ETHIOPIAN JOURNAL OF SCIENCE AND TECHNOLOGY …bdu.edu.et/sites/default/files/journal/Vol.7 No. 2.pdf · ETHIOPIAN JOURNAL OF SCIENCE AND TECHNOLOGY VOLUME 7, NUMBER 2 (JUNE, 2014)

ETHIOPIAN JOURNAL OF SCIENCE AND TECHNOLOGY VOLUME 7, NUMBER 2 (JUNE, 2014)

EDITORIAL BOARD

EDITOR-IN-CHIEF Professor Mulugeta Kibret Bahir Dar University, Department of Biology Email: [email protected], [email protected]: +251-58-226-6597Fax: +251-58-226-4066Mailing address: P.O. Box 79Bahir Dar, Ethiopia

ASSOCIATE EDITORS Dr Amare Benore, Department of Physics, Science College, BDUDr Awoke Andargie, Department of Mathematics , Science College, BDUMr Bayeh Abera, Department of Microbiology, Immunology and Parasitology, College of Medicine and Health Sciences, BDUDr Eneyew Tadesse, School of Chemical and Food Engineering, BDUDr Essey Kebede, Department of Statistics, Science College, BDUDr Hailu Mazengia, Department, of Animal Science College of Agriculture and Environ-mental Sciences, BDUDr Meareg Amare, Department of Chemistry, Science College, BDUDr Solomon Tesfamariam, School of Mechanical and Industrial Engineering, BDUProfessor Thakor Krushang,Ethiopian Institute of Textile and Fashion Technology, BDUDr Wassie Anteneh, Department of Biology, Science College, BDUMr Zelalem Liyew, Department of Earth Science, Science College, BDU

ADVISORY BOARD

Professor Gezahegn Yirgu, Addis Ababa University, EthiopiaDr Gizaw Mengistu, Addis Ababa University, EthiopiaDr Habtu Zegeye, Botstwana University, Botswana Dr Melaku Walle, Bahir Dar University, EthiopiaDr Mesfin Tsige, University of Akron, Ohio, USADr Mohammed Tesemma, Spelman College, Atlanta, USADr Mulugeta Bekele, Addis Ababa University, EthiopiaDr Mulugeta Gebregziabher, University of Medical South Carolina, USAProfessor Murali Mohan, SP-Mahila University, India Professor Robert McCrindle, Tshwane University of Technology, South AfricaDr Semu Mitiku, Addis Ababa University, EthiopiaDr Solomon Harrar, University of Montana, USA Dr Solomon Libsu, Bahir Dar University, EthiopiaProfessor Srinivas Rao, Andhra University, IndiaProfessor Teketel Yohannes, Addis Ababa UniversityProfessor Temesgen Zewotir, University of KwaZulu Natal, South Africa Dr Tesfaye Baye, University of Cincinnati, USA

Dr Abebe Getahun, Addis Ababa University, Ethiopia Professor Abera Mogessie, Karl-Franzense University of Graz, AustriaProfessor Afework Bekele, Addis Ababa University, EthiopiaDr Berahnu Abraha, Bahir Dar University, EthiopiaProfessor Bhagwan Singh Chandravanshi, Addis Ababa University, EthiopiaDr Emmanuel Vreven, Royal Museum of Central Africa, BelgiumDr Endawoke Yizengaw, Institute for Scientific Research, Boston College, USADr Eshetie Dejen, FAO East African Regional Office, EthiopiaDr Firew Tegegne, Bahir Dar University, EthiopiaDr Gebregziabher Kahsay, Bahir Dar University, EthiopiaProfessor Geog Güebitz, University of Natural Resources and Life Science, Vienna, AustriaDr Getachew Adamu, Bahir Dar University, Ethiopia

© Science College, Bahir Dar University, 2014


CONTENTS


Effect of ultrasound on protein metabolism in the silkworm

Murali Mohan, P., Siva Prasad, S., Sahitya Chetan, P.

67

Variations in body shape of European seabass larvae (Dicentrarchus labrax) reared under xenic and axenic conditions

Eyasu Shumbulo Shuba, Dominique Adriaensand, Peter Bossier

85

Assessment of antioxidant potential of Moringa stenopetala leaf extract Tesfaye Tebeka, Solomon Libsu

93

Ethnobotanical study of medicinal plants in Ankober woreda, central Ethiopia

Daniel Kalu, Ali Seid

105

Second degree generalized gauss-seidel iteration method for solving linear sys-tem of equations

Tesfaye Kebede

115

Author Guidelines

Ethiop. J. Sci. & Technol. 7(2) 67- 84, 2014 67

Effect of ultrasound on protein metabolism in the silkworm, Bombyx mori (L.)

Murali Mohan, P.*1, Siva Prasad, S.2, and Sahitya Chetan, P.3

1Department of Sericulture, SP Mahila University, TIRUPATI–517502, India, Email: [email protected] 2Department of Zoology, Smt. N.P.S. Government College for Women, Chittoor– 517002, India

3Scuola Internazionale Superiore Studi Avanzati (SISSA), Via Bonomea, No. 265 Trieste – 34136, Italy

ABSTRACTThe parameters of protein metabolism, such as the levels of soluble, structural and total proteins, free amino acids, and the activity levels of protease, aspartate and alanine aminotransferases and glutamate dehydrogenase were as-sayed in the hemolymph, silk-gland, muscle and fat-body on different days of the 5th instar larva of the silkworm, Bombyx mori, following exposure of the silkworm eggs to 1 MHz continuous wave of ultrasound at an intensity of 9W/cm2 for 2 minutes. Ultrasound was found to promote the accumulation of proteins, which include silk proteins as well, while retarding proteolysis and turnover of proteins towards the release of amino acids, keto-acids etc. Changes in the levels of these biochemical constituents are correlated with the events of histogenesis and histolysis associated with metamorphosis. It may be inferred that protein metabolism is stimulated by ultrasound, resulting in greater turn-over of silk proteins, spinning activity and silk output.

Key words: Ultrasound, Silkworm, Bombyx mori, Proteins, Amino Acids, Protease, Aminotransferases, Glutamate dehydrogenase.DOI: http://dx.doi.org/10.4314/ejst.v7i2.1

INTRODUCTIONInsect metamorphosis is a dynamic biochemical ac-tivity. Chen (1971) highlighted the role of biochem-ical constituents in insect metamorphosis. Since the silkworm is an economically important insect, sever-al insect physiologists attempted to elucidate the role of biochemical constituents in silk protein synthesis and egg formation (Horie et al., 1971; Mathavan et al., 1984). The growth of silkworm during metamor-phosis is accompanied by the increase in the body weight and accumulation of various biochemical con-stituents like proteins, amino acids and enzymes like proteases, aminotransferases and glutamate dehy-drogenase (Pant and Jaiswal, 1981; Bannikov et al., 1982; Wangerli and Xuting-Sen, 1982; Bose et al., 1989; Siva Prasad and Murali Mohan, 1990). More importantly, the parameters of protein metabolism have been extensively examined because of their

role in development, morphogenesis and intermedi-ary metabolism (Tojo et al., 1980; Ogawa and Tojo, 1981; Sasaki et al., 1981; Robert and Rutt, 1982; Sa-rangi, 1985; Ravikumar and Sarangi, 2004).

A novel approach in silkworm research is the ma-nipulation of biochemical machinery through exoge-nous modulators that could boost the silk production. This included the administration of certain neurohu-moral factors, vertebrate hormones and various oth-er chemicals like cyclic AMP and prostaglandins, which could have a profound influence on the growth rate, larval life cycle and fecundity (Singh and Dut-ta, 1980; Bharathi and Govindappa, 1987; Thyagara-ja et al., 1991; Bharathi, 1993). Significant positive impact of vertebrate thyroxine on silkworm biology, especially in improving the pre- and post-cocoon pa-rameters is well documented (Bharathi et al., 1986;

______________________________*Correspondind author

©This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/CC BY4.0).

68 Murali Mohan et al.

Krishnamurthy et al., 1987; Chaudhuri and Medda, 1992). Another vertebrate hormone, namely prolac-tin, induced improvement in the growth and repro-ductive potential of silkworms (Bhaskar et al., 1983; Bharathi et al., 1984, 1986). The dietary administra-tion of vertebrate sex hormones like ethynyl estradi-ol (EE) and norethindrone (NE) to the silkworm in-creased the larval weight, cocoon and shell weights, female pupal and adult weights, but the larval, pupal periods and the egg-hatchability were significantly reduced (Saha and Khan,1977).

These investigations opened up alternative strate-

gies for improving the economic parameters of the

sericulture industry by regulating the biochemical

machinery. One such option is the ultrasound, whose

impact on larval life in Drosophila has been report-

ed (Child et al., 1981). Further, it was reported that

ultrasound irradiation does not cause any detectable

deterioration in behavioral responses such as mat-

ing, oviposition, larval development and pupation in

insects (Koehler et al., 1986). Ultrasound has been

used as an exogenous modulator of several param-

eters in both vertebrate and invertebrate models.

However, the mechanism of action of ultrasound in

these models has not yet been resolved. A few stud-

ies are also available on ultrasound effects on silk-

worm. Samal et al. (2013) examined ultrasound son-

ication effects on the properties of silkworm fibroin

protein. Murali Mohan and Siva Prasad (2013) stud-

ied the effect of ultrasound on economic characters

of the silkworm and found improvement in several

characters including silk output upon exposure to

ultrasound. Since the silk fiber is composed of the

protein fibroin, it is important to examine the effect

of ultrasound on the silkworm protein metabolic

profile. Taking this cue, ultrasound was used in the

present investigation for the manipulation of protein

metabolism in the silkworm and to examine the pos-

sibility of its utility in sericulture.

MATERIALS AND METHODSThe present investigation was carried out on the hybrid of Pure Mysore variety (Multivoltine) and NB4D2 variety (Bivoltine) of the silkworm Bombyx mori. The silkworm eggs in blue-egg stage (10-12 h old) were exposed to ultrasound at a frequency of 1 MHz and an intensity of 9 W /cm2 for 2 min, using an ultrasound equipment, model WIPRO–GE Al-pha–Logic–100. Prior to exposure, the egg-card was kept in a sealed, water-filled polythene bag, smeared with gel so as to prevent the diversion of ultrasonic waves. The bag was suspended in the ultrasonic bath tank. Water can be continuously circulated inside the tank, and its level can be maintained more or less at a fixed height. A transducer was kept at the base of the tank, and it transmits ultrasonic energy to the eggs in a focused way. The duration of exposure was standardized by exposing the eggs to varying inten-sities of ultrasound waves at different time intervals, viz. 2, 5, 10, 15, 20, 25, and 30 minutes. Promising results were obtained at 1 MHz continuous wave of ultrasound at an intensity of 9W/cm2 for 2 minutes. The larvae that emerged from the exposed (experi-mental) and unexposed (control) eggs were used in the investigation in the 5th instar, which accounts for all the silk biosynthesis and secretion into the lumen of the silk-gland. The treatment of eggs with ultra-sound was replicated six times separately. Tissues from at least ten silkworms that emerged from each treated lot of eggs were pooled for biochemical esti-mations. Daily changes in biochemical parameters of protein metabolism such as proteins, free amino ac-ids, and the activity levels of the enzymes protease, aspartate and alanine aminotransferases, and gluta-mate dehydrogenase were observed in the 5th instar larvae. Tissues such as hemolymph, silk-gland, mus-cle and fat-body, isolated by dissecting the larvae in ice-cold silkworm Ringer (Yamaoka et al., 1971) were used for the biochemical assays. The total pro-tein content as well as the soluble protein content was estimated by the method of Lowry et al. (1951). The trend of changes in the structural protein levels


was obtained by subtracting the soluble protein lev-els from the total protein levels in both control and experimental worms and then calculating the percent changes for each day of the 5th instar against the re-spective controls. The free amino acid content was estimated by the method of Moore and Stein (1954) as described by Colowick and Kaplan (1957) and the protease activity by the method of Davis and Smith (1955). The activity levels of aspartate aminotrans-ferase (AAT) and alanine aminotransferase (AlAT) were estimated by the method of Reitman and Fran-kel (1957) as described by Bergmeyer and Bruns (1965) and the activity of glutamate dehydrogenase (GDH) was assayed by the method of Lee and Lardy (1965).

