peroxisome proliferator-activated receptor gamma (PPARG) is now recognized to play a main PPARG entered the spotlight

as a major player in metabolic regulation. Regarding to its functional role in metabolic pathways, PPAR-γ2 has been one of the most commonly studied candidate gene for metabolic disorders, such as obesity. Among several polymorphisms reported in the PPAR-γ2 gene, two common variant includ-ing a missense Pro12Ala polymorphism in exon B and a silent mutation C1431T in exon 6 have been studied in several popu-lations and the results have been inconsis-tent. Many epidemiological studies reported these two common variants of PPAR-γ2 are associated with obesity risk and measures of it, however, some other studies have showed no association with obesity4, 5. The


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allele frequencies for a Pro123Ala substi-tution in peroxisome proliferator–activated receptor-g differ among ethnic groups, and its relationship with diabetes and associ-ated diseases is controversial6.

Studies also show that about half of the SNP mutations occurring in the cod-ing regions are missense while the rest are silent7. Since missense mutations are known to be one the main causes for major genetic disorders, many of these are the single causative factors for rare single gene inherited disorders. It is also expected that some more frequent missense mutations arising from SNPs in the coding regions will be associated with common genetic disorders8.

DbSNP contains 1,463,178 submissions from 97 registered groups describing vari-ation in five species human, mouse rat, chimpanzee, and the malaria parasite. Sub-mission can be divided into four general categories with the following percentages of the total database size: (i) SNP mining from the human genome project sequences, 65%; (ii) private investigator/corporate exper-imental result, 28%; (iii) mined from EST databases, e.g. (Masood, 1999), 6%; (iv) con-tinuing result of the NHGRI SNP discovery RFA, 1%. Public and private initiatives to discover new SNP in human identified over 306,000 variations in the period 1999-20009

Materials and MethodsNational Centre for Biotechnology

dbSNP (SNP database)

The Single Nucleotide Polymorphism Data-base (dbSNP) is a free public archive for genetic variation within and across different species developed and hosted by the National Center for Biotechnology Information (NCBI) in col-laboration with the National Human Genome Research Institute (NHGRI). Although the name of the database implies a collection of one class of polymorphisms only (i.e., single nucleotide poly-morphisms(SNPs), it in fact contains a range of molecular variation: (1) SNPs, (2) short deletion and insertion polymorphisms (indels/DIPs), (3) microsatellite markers or short tandem repeats (STRs), (4) multi nucleotide polymorphisms

(MNPs), (5) heterozygous sequences, and (6) named variants10.

dbsnp database from NCBI was used for data curation. Gene name and disease name was typed in search engine to identify all SNPs related to gene.

PREDICT SNPConsensus classifier that combines six of

the top performing tools for the prediction of the effects of mutation on protein func-tion. The obtained results are provided together with annotations extracted from the Protein Mutant Database and the Uni-Prot database. PredictSNP consensus pre-diction was calculated using the following equation:

Where N is the number of integrated tools rep-resents the overall prediction (+1 for the delete-rious prediction, −1 for the neutral prediction) and expresses the transformed confidence scores. The output value of PredictSNP score belongs to the continuous interval <−1, +1>. The mutations are considered to be neutral for the values in the interval <−1, 0> and deleterious for the values in the interval 0, +1>. The absolute distance of the PredictSNP score from zero expresses the con-fidence of the consensus classifier about its pre-diction. For easy comparison with the confidence scores of individual integrated tools, we trans-formed the confidence of the PredictSNP consen-sus classifier to the observed accuracy in the same way as described for confidence scores of the inte-grated tools11.

Results and DiscussionSNP Analysis

The present study determined the fre-quency of the proline to alanine susbsti-tution in the human PPAR γ2 gene from insilico study to prove its role on diabetes mellitus and obesity. Type 2 diabetes has a strong genetic component. There is evi-dence that the Pro12Ala polymorphism is linked to obesity and T2DM12. In recent years many studies have focused on the

Santhi and Jayasree: Insilico Study on PPAR γ2 Gene

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association between PPAR-γ2 gene variants and obesity to may clarify the molecular mechanism of obesity, but the results has been inconsistent. The present study set out to assess the association of most common variants of PPAR-γ2 gene with obesity and obesity related markers using Insilco tools. Our results showed significant association of PPAR- Pro12Ala polymorphisms with risk of obesity, but suggest that Ala risk allele of

Pro12Ala polymorphism is correlated with predisposition to obesity related markers. Total of 36564 SNPs ids were retrieved for PPARG gene, Limits was used for Obesity and T2DM the result got only 8 SNPs. The table shows the SNP result from dbSNP (Table.1).