Statistical treatment of dataThe difference in physiological activity between the

control and ultrasound-treated (experimental) groups

was statistically tested. All the assays were carried

out with six separate replicates from each group.

Values were expressed as mean ± standard deviation

(SD) from six replicates. The mean and SD were

worked out using INSTAT statistical software and

Dunnett’s multiple comparison test followed by one-

way Analysis of Variance (ANOVA) using the SPSS

statistical tool (SPSS for windows, release 17.0.1,

2008, SPSS Inc., Chicago, IL) to assess the differ-

ences. P values of <0.05 were considered as statisti-

cally significant. Percentage changes were calculated

from control to treated and presented along with the

statistical test.

RESULTS

Tables 1 to 8 and Figures 1 to 8 highlight the chang-

es in the biochemical parameters, viz., proteins,

free amino acids, protease, aminotransferases and

glutamate dehydrogenase and the effect of ultra-

sound on these parameters in the 5th instar larvae of

Bombyx mori.

The levels of total and soluble proteins recorded an increasing trend in all the tissues from the 1st day to the 6th day of the instar. The extent of increase var-ied from tissue to tissue. The levels of total proteins registered a maximum increase of 15.4% on 1st and 4th days in hemolymph, 92.4% in silk-gland, 71.4% in muscle and 28.6% in fat-body on the 6th day of the 5th instar. Thus, ultrasound treatment elevated the total protein level in all the tissues. However, the increase was not progressive and consistent in hemolymph on different days of the instar (Table

1).

The levels of soluble proteins showed a steady in-crease from the 1st day to the 6th day of the instar. However, in the worms treated with ultrasound, lower increases in the levels of soluble proteins were evident in all the tissues. This trend was in-consistent in the hemolymph, with greater increas-es on certain days and lower increases on the other days. The changes in hemolymph were statistically not significant (Table 2).

The structural protein levels showed slight increas-

es in hemolymph on different days of the instar. In

the remaining tissues their levels increased pro-

gressively throughout, reaching the maximum by

the 6th day. High increases were recorded in the

silk-gland (169.7%) and muscle (332.7%) on the

6th day (Table 3).

Under the impact of ultrasound, the levels of to-

tal free amino acids showed a progressive increase

in different tissues on all days of the instar. When

the percent increases were calculated against the

controls for each day, the increases were found to

be relatively small and there was no discernible

trend. Except in hemolymph (from the 3rd day), the

increases in all other tissues were not statistically

significant (Table 4).


Tabl

e 1.

Day

-to-d

ay c

hang

es in

the

leve

ls o

f tot

al p

rote

ins (

expr

esse

d as

mg

prot

ein/

g w

et w

eigh

t of t

he ti

ssue

or 1

ml o

f hem

olym

ph) d

urin

g th

e 5th

inst

ar

evel

opm

ent o

f Bo

mby

x m

ori u

nder

the

impa

ct o

f ultr

asou

nd.

Tiss

ueIn

dice

s 1st

Day

2nd D

ay3rd

Day

4th D

ay5th

Day

6th D

ay

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Hem

olym

phM

ean

SD%

cha

nge

t-tes

t

77.8

5±3

.13

89.8

4±3

.18

82.3

9±1

.89

94.4

0±2

.48

84.4

7±2

.48

96.3

2±3

.12

86.1

2±3

.79

99.3

7±2

.48

89.0

2±1

.89

101.

86±2

.48

91.0

9±1

.89

104.

34±2

.48

15.

4 P <

0.05

14.6

P <

0.05

14.0 P <

0.05

15.4

P <

0.0

5

14

.4 P <

0.05

14.

6P

< 0.

05

Silk

gla

ndM

ean

SD%

cha

nge

t-tes

t

80.8

9±5

.97

124.

20±1

0.4

82.3

8±7

.013

6.45

±9.4

984

.88

±9.4

814

6.84

±9.4

899

.37

±6.2

117

5.86

±8.9

711

3.87

12.6

221

1.21

±8.9

712

4.22

±11.

423

8.96

±9.4

8

53.

5P

< 0.

001

65.

6P

< 0.

001

73.0

P <

0.00

1

7

6.9

P <

0.00

1

85

.5P

< 0.

001

92.4

P <

0.00

1

Mus

cle

Mea

nSD

% c

hang

et-t

est

37.2

7±6

.27

53.7

6±9

.48

43.4

8±6

.21

62.1

1±1

0.42

47.6

2±9

.48

72.4

6±9

.49

55.9

0±6

.21

86.9

5±1

1.42

62.1

1±1

0.42

99.3

2±6

.21

64.5

0±1

0.0

110.

53±1

2.00

38.9

P <

0.00

1

4

2.9

P <

0.00

1

52

.2P

< 0.

001

55.

6P

< 0.

001

5

9.9

P <

0.00

1

7

1.4

P <

0.00

1

Fat b

ody

Mea

nSD

% c

hang

et-t

est

43.4

8±6

.21

50.5

7±9

.48

55.9

0±6

.21

66.3

2±6

.21

35.1

9±9

.49

43.4

8±6

.21

49.6

9±6

.21

62.1

1±6

.21

64.5

4±1

0.0

81.0

7±5

.73

57.9

7±9

.48

74.5

3±6

.21

16.0 P <

0.05

18.6

P <

0.01

23.6 P <

0.01

25.0

P <

0.01

25.6 P <

0.01

28.6

P <

0.01

Not

e: E

xptl.

† st

ands

for e

xper

imen

tal (

treat

ed w

ith u

ltras

ound

). Ea

ch v

alue

is th

e m

ean

± St

anda

rd D

evia

tion

(SD

) of s

ix se

para

te o

bser

vatio

ns. F

or e

ach

obse

rvat

ion,

tiss

ue fr

om a

t lea

st 1

0 la

rvae

was

poo

led.

The

per

cent

cha

nges

for a

ll da

ys w

ere

calc

ulat

ed b

y ta

king

the

resp

ectiv

e co

ntro

ls a

s the

refe

renc

e.

Cha

nges

are

sign

ifica

nt a

t lea

st a

t 5%

leve

l.


Tabl

e 2.

Day

-to-d

ay c

hang

es in

the

leve

ls o

f sol

uble

pro

tein

s (ex

pres

sed

as m

g pr

otei

ns/g

wet

wei

ght o

f the

tiss

ue o

r 1m

l of h

emol

ymph

) dur

ing

the

5th in

-st

ar

de

velo

pmen

t of B

omby

x m

ori u

nder

the

impa

ct o

f ultr

asou

nd.

Tiss

ueIn

dice

s1st

Day

2nd D

ay3rd

Day

4th D

ay5th

Day

6th D

ay

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Hem

olym

phM

ean

SD %

chan

get-t

est

21.1

1±2

.48

23.1

8±1

.89

26.0

8±2

.48

28.1

5±3

.12

28.5

7±2

.48

31.0

5±2

.48

31.4

7±1

.55

33.5

4±2

.03

35.6

1±1

.55

37.6

8±1

.89

39.7

5±2

.48

44.7

2±2

.48

9

.8

NS

7

.9

NS

8

.7

NS

6

.2

NS

5

.8

NS

12.

5

NS

Silk

gla

ndM

ean

SD %

chan

get-t

est

31.0

5±5

.12

55.9

0±6

.21

35.1

9±4

.63

64.1

8±9

.48

43.4

8±6

.21

68.3

2±6

.12

49.6

9±5

.07

72.4

6±7

.75

60.0

4±1

0.55

86.9

6±6

.21

72.4

6±1

1.63

99.3

8±6

.2

80.0

P <

0.00

1

82.4

P <

0.00

1

5

7.1

P <

0.00

1

45.8

P <

0.0

01

44.8

P <

0.00

1

37.

2P

< 0.

001

Mus

cle

Mea

nSD %

ch

ange

t-tes

t

30.2

4±7

.13

45.0

5±4

.21

31.0

5±4

.12

45.5

5±8

.48

37.2

7±4

.21

47.6

2±5

.48

43.4

8±3

.10

54.4

9±5

.07

51.7

6±5

.75

64.1

8±5

.48

56.1

8±6

.18

74.5

3±8

.42

4

8.9

P

< 0

.001

46

.7P

< 0.

001

27

.8P

< 0.

05

25.3

P

< 0

.05

24

.0P

< 0.

05

32.

7

P <

0.0

01

Fat b

ody

Mea

nSD %

ch

ange

t-tes

t

18.6

3±3

.21

27.7

7±4

.49

24.8

4±4

.21

35.9

8±5

.11

28.9

8±4

.49

35.1

9±5

.63

31.0

6±3

.21

37.4

7±4

.21

43.4

8±5

.07

50.9

7±5

.48

55.9

±5.2

171

.04

±6.6

2

49.1

P <

0.00

1

44.

84P

< 0.

001

2

1.4

P <

0.05

20

.6P

< 0.

05

17.2

P <

0.05

27.

1

P

< 0.

01

Foot

note

is th

e sa

me

as in

Tab

le 1

. N

S –

Not

sign

ifica

nt


Tabl

e 3.

Day

-to-d

ay c

hang

es in

the

leve

ls o

f stru

ctur

al p

rote

ins

(exp

ress

ed a

s m

g pr

otei

n / g

wet

wei

ght o

f the

tiss

ue o

r 1m

l of h

emol

ymph

) dur

ing

the

5th in

-st

ar

o

f Bom

byx

mor

i und

er th

e im

pact

of u

ltras

ound

.

Tiss

ue1st

Day

2nd D

ay3rd

Day

4th D

ay5th

Day

6th D

ayC

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†

Hem

olym

ph56

.74

66.6

6(1

7.5)

56.3

166

.32

(17.

8)55

.90

65.2

7(1

8.5)

54.6

565

.83

(20.

4)53

.41

64.1

8(2

0.2)

51.3

459

.62

(16.

1)

Silk

gla

nd49

.84

68.3

0(3

7.0)

47.1

972

.27

(53.

1)41

.40

78.5

2(8

9.7)

49.6

810

3.4

(108

.1)

53.8

312

4.25

(130

.8)

51.7

613

9.58

(169

.7)

Mus

cle

7.03

8.71

(23.

9)12

.43

16.5

6(3

3.2)

10.3

524

.84

(40)

12.4

232

.46

(161

.3)

10.3

535

.14

(239

.5)

8.32

36.0

0(3

32.7

)

Fat b

ody

24.8

522

.80

(-8.

2)31

.06

30.3

4(-

2.3)

6.21

8.29

(33.

5)18

.63

24.6

4(3

2.2)

21.0

630

.1(4

2.9)

2.07

3.49

(68.

6)

Not

e: E

xptl.

† st

ands

for e

xper

imen

tal (

treat

ed w

ith u

ltras

ound

). Ea

ch v

alue

was

obt

aine

d by

sub

tract

ing

the

valu

es o

f sol

uble

pro

tein

s fr

om th

e va

lues

of t

otal

pr

otei

ns. T

he d

ata

was

not

giv

en s

tatis

tical

trea

tmen

t as

it re

pres

ents

der

ived

dat

a. H

owev

er, p

erce

nt c

hang

es in

thei

r lev

els

in th

e ul

traso

und-

treat

ed la

rvae

wer

e ca

lcul

ated

and

pre

sent

ed in

par

enth

eses

.