There are total of 8 SNPs were retrieved from the PPARG gene, (rs1800571, rs1801282, rs1805192, rs17241090, rs17749580, rs36206375, rs56460253, rs57327233). In that the last 5 SNPs

(rs17241090, rs17749580, rs36206375, rs56460253, rs57327233) were merged to (rs1801282), so at last we got only 3 SNPs here these all 3 SNPs shows Fwd direc-tion and Missense function, but rs1801282, rs1805192 SNPs have same allele change, Residue change and Residue Position but with different chromosome position, so for Further analysis of Predict SNP we can take the rs1800571, rs1801282 only because

it can have the different Residue posi-tion change rather than, rs1805192, thus PPARG gene SNPs is filtered and it can be used for further analysis.

PREDICTSNP

Human active SNPs obtained from dbSNP database were uploaded in Pre-dictSNP to find the deleterious SNPs. rs1801282 SNPs were found to be delete-rious. The table indicating the deleterious mutations (Table. 2)

Table.1.SNP results from dbSNP

SNP ID Gene ID

Chromosome position

Allele change Function SNP to

mRNA Position Residue change

1 rs1800571 PPARG 3:12381349 CCA ⇒ CAA missense Fwd 85 P [Pro] ⇒ Q [Gln]

2 rs1801282 PPARG 3:12351626 CCA ⇒ GCA missense Fwd 12 P [Pro] ⇒ A [Ala]

3 rs1805192 PPARG 3:12379739 CCC ⇒ GCC missense Fwd 12 P [Pro] ⇒ A [Ala]

Chromosome 3 - NC_000003.12[120043604 [120043604

SYN2 GSTM5P1 RNA5SP123

MTC01P5 PPARG

TSEN2 RNU6-377PFig.1. Chromosome Position of SNP ID rs1801282

Table: 2 Table indicating the highly deleterious mutations screened by predictsnp

rs ID Mutation Nucleotide change

Polyphen 1

Polyphen 2 MAPP PhD

SNP SNAP SFT Predict SNP

rs1801282 P12A G/C 67% 75% 77% 89% 62% 46% 65%


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Detection of deleterious nsSNPs is a major and challenging task. Here we identified del-eterious mutations screened by predictsnp is rs1801282 SNP id with P12A as nucleotide change as G/C that shows the highest percent-age of 65% predictsnp. Most of the deleterious nsSNPs are well supported for the significant finding of the analysis. Significant association of Pro12Ala polymorphism of PPARγ2 gene with T2DM was found in ethnic Kashmiri population12. In the obese group, the carriers of 12Ala risk allele had higher body weight (P=0.019), waist circumference (P=0.003), and waist/hip ratio (P=0.048) compared to the wild-type homozygotes Iran population13. The Iranian population study suggests that Pro12Ala but not C1431T polymorphism of PPAR-γ2 gene is correlated with predisposi-tion to obesity related markers13.

Obesity as a chronic medical disease has shown increases in prevalence during recent years worldwide and requires enhanced research, treatment and prevention efforts. A growing body of evidence has demonstrated not only environmental factors including life-style of bad diet, inactivity as well as meta-bolic and endocrine abnormalities are the most important players in the etiology of obe-sity, but genetic factors also have a major role. Genome-wide association studies have clearly revealed that there is a genetic contribution to obesity14. Until now several candidate genes, including PPAR-γ2 gene have been searched in term of their involvement in the etiology of obesity and body fat accumulation. The PPAR-γ2 which has a key role in adipocyte differentiation, energy storage, insulin resis-tance and lipid metabolism, has gained inter-est as a good candidate gene for the metabolic related disorders15. The association of the vari-able rs1801282 locus of the PPARG2 gene (peroxisome proliferator-activated receptor gamma) with type 2 diabetes mellitus and its complications was analyzed in inhabitants of the Republic of Bashkortostan. The genotype frequencies of the variable rs1801282 locus of the PPARG2 gene did not significantly differ in groups of healthy persons and patients with type 2 diabetes in all three considered inher-itance models (codominant, dominant, and recessive). At the same time, it was demon-strated that the risk of one of the diabetic

complications, i.e., diabetic nephropathy, was associated with the variable rs1801282 locus of the PPARG2 gene. Diabetic nephrop-athy was more common in patients with the C/C genotype (62.7%) compared to the C/G and G/G genotypes (37.5%), P = 0.036. The G allele is protective in regard to diabetic nephropathy (OR = 0.36) in patients with type 2 diabetes mellitus16.