Tabl

e 4. D

ay-to

-day

chan

ges i

n th

e lev

els o

f tot

al fr

ee am

ino

acid

s (ex

pres

sed

as m

g of

tyro

sine

equi

vale

nts/

g w

et w

eigh

t of t

he ti

ssue

or 1

ml o

f hem

olym

ph)

dur

ing

the

5th in

star

of B

omby

x m

ori u

nder

the

impa

ct o

f ultr

asou

nd.

Tiss

ueIn

dice

s1st

Day

2nd D

ay3rd

Day

4th D

ay5th

Day

6th D

ay

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Hem

olym

phM

ean

SD%

cha

nge

t-tes

t

4.16

±0.3

64.

58±0

.37

4.99

±0.7

55.

81±0

.24

5.26

±0.5

97.

00±0

.14

5.99

±0.1

08.

00±0

.09

6.10

±0.0

88.

16±0

.05

6.25

±0.0

78.

25±0

.04

10

.1

NS

16

.4

NS

33

.1P

< 0.

001

33

.6P

< 0.

001

33

.8P

< 0.

001

32

.0P

< 0.

001

Silk

gla

ndM

ean

SD%

cha

nge

t-tes

t

30.4

8±2

.34

34.0

7±2

.17

33.7

6±2

.93

36.1

1±3

.94

35.6

3±1

.94

37.7

0±2

.70

37.0

5±1

.38

38.4

5±1

.77

37.9

8±1

.38

39.3

9±1

.94

38.9

2±1

.47

40.8

0±2

.74

11

.8

NS

6

.9

NS

5

.8

NS

3

.8

NS

3

.7

NS

4

.8

NS

Mus

cle

Mea

nSD

% c

hang

et-t

est

24.3

8±1

.98

26.2

6±1

.94

15.8

1±0

.97

17.2

±0.9

926

.73

±1.4

728

.60

±2.4

627

.70

±2.4

329

.54

±2.3

828

.14

±1.8

330

.48

±2.4

729

.22

±2.7

131

.89

±2.9

4

7.7

N

S

8.8

N

S

7.0

NS

6

.6

NS

8

.3

NS

9

.1

NS

Fat b

ody

Mea

nSD

% c

hang

et-t

est

22.5

1±0

.94

24.2

2±1

.71

22.5

0±1

.54

25.3

2±2

.23

23.9

1±1

.46

25.4

7±2

.97

24.8

5±1

.38

26.2

6±2

.38

25.3

2±0

.98

26.3

8±0

.96

26.2

6±0

.97

27.6

7±1

.07

7

.6

NS

12

.5

NS

6

.5

NS

5

.7

NS

4

.2

NS

5

.4

NS

Foot

note

is th

e sa

me

as in

Tab

le 1

. N

S –

Not

sign

ifica

nt


The activity levels of protease recorded slight el-evations in all the tissues throughout the instar, both in the control and experimental silkworms. In general, the elevations were found to be progres-sively lowered as the instar progressed from the 1st day to the 6th day in the case of hemolymph, silk-gland and muscle, while the percent increase was progressive with the days of the instar. The basic levels of protease activity were more or less similar in all the four tissues. Except in the muscle, the increases in activity in all the other tissues were not statistically significant (Table 5).

The activity of aspartate aminotransferase (AAT) maintained relatively constant levels in the hemo-lymph both in the control and experimental silk-worms on all days of the instar without any percep-tible variation. In silk-gland and muscle the enzyme activity recorded marginal percent increases on all the days of the instar both in the control and ex-perimental silkworms, but without any particular trend. These small increases were not statistically significant. However, in the case of fat-body, the AAT activity showed progressive percent increas-es in the silkworms under the impact of ultrasound as the instar progressed, and the changes were sta-tistically significant (Table 6).

The alanine aminotransferase (AlAT) activity by and large showed a similar trend as of AAT, with marginal percent increases under the impact of ultrasound on all days of the instar. However, in hemolymph, silk-gland and muscle, these slight in-creases got progressively lowered as the instar pro-gressed. These percent increases were statistically not significant. However, in the case of fat-body, the AlAT activity showed a trend that was simi-lar to the one exhibited by AAT activity, with pro-gressive percent increases in the silkworms under the impact of ultrasound as the instar progressed. These changes were statistically significant from the 4th day onwards (Table 7). Although not statis-

tically significant, the percentage changes in gluta-mate dehydrogenase (GDH) activity progressively rose in all the tissues of the silkworms under the influence of ultrasound. These increases were mar-ginal and statistically not significant in the case of silk-gland and fat-body, and significant only on the 5th and 6th days of the instar in the muscle. In the hemolymph the percent increases were statistically significant from the 3rd day onwards (Table 8).

DISCUSSIONIn the present study, ultrasound was in general found to have a stimulatory effect on the protein metabolism. Since proteins are the chief organic constituents regulating the biochemical events in the cell, it appears logical that ultrasound has a stimulatory effect on the protein metabolism. Al-though increased levels of proteins were observed in silkworm tissues (Zaidi and Khan, 1979: Tojo et al., 1980; Siva Prasad and Murali Mohan, 1990), these parameters have not been hitherto analyzed with reference to ultrasound in the silkworm. However, reports are available on the effect of ultrasound on protein synthesis with reference to differential tissue response in other animals (Lele et al., 1973).

Proteins perform multiple functions. The hemo-lymph proteins are implicated in ecdysis, growth of reproductive organs and salivary glands, formation of hemocytes and chitin (Zaidi and Khan, 1979; Gakhar and Maleyvar, 1985). In muscle, most of these proteins are contractile that facilitate feeding and spinning behaviors of silkworm (Siva Prasad and Murali Mohan, 1990). Obviously, the intensi-fication of these two behaviors is of paramount im-portance in 5th instar larvae. The feeding behavior is more pronounced in early stages and is responsi-ble for the uptake of the nutrients, while the spin-ning behavior manifests at the end of the 5th instar and is responsible for spinning of the cocoon. As such, the prevalence of higher levels of structural


Tabl

e 5. D

ay-to

-day

chan

ges i

n th

e lev

els o

f pro

teas

e act

ivity

(exp

ress

ed as

µm

oles

of t

yros

ine e

quiv

alen

ts/m

g pr

otei

n/h)

dur

ing

the 5

th in

star

of B

omby

x mor

i

un-

der t

he im

pact

of u

ltras

ound

.

Tiss

ueIn

dice

s1st

Day

2nd D

ay3rd

Day

4th D

ay5th

Day

6th D

ay

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Hem

olym

phM

ean

SD % c

hang

et-t

est

0.03

1±0

.01

0.03

6±0

.02

0.03

3±0

.01

0.03

7±0

.01

0.03

6±0

.02

0.04

±0.0

20.

04±0

.02

0.04

4±0

.02

0.04

±0.0

20.

048

±0.0

20.

048

±0.0

20.

054

±0.0

2

16.

1

NS

1

2.1

N

S

11.1

NS

1

0.0

N

S

20

.0P

< 0.

05

1

2.5

NS

Silk

gla

ndM

ean

SD % c

hang

et-t

est

0.64

±0.0

10.

70±0

.01

0.67

±0.0

15

0.73

±0.0

20.

67 ±

0.03

0.73

±0.

020.

7±0

.02

0.76

±0.

020.

74±0

.02

0.8

±0.0

20.

8±0

.02

0.84

±0.0

2

9.8

NS

8

.9

NS

8.7

NS

8

.6

N

S

8.1

N

S

5

.0

NS

Mus

cle

Mea

nSD %

cha

nge

t-tes

t

0.06

±0.0

02

0.08

±0.0

030.

06 ±

0.00

10.

075

±0.0

020.

06±0

.001

0.07

±0

.002

0.06

±0

.002

0.07

±0.

002

0.07

±0

.005

0.08

±0.0

060.

07 ±

0.00

40.

08

±0.0

07

33.

3

P

< 0

.01

25.

0P

< 0.

05

1

6.7

P <

0.05

16.

7P

< 0.

05

14

.3

N

S

1

4.3

NS

Fat b

ody

Mea

nSD %

cha

nge

t-tes

t

0.05

±0.0

010.

053

±0.0

020.

055

±0.0

020.

058

±0.0

020.

059

±0.0

030.

06±0

.005

0.06

5 ±0

.002

0.07

±0.0

030.

07±0

.006

0.08

±0.0

050.

07±0

.005

0.08

±0.0

05

6

.0

N

S

5.4

N

S

1

.7

N

S

8

.33

NS

1

4.3

N

S

1

4.3

NS

Foot

note

is th

e sa

me

as in

Tab

le 1

. N

S –

Not

sign

ifica

nt


Tabl

e 6. D

ay-to

-day

chan

ges i

n th

e lev

els o

f asp

arta

te am

inot

rans

fera

se (A

AT) a

ctiv

ity (e

xpre

ssed

as µ

mol

es o

f pyr

uvat

e for

med

/mg

prot

ein/

h) d

urin

g th

e 5th

in-

star

of B

omby

x m

ori u

nder

the

impa

ct o

f ultr

asou

nd.

Tiss

ueIn

dice

s1st

Day

2nd D

ay3rd

Day

4th D

ay5th

Day

6th D

ay

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

H

emo-

lym

ph

Mea

nSD %

cha

nge

t-tes

t

5.06

±0.0

25.

11±0

.02

5.1

±0.0

25.

17±0

.03

5.06

±0.0

45.

11±0

.04

5.04

±0.0

25.

1±0

.02

5.02

±0.0

25.

08±0

.02

5.00

±0.0

25.

05±0

.03

1.0

N

S

1

.4

N

S

1

.0

N

S

1.2

N

S

1

.2

N

S

1

.0

NS

Silk

gla

ndM

ean

SD % c

hang

et-t

est

0.7

±0.0

20.

74±0

.03

0.74

±0.0

30.

77±0

.02

0.77

±0.0

20.

79±0

.02

0.82

±0.0

20.

84±0

.02

0.84

±0.0

30.

87±0

.03

0.88

±0.0

20.

92±0

.02

5.7

NS

4.1

NS

2.6

NS

2.

4

NS

3.6

NS

4.6

NS

Mus

cle

Mea

nSD %

cha

nge

t-tes

t

1.47

±0.0

21.

52±0

.02

1.40

±0.0

21.

48±0

.02

1.38

±0.0

21.

46±0

.02

1.34

±0.0

31.

4±0

.02

1.32

±0.0

21.

38±0

.02

1.30

±0.0

21.

34±0

.02

3.4

NS

5.7

NS

5.8

NS

4.

5

NS

4.5

NS

3.1

N

S

Fat b

ody

Mea

nSD %

cha

nge

t-tes

t

0.52

±0.0

20.

58±0

.04

0.56

±0.

020.

64

±0.0

20.

54 ±

0.02

0.62

±0.

020.

48 ±

0.02

0.56

±0.0

20.

43 ±

0.03

0.52

±0.

020.

38 ±

0.02

0.46

±0.0

2

11

.5

N

S

14.

3

P <

0.0

5

14

.8

P

< 0

.05

16.

7

P

< 0

.05

20.9

P <

0.0

1

21

.1

P

< 0.

01

Foot

note

is th

e sa

me

as in

Tab

le 1

. N

S –

Not

sign

ifica

nt


Tabl

e 7.

Day

-to-d

ay ch

ange

s in

the l

evel

s of a

lani

ne am

inot

rans

fera

se (A

lAT)

activ

ity (e

xpre

ssed

as µ

mol

es o

f pyr

uvat

e for

med

/mg

prot

ein/

h) d

urin

g th

e 5th

in-

star

of B

omby

x m

ori u

nder

the

impa

ct o

f ultr

asou

nd.

Tiss

ueIn

dice

s1st

Day

2nd D

ay3rd

Day

4th D

ay5th

Day

6th D

ay

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Hem

olym

phM

ean

SD%

cha

nge

t-tes

t

1.43

±0.1

1.49

±0.

131.

32±0

.09

1.37

±0.0

81.

26±0

.03

1.29 ±0.0

21.

21±0

.04

1.23

±0.0

41.

15±0

.05

1.17

±0.

051.

11±0

.03

1.13

±0.0

3

4.2

N

S

3.8

N

S

2.4

N

S

1.7

N

S

1.7

N

S

1

.8

NS

S

ilk g

land

Mea

nSD

% c

hang

et-t

est

2.33

±0.0

22.

38 ±

0.02

2.42

±0.0

22.

47 ±

0.02

2.54

±0.0

42.

57±0

.03

2.62

±0.0

22.

67±0

.03

2.76

±0.0

22.

78 ±

0.02

2.82

±0.0

32.

95±0

.04

2.

2

NS

2

.1

NS

1.

2

NS

1.

9

NS

0.

7

NS

4.6

N

S

Mus

cle

Mea

nSD

% c

hang

et-t

est

1.96

±0.0

21.

98 ±

0.02

1.9

0.02

1.93

±0.0

31.

72±0

.02

1.86

±0.0

61.

64±0

.03

1.75

±0.0

51.

55±0

.02

1.67

±0.

041.

5±0

.02

1.58

±0.

02

1.0

N

S

1.6

N

S

8.1

N

S

6.7

N

S

7

.7

NS

5.

3

NS

Fat b

ody

Mea

nSD

% c

hang

et-t

est

2.28

±0.0

22.

33 ±

0.03

2.16

±0.0

52.

22±0

.02

2.10

±0.0

92.

17±0

.01

1.92

±0.0

62.

12 ±

0.02

1.82

±0.0

22.

02 ±

0.06

1.74

±0.0

41.

96±0

.02

2.

2

NS

2.8

NS

3.

3

NS

10.

4P

< 0.

02

1

0.9

P <

0.02

12.6

P <

0.02

Foot

note

is th

e sa

me

as in

Tab

le 1

, exc

ept t

hat c

hang

es a

re si

gnifi

cant

at 2

% le

vel.

NS

– N

ot si

gnifi

cant


Tabl

e 8. D

ay-to

-day

chan

ges i

n th

e lev

els o

f glu

tam

ate d

ehyd

roge

nase

(GD

H) a

ctiv

ity (e

xpre

ssed

as µ

mol

es o

f for

maz

on fo

rmed

/mg

prot

ein/

h) d

urin

g th

e 5th

in-

star

of B

omby

x m

ori u

nder

the

impa

ct o

f ultr

asou

nd.

Tiss

ueIn

dice

s1st

Day

2nd D

ay3rd

Day

4th D

ay5th

Day

6th D

ay

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Con

trol

Expt

l.†C

ontro

lEx

ptl.†

Hem

olym

phM

ean

SD%

cha

nge

t-tes

t

0.48

±0.0

20.

51

±0.0

20.

43±0

.02

0.48

±0.0

30.

36±0

.02

0.44

±0.

020.

32±0

.02

0.40

±0.0

30.

25±0

.02

0.38

±0.0

20.

17±0

.02

0.32

±0.

02

6

.2

N

S

11.

6

NS

22.

2

P <

0.01

25.

0

P

< 0

.01

5

2.0

P

< 0

.001

88.

2P

< 0.

001

Silk

gla

ndM

ean

SD%

cha

nge

t-tes

t

1.51

±0.0

21.

56±0

.02

1.61

±0.0

41.

67

±0.0

21.

68±0

.01

1.74

±

0.02

1.74

±0.0

21.

8±0

.02

1.82

±0.0

21.

89

±0.0

41.

93 ±

0.02

2.01

±0.5

6

3

.3

N

S

3.7

N

S

3.6

N

S

3.5

N

S

3.8

N

S

4.1

N

S

Mus

cle

Mea

nSD

% c

hang

et-t

est

0.51

±0.0

20.

57±0

.03

0.46

±0.0

30.

52

±0.

030.

43±0

.03

0.48

±0.

020.

38±0

.02

0.42

±0.0

30.

34±0

.02

0.40

±0

.02

0.28

±0.0

20.

38

±0.0

2

11

.8

N

S

13.

0

NS

11.

6

N

S

1

0.5

NS

17.

7P

< 0.

05

35.7

P <

0.00

1

Fat b

ody

Mea

nSD

% c

hang

et-t

est

0.41

±0.0

20.

44±0

.02

0.35

±0.0

20.

38±0

.02

0.32

±0.0

20.

34±0

.02

0.27

±0.0

20.

30±0

.02

0.22

±0.0

20.

26 ±

0.02

0.18

±0.0

20.

22 ±

0.02

7.3

NS

8.6

NS

6.3

NS

11.

1

N

S

1

8.2

NS

22.

2

N

S

Foot

note

is th

e sa

me

as in

Tab

le 1

. N

S –

Not

sign

ifica

nt


proteins during the 5th instar (Table 3) is indicative of strengthening of the muscle tissue for increased efficiency of feeding behavior during the larval de-velopment. Further, the increases in the levels of structural proteins coupled with the increase in the levels of total and soluble proteins is indicative of the ongoing histolysis associated with the degener-ative metamorphic changes and delegation of pro-teins towards the spinning activity as well as the formation of the silk proteins in the silk-gland. In silk-gland, the proteins are used for the synthesis of silk proteins, viz., fibroin and sericin (Horie et al., 1971). The phenomenal increase in the levels of structural proteins (Table 3) in silk-gland and mus-cle is indicative of the continuous synthesis and ac-cumulation of the silk proteins. The fat-body seems to act as the storage organ similar to that of liver in vertebrates (Price, 1973). Further, hemolymph pre-sumably acts as a transitory repository of biochemi-cal constituents, into which the tissues leak out their biochemical constituents and retrieve them when needed. Thus, a dynamic biochemical exchange mechanism seems to operate in silkworm and oth-er insects, facilitating the exchange of substances between the fat-body and hemolymph (Martin et. al., 1971; Noguchi et al., 1974; Sarangi, 1985; Na-gata and Kobayashi, 1990; Ravikumar and Sarangi, 2004). Ultrasound presumably enhances the ab-sorption of soluble proteins from the hemolymph and fat-body. The mechanism by which ultrasound causes changes in silkworm is not yet known. The Increased levels of total, soluble and structural pro-teins in silkworm tissues indicate the growth-pro-moting nature of ultrasound when applied in low-er dosages and indicate a promising future for the sericultural industry. Apparently, ultrasound seems to enhance the protein synthesis in general, with a bias towards the silk proteins in the silk-gland and contractile proteins in the muscle. Interestingly, the greater accumulation of structural proteins in the muscle (332.7%) following the ultrasound treat-

ment deserves some attention, and it is indicative of the promising role of ultrasound in consolidating protein levels in tissues. Such consolidation could provide the required tensile strength to the muscles, facilitating active spinning of the cocoon at the end of 5th instar.

Amino acids are the building blocks of proteins. Ultrasound irradiation caused an elevation in the levels of free amino acids in all the tissues, although to a relatively smaller degree. The silkworm and other lepidopteran insects are known to contain unusually large amounts of free amino acids (Siva Prasad and Murali Mohan, 1990; Sinha et al., 1991). Insect metamorphosis is a dynamic process involving both histogenesis and histolysis (Horie and Watanabe 1983; Anderson 1984). The levels of structural, soluble and total proteins in hemolymph, silk-gland, muscle and fat-body indicate the pres-ence of histolytic activity in different tissues in the 5th instar larvae and the consequent build-up of pro-teins in the tissues. The constructive activity in one tissue appears to match the lytic activity in some other tissue. While histogenesis occurs in silk-gland, muscle and fat-body and proteins accumu-late in the hemolymph, histolysis seems to proceed in other tissues. Obviously, the amino acid pool in the silkworm is derived both from proteins through histolysis and from non-protein sources like carbo-hydrates and lipids through de novo synthesis. In-crease in the levels of free amino acids following ultrasound-treatment is attributable to the synthesis of amino acids from non-protein sources like glu-cose and fatty acids (Bose et al., 1989), although to a smaller degree. Given the importance of silk-worm, it is presumed that amino acids are crucial for the synthesis of fat-body, particularly during larval-pupal transition. It is likely that amino acids are mobilized from other tissues into the silk-gland and fat-body via the hemolymph, as suggested by Noguchi et al. (1974). Through its elevatory ef-fect, ultrasound seems to trigger certain metabolic

Effect of ultrasound on protein metabolism in silkworm


events (Pant and Jaiswal, 1981), such as transam-ination, lipogenesis, maintenance of homeostasis, energy metabolism, formation of hemocytes etc, by actively mobilizing the amino acid pool from hemolymph and fat-body during metamorphosis.

Proteases are a group of proteolytic enzymes that hydrolyze proteins into amino acids (Chen, 1971). Protease activity levels recorded an overall increase during the 5th instar development. Greater enzyme activity was observed in silk-gland followed by muscle, fat-body and hemolymph. Protease activi-ty has been reported in silkworm and other insects (Eguchi and Iwamoto, 1975; Bharathi and Miao Yungen, 2003). Further, ultrasound caused an ele-vation in its activity to varying degrees in different tissues. The presence of non-intestinal protease ac-tivity in silkworm tissues is attributed to its role in proteolysis, characterizing insect metamorphosis (Chen, 1971). The positive impact of ultrasound on enzyme activity indicates its ability to degrade pro-teins by activating proteolytic enzymes. Histolysis seems to be more pronounced in silk-gland, muscle and fat-body as evidenced by increased turnover of amino acids in these tissues. This could probably bring about the degeneration of silk-gland during pupal stage, leaving the fat-body that forms the bulk of pupal weight. The significance of protease activity vis-à-vis muscle degeneration needs to be elucidated with appropriate histochemical and bio-chemical investigations.

Aminotransferases enable the transfer of amino groups of all amino acids except lysine and thre-onine to 2-oxo-glutarate, oxaloacetate and py-ruvate to form glutamate and alanine respective-ly (Lehninger, 1993). The presence of aspartate (AAT) and alanine (AlAT) aminotransferase activ-ity was detected in hemolymph, silk-gland, muscle and fat-body of silkworm as reported in earlier in-vestigations (Pant and Jaiswal, 1981; Bannikov et

al., 1982; Urbesekf 1989; Siva Prasad and Murali Mohan, 1990; Vankata Rami Reddy et al., 1992). Ultrasound caused an elevation in the activity lev-els of both AAT and AlAT in silkworm tissues (Ta-bles 6, 7), indicating increased turnover of amino acids and glutamate-formation during metamor-phosis in the silkworm. The elevation in the levels of free amino acids, although to a moderate degree, observed in the present investigation (Table 4) sup-port this assumption. This indicates the crucial role for aminotransferases in protein synthesis in silk-worm tissues (Fukuda, 1960). The increase in the levels of both total and soluble proteins (Tables 1, 2), vis-à-vis aminotransferase activity (Tables 6, 7) highlights the role of both AAT and AlAT in protein metabolism of silkworm. The impact of ultrasound on aminotransferase activity and pro-tein synthesis is positive, but differs in sensitivity from tissue to tissue. Further, ultrasound seems to enhance protein synthetic activity more through AlAT than through AAT. Presumably AlAT is more sensitive to ultrasound compared to AAT. The role of ultrasound in the manipulation of various met-abolic events like glucogenesis, gluconeogenesis, biological oxidations, histolysis and histogenesis (Pant and Jaiswal, 1981; Venkata Rami Reddy et al., 1992) by elevating the levels of aminotrans-ferases needs to be ascertained. Such an approach could be used to shorten the life cycle of silkworm.