Conclusion These data shows a novel functional varia-

tion in Pro12Ala variant within the promoter of PPAR-γ2. This promoter variant is associ-ated with metabolic predictors of type 2 diabe-tes and obesity. These findings suggest that the PPAR-γ2 Pro12Ala variant may increases the risk factor for development of Type 2 Diabetes in Obese People.

References1. Mogre V., Salifu Z. S., Abedandi R., 2014.

Prevalence components and associated demographic and lifestyle factors of the met-abolic syndrome in type 2 diabetes mellitus. J Diabetes Metab Disord. 13:80.

2. Anthony F.S., Braunwald E., Kasper D. L., 2008. Harrison’s principles of internal Med-icine. 17th Ed, McGraw Hill Publication., New York, p. 2275.

3. Danawati C.W., Nagata M., Moriyama H., Hara K., Yasuda H., Nakayama M.,Kotani R., Yamada K., Sakata M., Kurohara M., Wiyono P., Asdie H., Sakaue M., Taniguchi H., Yokono K., 2005. A possible association of Pro12Ala polymorphism in peroxisome proliferator-activated receptor γ2 gene with obesity in native Javanese in Indonesia. Dia-betes Metab Res Rev.;21:465–9.

4. Prakash J., Srivastava N., Awasthi S., Agar-wal C., Natu S., Rajpal N., Mittal B., 2012. Association of PPAR-gamma gene polymor-phisms with obesity and obesity-associated phenotypes in North Indian population. Am J Hum Biol. 24: 454-459.

5. Stefanski A., Majkowska L., Ciechanowicz A., Frankow M., Safranow K., Parczewski M.,Pi-larska K., 2006. Lack of association between the Pro12Ala polymorphism in PPAR-gamma 2 gene and body weight changes, insulin

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resistance and chronic diabetic complications in obese patients with type 2 diabetes. Arch Med Res. 37: 736-743

6. Mato E.P., Pokam-Fosso P.E., Atogho-Tiedeu B., Noubiap J.J., Evehe M.S., Djokam-Dad-jeu R., Donfack O.S., Ngwa E.N.,Guewo-Fo-keng M., Mbacham W.F.,2016. The Pro12Al apolymorphism in the PPAR-gamma2 gene is not associated to obesity and type 2 dia-betes mellitus in a Cameroonian population. BMC obesity, 3: 26.

7. Halushka M.K., Fan J.B., Bentley, K., Hsie, L., and Shen N., 1999. Patterns of single-nu-cleotide polymorphisms in candidate genes for blood-pressure homeostasis. Nature Genetics, 22(3): 239-247.

8. Brookes A.J., 1999. The essence of SNPs. Gene, 234: 177-186.

9. Brookes A.J., Lehvaslaiho H., SiegfriedM., Boehm J.G., Yuan Y.P., Sarkar C.M., Bork P., and Ortigao F., 2000. HGBASE A Data-base of SNPs and other variations in and around human genes. Nucleic Acid Research, 28: 356-360.

10. Sherry S.T., Ward M. H., Kholodov M., Baker J., Phan L., Smigielski E.M., and Sirotkin K., 2000. DbSNP: the NCBI database of genetic variation. Nucleic Acids Research, 29: 308-311.

11. Bendl, J., Stourac, J., Salanda, O., Pavelka, A., Wieben, E., Zendulka, D. J., Brezovsky J., and Damborsky. J., 2014. PredictSNP: robust and accurate consensus classifier for prediction of disease-related mutations. PLoS Computational Biology, 10: 34-40.

12. Majid M, Masood A., Kadla S.A., Hameed I., Ganai B.A., 2017. Association of Pro12Ala Polymorphism of Peroxisome proliferator-Ac-tivated Receptor gamma 2 (PPARγ2) Gene with Type 2 Diabetes Mellitus in Ethnic Kashmiri Population. Biochem Genet.55 (1), 10-21.

13. Hassan Mehrad-Majd,, Majid Ghayour-Mo-barhan , Mohammad-Reza Zali., 2017. Effect of two common variants in PPAR-γ2 gene on susceptibility to obesity. Biomedical Research: 28 (13), 5671-5677.

14. Loos R.J., 2012. Genetic determinants of common obesity and their value in predic-tion. Best Pract Res Clin Endocrinol Metab, 26: 211-226.