Glutamate dehydrogenase (GDH) is an allosteric enzyme localized mainly in the mitochondrial part of the cell and facilitates the transfer of ami-no groups of amino acids to α-ketoglutarate by transamination, forming L-glutamate with the re-lease of ammonia (Lehninger, 1993). It ensures the availability of α-ketoglutarate to citric acid cycle, and thus connects protein metabolism with car-bohydrate metabolism. Ultrasound showed an el-evatory effect on GDH activity in all the tissues of silkworm (Table 8). Some reports are available


on GDH activity in Bombyx mori (Venkata Rami Reddy et al., 1992). The enhanced activity of GDH in different tissues in the present study is indicative of increased oxidative deamination of glutamate in these tissues. The α-ketoglutarate generated by this enzyme is probably used-up in ensuring sperm mo-bility in silkworm (Osanai et al., 1987). Ultrasound could activate this process and thus may increase the sexual potential of silkworm.

As stated earlier, the mechanism of ultrasound ir-radiation on silkworm is not yet known. However, ultrasound is known to cause an elevation in the temperature, which in turn alters the rate of protein synthesis by optimizing the activity of enzymes (Woeber, 1965). The increased activity levels of aminotransferases, protease and GDH under the impact of ultrasound indicate this possibility. This aspect warrants elucidation in silkworm.

The role of ultrasound in protein metabolism needs special mention in economically viable insects like the silkworm, in view of its profound and positive impact on biochemical parameters. Under its influ-ence the entire biochemical machinery in silkworm is apparently geared up to synthesise silk proteins in silk-gland and contractile proteins in muscle during 5th instar development. While the former are used up as the raw materials for the cocoon, the latter are used for generating a muscular mecha-nism necessary for spinning the cocoon at the end of 5th instar. The increase in the concentration of amino acids with concomitant increase in the lev-els of total, soluble and structural proteins in silk-gland and muscle under the impact of ultrasound reflects this fact. Metabolically, silk-gland seems to occupy a preeminent position during the meta-morphosis in silkworm, closely followed by the muscle. Through active deamination of amino ac-ids, facilitated by AAT, AlAT and GDH, silk-gland presumably meets some of its energy requirements by way of enhanced intermediary metabolism. The

growth and metamorphosis of silkworm are charac-terized by both constructive (histogenesis) and de-structive (histolysis) events, probably triggered by ultrasound, as evidenced by the increased protease activity coupled with increased titers of free amino acids in silkworm tissues. It may be concluded that under the impact of ultrasound protein metabolism is stimulated to achieve greater turnover of silk pro-teins, greater spinning activity, and consequently greater silk output.

CONCLUSIONSExposure of silkworm eggs to appropriate doses of ultrasound in terms of intensity and duration of ex-posure would lead to acceleration of protein metab-olism with reference to protein and free amino acid levels and the activity levels of protease, aspartate and alanine aminotransferases and glutamate dehy-drogenase. The enhancement in protein metabolism would in turn result in the improvement of economic characters of the silkworm, having a positive impact on the profitability of Sericulture.

ACKNOWLEDGEMENTSThe authors are highly thankful to SP Mahila Uni-versity, Tirupati for providing the necessary facil-ities for the investigation. They also express their gratitude to Dr. R.V. Suresh Kumar, Department of Surgery and Radiology, College of Veterinary Sci-ences, Tirupati, for extending his help in providing access to the ultrasound equipment for exposing the silkworm eggs to ultrasound.

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Ethiop. J. Sci. & Technol. 7(2) 85-92, 2014 85


Eyasu Shumbulo Shuba*1, Dominique Adriaens2 and Peter Bossier2

*1Arba Minch University P.O.Box 21, Arba Mich, Ethiopia, E-mail: [email protected] Tel: +251-923-0266992Ghent University Sint-Pietersnieuwstraat 25, B - 9000 Ghent, Belgium

ABSTRACT

High rates of body deformities is one of the most challenging problems in fish culture and is a common source of downgrading the product value of commercially raised fish. The aims of this study are explore the effects of xenic and axenic rearing conditions on the seabass larvae and check whether the rearing condition has a different ef-fect as larvae grow older. The larvae of seabass larvae, Dicentrarchus labrax L. (Teleostea, Moronidae), were reared under xenic and axenic rearing conditions up to fourteenth day after hatching (DAH). The size and shape were analyses for specimens of three age groups: DAH zero, four and 12. The growth comparison was done based on length measurements. For specimens of DAH zero and four, the total length, gut length, predorsal fin length, yolk sac length and height were measured. For specimens of DAH 12 the total length, standard length, gut length, dorsal fin fold length and height, anal fin fold length and height, preanal fin fold length, predorsal fin fold length, eye diameter, head height, hind gut length and notochord diameter were measured. The average measurements for these metric variables were lower in axenic specimens than xenic ones suggesting better performance in xenic treat-ment. The variations in body shape were studied and quantified using geometric morphometrics. The results showed significant differences in shape between the xenic and axenic specimens at all age groups. Thus, the importance of lar-val rearing conditions in determining seabass shape and quality is evident.

Key words/phrases: seabass, aquaculture, body deformity, geometric morphometrics DOI: http://dx.doi.org/10.4314/ejst.v7i2.2

INTRODUCTION The seabass, Dicentrarchus labrax, Linnaeus 1758, is primarily marine fish that sometimes enters brack-ish and freshwaters, tolerating wide range of envi-ronmental conditions: eurythermic (5-28°C) and eu-ryhaline (3‰ to full strength sea water). Historically it was cultured in coastal lagoons and tidal reservoirs before the mass-production of juveniles started in the late 1960s (FAO, 2007). Of all non-salmonid species, the seabass was the first marine species to be commercially cultured in Europe and at present it is the most important commercial fish widely cul-tured.

The high rates of occurrence of body deformi-ties is one of the most challenging problems in fish culture and are a common source

of downgrading the product value of commercially raised fish (Sfakianakis et al., 2006; Verhaegen et al., 2007). Various sources of body de-formities have been identified: pollutants (Bengtsson et al., 1988; Nguyen and Jansen, 2002), temperature (Polo et al., 1991), water currents (Divanach et al., 1997), nutrition (Dedi et al., 1995; Takeuchi et al., 1998; Haga et al., 2002), diseases (Breinholt and Heckmann, 1976), and genetics (Campbell, 1995). In general, according to Sfakianakis et al. (2006) they develop due to the lack of sufficient knowl-edge on the optimum environmental requirements of fish at the different stages of their life. Despite the impressive progress made in the rearing methods, nutrition and disease control, morpho-anatomical ab-normalities continue to affect the hatchery produc-tion (Koumoundouros et al., 2002, Koumoundouros

*Corresponding author© This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/CC BY4.0)

86 Eyasu Shumbulo Shuba et al.

et al., 2004, Planas and Cunha, 1999, Boglione et al., 2003, Cahu et al., 2003 and Sfakianakis et al., 2004). They mainly develop during the early life history of fish, and can affect external morphology, surviv-al, growth rate, behavior and finally production cost and effectiveness of the hatcheries (Divanach et al., 1996, Koumoundouros et al., 1997).

This study was intended to explore the effects of xen-ic and axenic rearing conditions on the seabass larvae in order to reveal the impact of these on the shape of the fish. Moreover, it was designed to see whether the rearing condition has a different effect as larvae grow older.

MATERIALS AND METHODSRearing conditionsEggs were brought from Gravelines, France in big plastic bag and transferred to 20 liter tank. In this tank, the eggs were acclimatized and portion of the eggs were disinfected (3 minutes in 100 ppm gutaraldehyde, 2400 eggs/L) for axenic set ups, so at this point there were xenic and axenic eggs. The disinfected ones were stored in incubation bot-tles for axenic rearing and the remaining eggs were kept in 20 liter tank for xenic treatment. From each treatment, 2 x 96 eggs are individually stored in two 96 multi-well plates: for this, eggs from one incubation bottle are taken. In this way, all eggs are exactly in the same medium, thus reducing the variation within a treatment. The eggs that re-mained in the tank are kept in a 20 L tank. The 20 L tank with UV sterilized water at 16°C was put on top of table for continuous flow-through and the tank was refilled several times a day. Microbiology test was done on samples 24 hrs after disinfection. This is performed on thirty eggs taken from the first eight bottles of incubation, homogenised and plated. To check tubes for contamination, a sample of one ml was taken from each incubation bottle and added to a sterile tube containing 9 ml marine broth (10%). After third day of incubation, the eggs hatched and

stocking was done as of three o’clock in the morn-ing of this day.

The larvae were reared under two setups: axen-ic and xenic. Twelve larvae from each axenic, i.e. the larvae from the disinfection, and xe-nic were stocked in vials containing 10ml of sterile seawater. Xenic larvae are taken from the tank. Axenic larvae are taken from bottles. From each bottle that is used for stocking the lar-vae in the vials, 10 larvae were homogenised and plated on 10% marine agar (MA) to check for axenity. The larval rearing continued up to the 14th day.

Samples A total of 45 samples were taken for each sampling dates. The larvae were anaesthetized with Tricaine

methanesulfonate, (TMS or MS-222) and kept in 8% formalin. After 72 hours the specimens were trans-ferred to 70% ethanol for storing.

Image acquisition

Images were taken using the following procedures:

The specimen was taken with plastic dropper and placed on a glass slide prepared for this purpose by applying hot wax with the use of metal frame heat-ed by Bunsen flame (Figure1). The specimen was washed three times with distilled water while it was on the slide. To this slide with the specimen, a drop or two of 1.6% alizarin solution was added and gently shacked for staining. This was washed three times again with distilled water to remove the re-mains of alizarin. At this time the right positioning of the larvae was checked under a light microscope and nail polish applied to it to fix the position. The specimen stained with alizarin and fixed with nail polish was placed in a series of baths starting from a bath of distilled water to the baths of glycerin at four different concentrations (i.e. 25%, 50%, 75% and 100%) for 30 minutes in each. At the end of bathing period, drops of 100% glycerin were added to the


slides with stained and fixed specimen, and covered with cover glass by applying heat from a hot metal frame. Finally, images were taken with a digital camera (colorveiw 8 SIS) attached to a micro-scope (Olympus 52x9) at 25X magnification with a lens of 0.5X magnification.

Length measurements

For length measurements of specimens from DAH zero and DAH four, TpsDig software was used (Rohlf, 2012). For this software the tps file was cre-ated by using tps file utility program. TpsDig was used for digitizing landmarks for geometric mor-phometric analyses (Figure 2). For DAH 12 spec-imens ImageJ software was used to measure the lengths. For specimens of DAH 0 and 4, five mea-surements were taken (Figure 3): total Length (TL), gut length (GL), yolk sac length (YSL), yolk sac height (YSH), and predorsal fin length (PDL). The yolk sac size was calculated based on the formula of ellipse area.

For specimens of DAH 12, a total of 13 mea-surements were taken (Figure 3) comprising the total length (TL), standard length (SL), gut length (GL), dorsal fin fold length (DFFL), anal fin fold length (AFFL), preanal fin fold length(PAF-FL), predorsal fin fold length (PDFFL), eye diame-ter (ED), head height (HH), hind gut length (HGL), notochord diameter (ND), dorsal fin fold height (DFFH) and anal fin fold height (AFFH).

For shape analyses the elliptic Fourier software was used (Iwata, 2010). This software extracts the contour shape from a full color bitmap image, delineates the contour shape with the elliptic Fou-rier descriptors, and performs a principal component analysis for summarizing the shape information. The original JPG image contours were redrawn manually in corel draw and saved as bitmap file (Figure 4). This was done in order to prepare files for loading in ellip-tic Fourier software so that appropriate contours were considered by the software.

The elliptical Fourier analysis was used to fit spec-imen outlines. A PCA was performed for each new data set. The first three principal component axes were plotted against total length as majority of varia-tion is explained by the first three PCs.

Statistical analyses

The mean, standard deviation, minimum and maximum were calculated from original length data in mm. The original length data were convert-ed to log transformed data in order to see the lin-ear relationships between variables. The regres-sion analyses were performed for log transformed length data to check for correlation and pattern of growth in the metric variables. The ANOVA and MANOVA were done to check for statistical vari-ation between groups. The principal component analyses (PCA) were performed to see the pattern of variation that exists. The CVA scatter plot was given for lengths measurements to identify the measured variable that best explains the differences among the treatment groups.

RESULTS

Growth analyses

While the total length of larvae increased by only 17% in axenic treatments at day 12 post-hatching, the increase was up to 33% in xenic ones. The av-erage value obtained for total length, gut length, yolk sac length and height are higher in xenically treated larvae whereas the average predorsal length was higher in axenically treated larvae at DAH 0 and 4 (Table 1). To test if these differences are statistical-ly significant, an ANOVA was performed. Accord-ingly, the axenic larvae have a significantly smaller total length (F1, 46 = 17.59, p <0.01), gut length (F1,

46 = 37.89, p <0.01), yolk sac length (F1, 46 = 28.64, p <0.01), and yolk sac height (F1, 46 = 17.59, p <0.01). However, the predorsal length was signifi-cantly higher in axenically treated larvae (F1, 46 = 18.32, p <0.01).

88 Eyasu Shumbulo Shuba et al.Ta

ble

1. M

etric

var

iabl

es m

easu

red

(in m

m) f

or la

rvae

of d

ay 0

and

4 a

fter h

atch

ing

Xen

icA

xeni

cD

AH

0D

AH

4D

AH

0D

AH

4M

ean

Min

Max

Mea

nM

inM

axM

ean

Min

Max

Mea

nM

inM

ax

Tota

l len

gth

3.67

±0.

143.

373.

874.

39±0

.35

3.80

5.10

3.48

±0.1

73.

223.

824.

35±0

.11

4.13

4.55

Gut

leng

th2.

02±0

.08

1.81

2.13

2.38

±0.1

1.99

2.74

1.86

±0.1

01.

692.

022.

36±0

.06

2.23

2.50

Yolk

sac

leng

th1.

14±0

.04

1.06

1.21

0.44

±0.0

60.

320.

551.

06±0

.06

0.93

1.19

0.38

±0.0

30.

320.

43

Yolk

sac

heig

ht0.

71±0

.06

0.58

0.86

0.37

±0.0

70.

270.

480.

66±0

.05

0.51

0.74

0.33

±0.0

30.

270.

38

Pred

orsa

l len

gth

0.52

±0.0

70.

440.

690.

44±0

.04

0.35

0.50

0.63

±0.1

00.

460.

870.

45±0

.03

0.37

0.51

DA

H=D

ays

afte

r hat

chin

g

Tabl

e 2

. Met

ric v

aria

bles

mea

sure

d (in

mm

) for

larv

ae o

f day

12

afte

r hat

chin

g

TL

SLG

LD

FFL

AFF

LPA

FFL

PDFL

EDH

HH

GN

DD

FFH

AFF

H

xenic

aver

4.94

4.73

2.75

4.33

2.22

1.88

0.59

0.29

0.70

0.45

0.18

0.30

0.2

Min

4.56

4.31

2.51

3.99

2.05

1.69

0.51

0.23

0.62

0.40

0.15

0.12

0.2

Max

5.26

5.05

2.92

4.78

2.41

1.95

0.63

0.32

0.80

0.50

0.21

0.36

0.3

SD0.

200.

210.

120.

230.

110.

080.

040.

030.

060.

030.

020.

060.

0

Axenic

aver

4.21

3.91

2.23

3.53

1.78

1.46

0.55

0.27

0.62

0.31

0.18

0.19

0.1

Min

3.79

3.39

1.94

3.06

1.50

0.99

0.48

0.22

0.52

0.23

0.15

0.11

0.1

Max

4.64

4.41

2.47

4.08

1.97

1.86

0.73

0.30

0.69

0.37

0.22

0.25

0.2

SD0.

240.

300.

190.

260.

130.

280.

070.

020.

040.

040.

020.

040.

0

TL=T

otal

leng

th, S

L=

Stan

dard

leng

th, G

L=

Gut

leng

th, D

FFL=

Dor

sal fi

n fo

ld le

ngth

,

AFF

L=A

nal fi

n fo

ld le

ngth

, PA

FFL

pre-

anal

fin

fold

, PD

FL=p

re-d

orsa

l fin

leng

th, E

D=E

ye d

iam

eter

, HH

=Hea

d he

ight

, HG

L=H

indg

ut le

ngth

, N

D=N

otoc

hord

dia

met

er, D

FFH

=Dor

sal fi

n fo

ld, A

FFH

=Ana

l Fin

Fol

d


At day 12 after hatching, axenic larvae have smaller average values (Table 2) for almost all metric vari-ables measured. The ANOVA test confirmed the sig-nificant differences for all variables measured except for the diameter of eye (F1, 34 = 2.87, p > 0.01) and the diameter of notochord (F1, 34 = 0.27, p >0.01).

The log transformed data of several metric variables over total length measurements of xenic and axenic specimens of DAH 4 were used for the purpose of

A C

B D

A= xenic DAH 4; B= Axenic DAH4; C= xenic DAH12 and D= Axenic DAH12

Figure 1. Plot of log transformed data of some metric variables over total length measurements

(Figure 2) for length data for both DAH0 and DAH4

reveals that the predorsal and yolk sac length are

better discriminators between the xenic and axenic

groups. But for day 12 after hatching total length,

anal fin fold and hind gut length are better discrim-

inators between the two treatments as revealed by

the CVA scatter plot (Figure 2).

comparison. The gut length exhibited a strong pos-itive correlation (R2 = 0.85) with respect to total length in xenic larvae of day 4 after hatching but the case is not true for axenically treated larvae. In xenic specimens the gut growth exhibited isometric growth patterns with the slope of the regression line 0.89, whereas negatively allometric in axenic larvae. Other metric variables displayed positive allometric growth pattern (Figure 1) with total length in both

treatments except the yolk sac height which is negatively allometric in axenic specimens (slope = -1.06).

The differences in the metric variables among the two treatments were statistically signifi-cant in all age groups as tested by MANOVA (F1, 46 = 86.32, p < 0.01), (F1, 49 = 31, p < 0.01) and (F1, 34 = 501.1, p < 0.01) in DAH0, DAH4 and DAH12, respectively. The CVA scatter plot


Analyses of shape

While most of the variations in shape within groups of treatments at day zero post hatching are observed at head region by folding upward or downward (Fig-ure 3 A and B), in larvae of day four after hatching it involves variation in the height of the larvae fol-

lowed by the bending at the head region (Figure 4 C and D). In the age group of day 12 after hatching, PC1 reflects bending whereas PC2 larval height, with the bending being more prominent in axenic larvae (Figure 3 E and F)

DAH0

DAH4

DAH12

Figure 2. The CVA scatter plot for metric variables of the specimens ( xenic = red: + and axenic = blue: □)

A

E

B

F

C

D

Figure 3. PC contours: A=DAH0-xenic, B=DAH0-axenic, C=DAH4-xenic, D=DAH4-axenic,

E=DAH12-xenic, F=DAH12-axenic


DISCUSSION

The average total length obtained in this study for xenic specimens were 3.67 ±0.14, 4.39 ±0.34 and 4.94 ± 0.19 mm at day 0, 4 and 12 after hatch-ing, respectively (the axenic ones are significantly lower than xenic). These values are slightly small-er but comparable with previously reported total length. Saillant et al. (2001) reported the total length of 8-day-old larvae ranged between 4·73 and 5·26 mm. Hatziathanasiou et al. (2002) reported a total length of 5.12 ±0.00 mm for a week old larvae after hatching; Anonymous (2004) about 4.5 mm at day 2 after hatching (this same author reported 4 mm total length for seabream at day 4 after hatching). The relatively smaller total length observed in this study may be due to low quality parents. Saillant et al. (2001) reported that egg size and total length were subjected to the influence of the female. These authors also noted that fertilization, survival during incubation and hatching were positively correlated and strongly influenced by the female parent and the interaction between both parents of the seabass.

In all the three age groups considered in this study, the gut length showed a strong positive correlation with respect to total length, except for the axenic larvae of day four after hatching. The growth pattern is nearly isometric. The differ-ence in growth pattern of gut length of axenic larvae of DAH four may be attributed to the relative low re-sorption of yolk sac and concomitant low total length. The yolk sac size diminished successively as lar-vae gets older age. These indicate the decrease in yolk sac size was also evident with increasing age as there is no exogenous feeding and the larvae depend on yolk sac as source of food at early stage of growth. This is in line with the Maneewongsa and Tat-tanon (1982) hypothesis that the rate of resorp-tion of the yolk is shown by decreasing diameter at older ages. According to Rønnestad (1998) the yolk is quickly resorbed during the embryonic and the early larval stages compared to later stages and

95% is depleted by 100 hours post fertilization in the seabass. Thus, the yolk sac exhibited negatively allo-metric growth pattern.

CONCLUSION

Based on this study we conclude that the axenic rearing conditions render a better survival but a larger deformity (folding) in-cidence of larvae. The folding of larvae is prominent at older age of axenically treat-ed ones as indicated by the PCA contours. The egg quality is most important for larval quality. In this study the low egg quality is evident from the observed low hatching rate and, later low survival of larvae and has resulted in higher bending of larvae. We sug-gest that the eggs are obtained from a single selected spawnners (not from wild as in this study) and same batch to compare the xenic and axenic conditions. Further investigations that consider more age groups from the same spawnners and the same batch is recommended to establish concrete knowledge about the effects of axenic rearing con-ditions on larval quality especially at later ages.

ACKNOWLEDGMENTS

I would like to gratefully acknowledge the invaluable cooperation of the staff of Evolutionary Morphol-ogy of Vertebrates, and Laboratory of Aquaculture and Artemia Reference Center, Ghent University. I am thankful to the Flemish Interuniversity Council (VLIR) for funding the study.


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Iwata, H. (2010). Elliptic Fourier version 1.3Hatziathanasiou, A., Paspatis, M., Houbart, M.,

Kestemont, P., Stefanakis, S and Kentouri M. (20020. Survival, growth and feeding in early life stages of European sea bass (Dicentrarchus labrax) intensively cultured under different stocking densities. Aquaculture 205: 89-102.

Koumoundouros, G., Oran, G., Divanach, P., Stefa-nakis, S and Kentouri, M. (1997). The opercu-lar complex deformity in intensive gilthead sea bream (Sparus aurata L.) larviculture: Moment of apparition and description. Aquaculture 156:165–177

Koumoundouros, G., Maingot, E., Divanach, P and Kentouri, M. (2002). Kyphosis in reared sea-bass (Dicentrarchus labrax L.): ontogeny and effects on mortality. Aquaculture 209: 49-58.

Nguyen, L.T.H and Janssen, C.R. (2002). Em-bryo-larval toxicity tests with the African cat-fish (Clarias gariepinus): Comparative sensi-tivity of endpoints. Archives of Environmental Contamination and Toxicology 42: 256-262.

Maneewongsa, S and Tattanon, T. (1982). Nature of eggs, larvae and juveniles of the seabass In: FAO Corporate Document Repository; Re-port of training course on seabass spawning and larval rearing songkhla, Thailand.

Polo, A., Yufera, M and Pascual, E., (1991). Effects of temperature on egg and larval development of Sparus aurata L. Aquaculture 92:367–375.

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Rønnestad, I., William, K., Amos, T., Mordechai, H and Hans J. F. (1998). Utilization of yolk fuels in developing eggs and larvae of European sea-bass (Dicentrarchus labrax). Aquaculture 162: 157-170.

Saillant, E., Chatain, B., Fostier, A., Przybyla, C and Fauvel, C. (2001). Parental influence on early development in the European seabass. Journal of Fish Biology 58: 1585-1600.