15. Auwerx J., 1999. PPARgamma, the ultimate thrifty gene. Diabetologia, 42: 1033-1049.

16. Avzaletdinova D.S., Sharipova .F., Kochetova O.V., Morugova T.V., Erdman V.V., Mus-tafina O.E., 2016. Association of variable rs1801282 locus of PPARG2 gene with dia-betic nephropathy, 52(8): 985-90.



Biochemical alterations in Silkworm Bombyx mori on exposure to a new generation Pesticide – λ Cyhalothrin

Shahila Ismail K.I. and Sreeranjit Kumar C.V.Research and PG Depatment of Zoology, Govt. Victoria College, Palakkad- 678006, India

AbstractSilkworm Bombyx mori is an important economic insects and a model species for Lepidopteran, being a primary producer of silk. Of the factors responsible for increasing the production of silk, the quality of mulberry plays the most important role. But silk production needs an improvement in quality as well as quantity and this reduction is mainly due to the environmental effects. The silkworm Bombyx mori feeds exclusively on mulberry leaves and is highly sensitive to pesticides in general. Although mulberry plantations are free of agrochemicals, pesticide drift can occur especially when mulberry trees grow next to cultivated plants. Aerial application of insectides to agricultural fields may cause damage to silkworm production when the chemicals drift to nearby mulberry plantations that provide food for the silkworms. Lambda cyhalothrin may have induced toxicity on economically important silkworms by disturbing their metabolisms. So in the present study we propose to demonstrate the efficacy of ascorbic acid against the degenerative and associated conditions on exposure to pesticides which may affect the quality and quantity of silk production. This provides a potential new method to improve the resistance of silk worm against new generation pesticides.

Key words: Bombyx mori, agrochemicals, Lambda cyhalothrin, ascorbic acid

IntroductionSericulture, the rearing and manage-

ment of silkworms for silk production, is a labour-intensive, agro-based industry which provides additional income to mar-ginal farmers in India. Sericulture plays a vital role in generating gainful employment opportunities to the rural people. Sericul-ture also provides self employment oppor-tunities to the educated unemployed youth and women of rural areas. One of the most economically important products of Sericul-ture is cocoon. Marketing value of cocoon is mainly depending upon its quality. The qual-ity of cocoon is determined by total cocoon weight, cocoon layer weight and cocoon layer ratio. It is in turn depending upon the availability of temperature, humidity and quality of mulberry leaves. The reduction in the quality and quantity of silk production is mainly due to the environmental effects. The environmental impacts of pesticides

consists of the effects on non- target species.

Bombyx mori is highly susceptible to insecticides and the applications of insec-ticide near sericulture project is not advis-able because the silkworms can be harmed by chemicals on the leaves, either through consumption of contaminated leaves or through other contact with the insec-ticides1,2. Hence, mulberry plantations intended for silkworms are free of pesti-cides. However, the complete elimination of pesticide drift is impossible and insecticides that are employed in other crops, especially those that are applied through aerial spray-ing can damage sericulture activity.

As a consequence of pesticide usage in the past couple of years, sericulture faces many common problems such as reduction in the quality and quantity of cocoon pro-duction and finally death of silkworm3. Now a day farmers are using new generation

Shahila Ismail and Sreeranjit Kumar: Biochemical alterations in Bombyx mori

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pesticide such as synthetic pyrethroids like KARATE® in major parts of Kerala due to its fast response and it is safe to verte-brates. Exposure to insecticides in mul-berry leaves could affect growth, reproduc-tion and the quality of economic character-istics of cocoon4, 5. Brain is the central part of nervous system and the important target of pyrethroid. These damages are mainly due to the oxidative stress of cells and ner-vous system and thereby destructing the reproductive system and other metabolic functions. But some substances can resist the activity of pesticide or oxidation dam-age. Ascorbic acid may have the antipesti-cidal effect. It is a highly effective antioxi-dant, acting to lesson oxidative stress6, 7.

Materials and MethodsCollection of Eggs, Rearing and Management of Silkworm

The eggs of Bombyx mori, bivoltine race, variety SK7 were collected from P2 Basic Seed Farm, National Silkworm Seed Organ-isation, Central Silk Board, Pallatheri, and reared in the laboratory. Optimum tem-perature and humidity were maintained.Tender mulberry leaves collected from the department mulberry garden was given ad-libitum and they were maintained in the light and dark cycle of 12hrs duration.