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Assessment of antioxidant potential of Moringa stenopetala leaf extract

Tesfaye Tebeka1 and Solomon Libsu* 2

1Department of Chemistry, Arba Minch University, Arba Minch, Ethiopia; E-mail: [email protected]

2Department of Chemistry, Bahir Dar University, Bahir Dar, Ethiopia; E-mail: [email protected]

ABSTRACTThis study was conducted to assess the antioxidant potential of Moringa stenopetala leaf obtained from a private gar-den in Bahir Dar City and powdered Moringa leaf purchased from a supermarket in Bahir Dar City by using ferric reducing antioxidant power, 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity, peroxide value and conjugated diene hydroperoxide assays. The powdered Moringa stenopetala leaf extract was invariably found to have a higher antioxidant capacity than the purchased Moringa powder. In the conjugated diene hydroperoxide and peroxide value assays, sunflower oil was used as an oxidation substrate. Both peroxide value and conjugated diene concentration for sunflower oil containing extracts of Moringa leaf and purchased Moringa powder were found to be lower than the corresponding values observed for the control showing the effectiveness of the extracts in delaying oxidation of the oil. The total phenolic content, in terms of mg gallic acid equivalent per 100 g of dry weight of sample was found to be 92.8 ±1.01 and 75.5 ± 2.28 for, respectively, powdered leaves of M. stenopetala and purchased powder of Moringa leaf. The antioxidant capacity of the powdered Moringa stenopetala leaf and purchased Moringa powder were found to be, respectively, 442.0±10.58 and 291.3±15.52 mg of ascorbic acid equivalent per 100 g of dry weight of plant sam-ple in the FRAP assay. The corresponding values for the powdered Moringa stenopetala leaf and purchased Moringa powder in the DPPH assay were found to be, respectively, 144.0±0.53, 138.8±1.05 mg of ascorbic acid equivalent per 100 g of dry weight of the sample. This difference in antioxidant capacity of these two samples can be attributed to differences in their total phenolic content. It is suggested that this antioxidant potential of the leaves of Moringa steno-petala may underlie the widespread use of the plant in traditional medicine.

Key words: Moringa stenopetala, Antioxidant potential, Ferric reducing antioxidant power (FRAP), 2,2-diphe-nyl-1-picrylhydrazyl (DPPH) radical, Folin-Ciocalteu’s phenol reagent (FCR), Peroxide value (PV).DOI: http://dx.doi.org/10.4314/ejst.v7i2.3

as significant interlinking risk factors that cause the majority of these diseases (WHO, 2003).

It is now widely believed that the main causes of these diseases are highly reactive chemical species known as free radicals. Notable among the free radi-cals are the reactive oxygen species (ROS) such as superoxide anion radical (O2¯

• ), hydroxyl radical (HO•) as well as the non-radical molecule hydro-gen peroxide (H2O2), all of which occur in the body either as a result of normal metabolic processes or enter the body from external sources. Cell damage caused by free radical-induced chain reactions ap-

INTRODUCTIONThe rapid rise of degenerative diseases is threat-ening economic and social development as well as claiming the lives of millions of people worldwide. It represents a major health challenge to mankind in the coming century. It is estimated that up to 80% of cardiovascular diseases, 90% of Type II diabetes, and one third of cancers can be avoided by chang-ing life-style, including diet (Stampfer et al., 2000; Hu, 2001; Key, 2002). Diet-related high cholesterol, high blood pressure, obesity, and insufficient con-sumption of fruits and vegetables have been cited


94 Tesfaye Tebeka and Solomon Libsu

pears to be a major contributor to aging and degen-erative diseases such as cataracts, immune system decline, brain dysfunction, cancer and many more (Halliwell, 1994; Halliwell, 1999; Temple, 2000; Lee et al. 2004; Sharma and Verma, 2013).

The buildup of ROS in the body is counteracted by various beneficial compounds known as anti-oxidants which stabilize or deactivate free radicals thereby safeguarding cells from oxidative damage (Tyagi et al., 2010). The health-beneficiary effect of antioxidants is ascribed to their ability to break up the free radical-induced chain reactions by provid-ing a hydrogen atom or an electron to the free radical and receiving the excess energy possessed by the ac-tivated molecule (Osawa and Namiki, 1981).

Several studies have shown that a number of plant products including polyphenolic substances (e.g. flavonoids and tannins) and various plant or herb extracts exert antioxidant actions (Osawa and Na-miki, 1981; Yen et al., 1996; Thea, 2012). The Mo-ringa species like M. stenopetala, M. peregrine, M. oleifera and M. concanensis are currently of wide interest because of their outstanding potential as nutritious vegetables, antibiotics and water clarifica-tion agents (Booth and Wickens, 1988; Nikon et al., 2003).

Moringa stenopetala, also known locally, amongst others, as Shiferaw or Aleko, belongs to the family Moringaceae which consists of a single genus, Mor-inga, which has about 14 different species (Edwards et al., 2002). The plant is native to the Horn of Af-rica particularly in Southern Ethiopia, North Kenya and Eastern Somalia (Mohammed Adefa, 2013). Of the 14 different species of Moringa the antioxidant activity of M. oleifera is extensively studied (Nwosu and Okafor, 1995).

Reported health benefits of M. stenopetala include its use for the treatment of ailments such as hyper-

tension, asthma, diabetes, and stomach pain as well as antifertility and antileishmanial effect (Yalemtse-hay Mekonnen and Amare Gessesse, 2002; Yalemtse-hay Mekonnen, 1999) and water clarification (Zen-ebe Shiferaw, 2011). However, a search into the literature made using the software SciFinder did not reveal any study made on the anti-oxidant capacity of the leaves of M. stenopetala grown in Ethiopia. In light of the expanding use of M. stenopetala in folk medicine, it is felt desirous to look into the antiox-idant capacity of its leaves using a variety of stan-dard methods as well as to investigate the relation-ship between antioxidant capacity and total phenolic content, which are believed to safeguard the body against pathogens and free radicals.

MATERIALS AND METHODSGallic acid, Folin-Ciocalteu’s phenol reagent (FCR), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical are all Sigma Aldrich products. Hydrochloric acid, po-tassium iodide and ferric chloride were obtained from BDH, England. Glacial acetic acid (NICE, laboratory reagent), chloroform (WINLAB limited), vitamin E, o-phosphoric acid (Fisher Scientific Lim-ited, UK) were used in this work. Sunflower oil used as an oxidation substrate was obtained from a local small-scale edible oil producer in Bahir Dar City, Ethiopia. All other chemicals and solvents were pro-cured from Blulux Laboratories Pvt. Ltd., India. All the chemicals used were of analytical grade and dis-tilled water was used throughout the experiments. Double Beam Perkin Elmer Lambda 35 UV- Visible and Single Beam UV- Visible (NV2003) spectropho-tometer were used for absorbance measurements.

Sample collection and preparationLeaves of Moringa stenopetala were collected from a garden in Bahir Dar City in February 2014 and fur-ther identified by comparison with a sample previ-ously authenticated (Edwrd et al., 2002). Soon after


collection, the leaves were wrapped up with alumi-num foil, taken to the laboratory and washed with tap water and then with distilled water in order to re-move dirt matter. The leaves were then air-dried for 10 days at room temperature away from the reach of sunlight. The dried samples were ground and 50 g of the resulting powder was placed in cellulose cone and defatted with petroleum ether (300 mL) for 7 h using a soxhlet extractor. The purpose of defatting is to remove fatty acids and lipids which will other-wise interfere with the intended antioxidant study. The residue was separated using suction filtration, dried and kept in air-tight container until use. In a parallel experiment, a 50 g portion of a green packed powder purchased from a supermarket in Bahir Dar City and claimed to be ground Moringa stenopetala leaves was defatted as described above. Residue was separated using suction filtration and dried at room temperature for one day.

The defatted powdered samples (20 g) were sepa-rately mixed with 200 mL of 80% aqueous metha-nol in Erlenmeyer flasks, completely wrapped with aluminum foil, and gently shaken (120 rpm) by an electrical shaker at room temperature for 24 hours and filtered. The filtrates were kept in dark place for further analysis.

Total phenolic content determinationThe amounts of phenolic compounds in the aqueous methanol extract of powdered Moringa stenopeta-la (MS) and purchased Moringa (MP) powder were determined by Folin-Ciocalteu reagent assay. To 1 mL of the extract (800 mg/L), 2 mL of Folin- Ciocal-teau reagent and 5 mL of distilled water were added. The mixture was kept at room temperature for 10 min, and then 20% sodium carbonate (w/v, 2 mL) was added and heated on a water bath at 40oC for 20 min and then cooled in an ice bath. Absorbance was read at 755 nm. The total phenolic content was calculated and expressed as gallic acid equivalent in

mg/g (GAE) per 100 g of dry weight of the plant matter (Dehshahri et al., 2012).

Ferric reducing antioxidant power (FRAP) assay The reducing power of the extracts was determined according to a literature procedure (Atawodi et al., 2010; Jayanthi and Lalitha, 2011). Variable concen-trations (200-1200 mg/L) of extracts (2.5 mL) were mixed with sodium phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide (2.5 mL, 1.0%). The mixtures were incubated at 50oC for 20 min. Then 10% trichloroacetic acid (2.5 mL) was added and centrifuged at1000 rpm for 10 min. The upper layer of the solution (5.0 mL) was decanted and diluted with 5.0 mL of water. Ferric chloride (0.1 mL, 0.1%) was added and absorbance was read at 700 nm. The antioxidant capacities of the extracts in terms of AAE (mg AAE per 100 g of dry weight of sample) were determined. Ascorbic acid was used as a standard in the range of (25-400 mg/L).

DPPH radical scavenging assay DPPH radical scavenging by Moringa extracts were estimated according to a previously reported meth-od (Irshad et al., 2012). 2 mL of DPPH solution in methanol (0.004%, 0.102 mM) was mixed with 2 mL of extracts with different concentrations (200-800 mg/L). For blank solution, the extracts were substituted by methanol and used for the correction of baseline at 515 nm. The tubes were allowed to stand at room temperature for 20 min. The anti-rad-ical activity was based on measurement of the reducing ability of the plant extract toward DPPH radical. Ascorbic acid was used as a standard in the range of (25-100 mg/L) and the scavenging effect of the leaf extracts were determined with a linear curve

of ascorbic acid standard.

Oil storage studies

The storage tests and the storage methods were car-

ried out as described by Chang et al. (2013) on re-

fined sunflower oil. The methanolic extracts of both


plant materials were separately added to pre-heated

(50°C) 100 mL refined sunflower oil at concentra-

tions of 1000 and 2000 mg/L. The oil samples were

stirred with a magnetic stirrer for 30 min at 400C

for uniform dispersion. Vitamin E (1000 mg/L) was

used as reference substance for comparative purpos-

es and a control set without added extracts was pre-

pared under the same set of analytical conditions.

The oils were then stored in an oven at 65°C for 6

days (to accelerate the deterioration of the oil) and

at room temperature for 18 days. Approximately 25

mL of the oils were withdrawn and pipetted in to

Erlenmeyer flask at intervals of 0, 2, 4 and 6 days

and 0, 6, 12 and 18 days after storage in oven and

at room temperature, respectively, for monitoring

peroxide value and conjugated dienes (at 232 nm)

resulting from the oxidative changes.

Peroxide value determination

Peroxide value was evaluated according to stud-

ies reported elsewhere (White and Crowe, 2001).

Thus, 5 g of oil with or without added antioxidant

(Moringa leaves extracts and vitamin E) and 30 mL

of the solvent (glacial acetic acid-chloroform, 3:2)

were added into a 250 mL glass-stoppered Erlen-

meyer flask. The solution was swirled until the sam-

ple is dissolved. Fresh 0.5 mL saturated KI (10%)

solution was added and the mixture was shaken for

l min. Thereafter, 30 mL of water was added and

titrated with 0.01N Na2S2O3 solution using 0.5 mL

of 1% starch indicator until the yellow color was

discharged. A blank was prepared alongside the oil

samples.