Supplementations0.001ppm synthetic pyrethroid Lambda

cyhalothrin (KARATE®) exposed to the feeding mulberry leaves by leaf dipping method of Zhang.et. al8 and 0.02% of Ascor-bic acid also supplemented during the study period.

Experimental protocolSoon after the fourth moult, the larvae

were randomly selected and grouped into 3 sets as follows:

Group 1 – ControlGroup 2 – Pesticide exposedGroup 3 – Pesticide + Vit.C supplementedBased on the method of Zhang et al. we

used leaf dipping bioassay8. Commercial grade insecticide which is in a liquid form

was first diluted in 1000 ml distilled water and accomplished with water to prepare solution of required concentration at 0.001 ppm. This concentration was tolerated by B.mori larvae, which are confirmed by our previous bioassay study.

Twenty numbers of silk worms were placed in each group. They were kept in room temperature and a light and dark cycle of 12 hour duration throughout the study. The Group 1 and Group 2 were fed with fresh mulberry leaves while group 3 was fed with fresh mulberry leaves soaked in 0.2% ascorbic acid solution .By the com-pletion of 4th instar, group 2 and group 3 were fed with pesticide treated mulberry leaves. The exposure was continued for five days and after the vth day the silk worms were collected and weighed and were sacri-ficed for the estimation of various biochemi-cal and histological parameters.

Collection of haemolymph and tissuesHaemolymph was collected by amputat-

ing the thoracic legs by using sterile instru-ment and was pooled and stored in pre-chilled eppendorf tubes containing a few crystals of phenyl thiourea. The silkgland, fat body, midgut and brain were dissected out by using sterile instruments and they were weighed and stored in pre chilled con-tainers and tissues were kept in fixatives for biochemical and histopathological studies. Protein concentration of haemolymph was determined following the method of Lowry et al. (1951)9.

Results Silkworm larvae fed with pesticide

treated foliages resulted in persistent growth inhibition at different levels during larval metamorphosis. It also caused growth disruption in larval and pupal morphogen-esis when compared to vitamin c supple-mented along with pesticide and control.

The concentration of total protein of hae-molymph, silk gland and cocoon of larvae exposed to pesticide showed a significant decrease than the control and pesticide + vit.c supplemented groups.


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DiscussionApplication of large quantity of pesti-

cides can certainly control varied types of pests, but it is also polluting the environ-ment. Silkworms are very sensitive to pes-ticides, thus pesticide pollution causes seri-ous losses to sericulture. How to increase

silkworm’s pesticide resistance to mitigate economic losses has become an urgent prob-lem. In this study we found that addition of an appropriate amount of ascorbic acid (Vit.C) onto mulberry leaves can reduce pesticide induced damages. The mechanism

of ascorbic acid’s effects on pesticide induced dam-ages was also investi-gated in order to seek for methods to better improve silkworm’s resistance to pesticide.

Silkworm larvae fed with pesticide treated foliage resulted in per-sistent growth inhibition

at different levels during metamorphosis. The silkworm larvae fed with pesticides treated foliage caused growth disruption in pupal and adult morphogenesis (Table 1). The present finding showed that pesticide is effective in inhibiting larval growth and inducing pupal deformities in silkworm. Such growth inhibitory effect of pesticide ranging from delay in moulting with the production of deformed insects, to the com-plete inhibition of growth at high doses1.

Growth and differentiation are closely related to their biochemical activities. Pro-tein synthesis during 5th instar is mainly depended on their food intake. Food intake reaches its peak in the 5th instar and its shows the quantity and quality of silk pro-duction. Body growth and silk gland devel-opment are related to the metabolism of silkworm. Pesticide reduces the protein content of silk worm in various tissues and haemolymph. But ascorbic acid pretreat-ment can enhance the ability of biochemi-cal metabolism thereby increase the protein contents. From the data obtained, the silk gland and cocoon registered more protein content when compared to haemolymph,

Table 1: Metamorphosis of silkworm Bombyx mori of different groups during experimental period (changes in larval number)

Groups Initial larvae

Survived larvae

Normal pupae

Deformed pupae

No: of emerged moth

Control 20 19 16 1 15

Pesticide 20 12 0 7 0Pesticide +vit.C 20 16 10 2 8

Fig.1. Deformed larvae on exposure to pesticide Fig.2. Deformed cocoon on exposure to pesticide

Fig.3. Concentration of total protein in Haemolymph

HAEMO

LYMPH

Shahila Ismail and Sreeranjit Kumar: Biochemical alterations in Bombyx mori

89

digestive system and fat body in both con-trol and ascorbic acid with pesticide treated groups. Silkgland is a reservoir for two important silk proteins such as fibroin and serisin10 and other proteins such as chap-erons, metabolic enzymes, heat shock pro-teins, immune proteins, serpins, transport proteins and those involved in gene expres-sion. Similarly the haemolymph of B. mori is the chief circulating fluid and flowing res-ervoir of about 241 to 298 proteins that pro-mote normal growth, metamorphosis, silk productions, chitin and haemocyte forma-tion, growth of salivary gland , reproductive organs and ecdysis. The major damages of pesticide come from its destroy to nervous. We also performed investigation on silk worm brains after pesticide treatments. Lambda cyhalothrin, caused serious brain tissue damages and cell death. Analysis of ultra structure revealed severely dam-aged cells and cell layers. This damages or shrinkage of cells create a large cavity between the cells which shows the defen-sive mechanism by the cells due to the effect of pesticides. Pesticidal treated larvae showed broad outer layer than the control to

prevent damages. When ascorbic acid was given to the larvae, such changes were sig-nificantly relieved. Binbin Wang et.al; 2015 also showed CeCl3 can reduce the brain damages caused by OP pesticide11.

From the study it is came to know that, the pesticide exposed larvae with dietary supplementation of ascorbic acid differ-entially influenced the Morphometric, metabolic and biochemical parameters of silkworm Bombyx mori. Ascorbic acid pre treatment could reduce pesticide induced damages. It also enhance biochemical metabolism in silkworm to protect oxida-tive damages. This study illustrated the potential mechanisms of ascorbic acid’s effect to enhance pesticide metabolism in silkworm brains and provided a theoretical basis to use ascorbic acid as an additive to improve silkworm’s pesticide tolerance in sericulture.

References1. Padmalatha C., Kumutha P., Chairman K.,

Ranjith Singh A. J. A., 2013. Effect of pes-ticides on the metamorphosis of silkworm, Bombyx mori; International Journal of Molecular Zoology; Vol 3, No.3.

2. Kayvan Etebari., Bizhannia A. R., Sorati R., Leila Matindoost., 2007. Biochemical changes in haemolymph of silkworm larvae due to pyriproxyfen residue; Pesticide bio-chemistry and Physiology, 88(1), 14-9.

3. Nath B. S., Suresh A., Varma B. M., Kumar R. P., 1997. Changes in protein metabolism in haemolymph and fatbody of the silk-worm, Bombyx mori in response to organo-phosphorous insecticides toxicity; Ecotoxi-col Environ saf; 36(2), 169-173.

4. Kumutha P., Padmalatha C., Chairman K., Ranjith Singh A. J. A., 2013. Effect of pes-ticides on the reproductive performance and longevity of Bombyx mori; ISSN, vol 2, 74-78.

5. Shigeharu Kuribayashi., 1981. Studies on the effect of pesticides on the reproduction of the silkworm, Bombyx mori L; The jour-nal of toxicological sciences, vol 6, 169-176.

6. Javed Humayun., 2002. Effect of 0.2% N with various combinations of ascorbic acid

Fig.4. Concentration of total protein in Silk gland

Fig.5. Concentration of total protein in Cocoon


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on growth and silk production of silkworm; Asian Journal of Plant Science 1, 650-651.

7. Jackson M. J., Khassaf M., Mc Ardle A., 2002. Effect of vitamin C supplements on antioxidant defence and stress proteins in human lymphocytes and skeletal muscle; J Physiol, 549.

8. Zhang J. Z., Li Z. M., Liu L., Mei L., Yao J. L., Hu C. G., 1951. Identification of early flower related ESTs in an early flowering mutant of trifoliate orange by suppression subtractive hybridisation and microarray analysis; Tree Physiol, 28(10), 1449-57.

9. Lowry O. H., Resenbrough N. J., Farr A. L., Randall R. J., 1951. Protein measure-ment with the Folin phenol reagent. J Biol Chem, 193(1), 265-75.

10. Komatsu K. 1980. Recent advances in seri-cin research. J, Sericult. Sci. Japan. 69: 457- 465.

11. Binbing Wang., Fanchi Li., Hua Zhang., Kaizun Xu., Jianghai Tian., Jingsheng Hu., Weide Shen., and Bing Li., 2015. Molecular signatures of reduced nerve tox-icity by CeCl3 in phoxim exposed silkworm brains; Scientific Reports 5, 12761.

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