Statistical Analysis

All the experiments were performed in triplicates

and the data have been presented as Mean ±

Standard deviation. The significance of the data

was assessed using one way ANOVA with sig-

nificance level set as r 0.05 by applying Tukey’s

post hoc test using SPSS 20 software package (IBM

Corporation,1989) and graphs as well as regression

coefficients were displayed using Origin 7 and Excel

2007.

RESULTS AND DISCUSSIONTotal phenolic contents

Appearance of a blue colored solution due to the

reduction of phospho-molybdic and phospho-tung-

stic acids contained in the Folin-Ciocalteu reagent

indicates the presence of phenolic compounds in

the samples (Rusak et al., 2008). Absorbance of the

blue complex was measured at 755 nm. To estimate

the total phenolic content, a calibration curve of gal-

lic acid absorbance (at 755 nm) versus concentration

was constructed (y = 3.745x + 0.0188; R2 = 0.99812).

Total phenolic content of the aqueous methanol ex-

tract of powdered leaves of M. stenopetala and the

purchased Moringa powder, expressed as mg gallic

acid equivalents (GAE) per 100 g dry weight (DW)

of the Moringa material, were found to be 92.8±1.01

and 75.5±2.28 mg, respectively (Table 1). These re-

sults are within the range of the values (68.8-200 mg

GAE/100 g DW) reported for the methanolic extract

of different parts of M. oleifera in winter and sum-

mer seasons (Shih et al., 2011). Reports in the liter-

ature reveal that the drying conditions such as the

drying temperature and circumstances might induce

changes in the composition of chemical constituents

which, in turn, might affect the antioxidant activities

of plant materials. Very high phenolic content from

Moringa oleifera species in freeze-dried sample was

reported (Siddhuraju and Becker, 2003). In contrast

to this, a significant decrease of up to 33% in the to-


tal phenolic contents of tomatoes when dried at 42oC

was reported by Kerkhofs and coworkers (2005).

The amount of phenols in fruits and vegetables are

also affected by genetic factor, environmental condi-

tions, such as light, temperature and growing season

and analytical methodology (Wasim et al., 2013).

The antioxidant effects of the Moringa samples in-

Table 1: Total phenolic contents of MS and MP leaf extract (80% methanol extracts)

Moringa samples TPCa mg GAE/100 g d w

Moriniga stenopetala 92.8 ±1.01b

Purchased Moringa powder 75.5 ± 2.28b

Values are means of triplicate determination of mean ± SD, there was no significant difference in the mean mg GAE/100g of the samples with same superscript at p<0.05.TPCa is total phenolic content.

vestigated in the present study can be ascribed to

their phenolic constituents whose antioxidant char-

acter is reported to emanate from their ability to do-

nate hydrogen atoms and chelate metal ions (Costa

et al., 2012; Shahriar et al., 2013).

Ferric Reducing Antioxidant Power

The ferric reducing antioxidant power assay mea-

sures the ability of antioxidants in the Moringa sam-

ples to reduce potassium ferricyanide, K3[Fe(CN)6],

to potassium ferrocyanide, K4[Fe(CN)6]. Addition

of free Fe3+ to the reduced product leads to the for-

mation of ferric ferrocyanide, Fe4[Fe(CN)6]3, also

known as Perl’s Prussian blue complex which has a

strong absorbance at 700 nm. In this regard, increase

in Fe3+ to Fe2+ transformation in the presence of test

sample implies that the sample contains electron-do-

nating compounds and can thus cause reduction of

the oxidized intermediates (Sayed et al., 2011).

As shown in Figure 1, an increase in absorbance of

the reaction mixture is accompanied by an increase

in the reducing capacity due to an increase in the

formation of the complex. This observation suggests

that some polyphenolic compounds in the sample

extracts are electron donors. Similar increase in re-

ducing power with increase in concentration was

observed in a study conducted on methanolic

extracts of pulp and seed of Cassia fistula (Irshad

et al., 2012). It has been reported that crude pheno-

lic extracts of blueberry leaves displayed a con-

siderable reducing power, primarily due to their

effect as electron donors and thereby halting

radical chain reactions by converting free radi-

cals to more stable products (Naczk et al., 2003).

The ferric reducing capacity of MS extract was ob-

served to be higher than MP (Table 2). The higher

antioxidant activity of MS leaf extract might be at-

tributed to its higher phenolic contents relative to

the MP extract as described in the aforementioned

FC assay.

Free radical scavenging activity (DPPH) Scavenging of the stable 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical, which shows a strong absorption at 515 nm, is a widely used method to


evaluate antioxidant activities in a relatively short time compared with other methods (Rajesh et al., 2011). Substances capable of donating hydrogen atoms or electrons are able to convert DPPH (Pur-ple) free radical into the non-radical reduced form, a reaction that can be monitored spectrophotomet-rically (Rajesh et al., 2011). The free radical scav-enging activity of the extracts in the present work was thus also evaluated by investigating their ability to quench the DPPH free radicals through donation of hydrogen atoms or electrons. The bleaching of DPPH color is indicative of the capacity of the test extract to scavenge free radicals.

The scavenging effects of extracts increased with their concentration and the percentage inhibitions values are presented in Table 3. The present study showed that scavenging of DPPH radicals by aque-ous methanol extracts of powdered Moringa leaves occurred in a dose-dependent manner, an observa-tion that has also been reported for leaf extracts of M. oleifera (Kumar et al., 2012) and M. peregrine (Dehshahri et al., 2012).

In the present study, the scavenging ability of the aqueous methanol extract of purchased Moringa powder (MP) was found to be lower than that of

Table 2: Antioxidant activities of Moringa leaf extract in reducing power assay

Moringa sample AOAa in terms of mg AAEb/100 g dry weightMSMP

442.0±10.58291.3±15.52

Values are mean ± SD for triplicate determinations AOAa is antioxidant activity, AAEb is ascor-bic acid equivalent.

Figure 1: Absorbance versus concentration of extracts in reducing power assay.

(MS: Moringa stenopetala; MP: Moringa powder purchased).


powdered M. stenopetala (MS) leaves extract (Table 4). This is also in line with the higher total pheno-lic content of powdered M. stenopetala (MS) leaves extract relative to that of purchased Moringa pow-der (MP) extract. The difference in the proportion of phenolic compounds between the two plant samples can be traced to differences in their drying condi-tions as well as differences in the time lapse between collection of the plant leaves and antioxidant studies.

Correlation between total phenol content and antioxidant capacityThe extents of antioxidant capacity of the Moringa extracts were correlated with their total phenolic content. A linear correlation appeared between the antioxidant capacity and the total phenolic content of the extracts (R2 = 0.98628, 0.99033 for FRAP and DPPH, respectively) suggesting that the antioxidant capacity may be due to the phenolic compounds as well as other constituents like flavonoids present in

Table 3: Antioxidant activity of MS leaf and MP extract with different concentra-tions against DPPH radical.

Concentration of extracts (mg/mL) % inhibition of MS % inhibition of MP

0.2 54.8±0.09a 51.4±0.29a

0.4 75.5±1.90b 71.2±0.54b

0.6 80.8±0.17c 78.4±0.34c

0.8 85.8±0.49d 83.6±0.45d

Values are mean ± SD of triplicate analysis. Different superscript letters within columns showed significant differences at (P<0.05), MS: Moringa stenopetala leaf, MP: Moringa leaf purchased.

Table 4: The antioxidant activity of Moringa leaf extract in mg of AAE/100g DW in DPPH.

Moringa sample AOA in terms of mg AAE/100g dry weight MS 144.0±0.53MP 138.7±1.05

Values are mean ± SD of triplicate analysis.

the Moringa leaves extracts. Flavonoids like querce-tin-3-O-rhamnoglucoside and quercetin 3-O-gluco-side have been reported as constituents of M. steno-petala (Bennett et al., 2003).

Stabilization of sunflower oil by extract of Moringa leaves

Peroxide value (PV) Peroxide value (PV) is often used as a measure of oxidation status of oils, fats and fatty foods (Bushra

et al., 2007). Table 5 (a and b) depicts the relative in-crease in peroxide value of sunflower oil (SFO) con-taining extracts of powdered leaves of M. stenopetala (MS), purchased Moringa powder (MP) and vitamin E kept at 650C and at room temperature for six and eighteen days, respectively. The highest PV was ob-served for the control sample (C-SFO) while lower peroxide values were observed for SFO containing extracts from Moringa and vitamin E at the end of the experimental period in both conditions, indicat-


ing a higher rate of oxidation for the control sample. A slow rise in the PV of the oil containing the plant extracts as compared to those of the control clearly indicates that the compounds in the plant extracts are able to deter oil oxidation. It may be noted from Table 5 (a and b) that Moringa leaf extract is able to prevent oxidative deterioration of sunflower oil to a comparable extent as the familiar antioxidant vita-min E, a finding in agreement with reported data for M. oleifera (Akhtar and Tsao, 2005; Atawodi et al., 2010). In a related study, vitamin E was shown to be a poor protector against oil oxidation compared with ethanol extracts of grape waste whose pheno-lic constituents are reported to be responsible for the antioxidant behavior (Theodora-Ioanna et al., 2007).

Table 5: Relative increase in peroxide value (PV) of SFO stabilized with the extracts of MS, MP and vitamin E under room temperature and oven (650C) storage.

a) Peroxide value of SFO stored at 650Cfor six daysST C-SFO Vit ES-SFO MSS-SFO MS*S-SFO MPS-SFO MP*S-SFO

0 5.2±0.56a 5.2±0.56b 5.2±0.56c 5.2±0.56d 5.2±0.56e 5.2±0.56f

2 11.07±0.61a 7.5±0.83b 7.3±1.00c 7.2±2.11d 7.5±2.00e 7.3±1.89f

4 18.3±1.00a 12.3±0.61b 11.87±1.2c 10.8±1.44d 12±0.35e 11.5±0.70f

6 28.5±1.02a 17.3±2.01b 17±2.58c 15.2±1.83d 17.3±2.4e 15.3±0.42f

% inhibition 47.9 49.4 57.0 47.9 56.5b) Peroxide value of SFO stored at room temperature for 18 days

ST C-SFO Vit ES-SFO MSS-SFO MS*S-SFO MPS-SFO MP*S-SFO

0 5.2±0.56a 5.2±0.56b 5.2±0.56C 5.2±0.56D 5.2±0.56E 5.2±0.56f

6 6.6±0.20a 6.27±1.90b 6.2±0.53c 6.1±0.94d 6.3±0.61e 6.1±1.30f

12 8.3±0.85a 7.5±2.20b 7.1±1.70c 7.0±1.40d 7.3±0.42e 7.1±0.28f

18 9.9±0.60a 8.8±0.40b 8.4±0.80C 8.3±0.80D 8.5±0.61E 8.3±0.57f

% inhibition 22.9 31.4 34.3 28.67 32.9

Values in the same column that are followed by different letters (a-f) are significantly different p < 0.05 in table a, and in table b (a-f) are insignificant p < 0.05 but capital letters in each column are significant at p<0.05 by Tukey’s multiple range tests. ST is storage time, C-SFO is control sunflower oil ,vit ES-SFO is vitamin E sta-bilized sunflower oil, MSS-SFO is Moringa stenopetala at 1000 mg/L stabilized SFO , MS*SSFO is Moringa stenopetala at 2000 mg/L stabilized SFO , MPS-SFO is Moringa purchased at 1000 mg/L stabilized SFO and MP*S-SFO is Moringa purchased at 2000 mg/L stabilized SFO.

a) Peroxide value of SFO stored at 650Cfor six days.b) Peroxide value of SFO stored at room temperature for 18 days.

Conjugated dienes (CD)

Free radicals formed by abstraction of a hydrogen

atom from a bis-allylic position of unsaturated fatty

acids readily undergo allylic rearrangement to pro-

duce conjugated dienes (Chatha et al., 2006, Valko,

et al., 2006). Molar absorptivities or molar extinc-

tion coefficients pertaining to absorption at 232 nm

by conjugated dienes can be measured for their de-

termination. As can be seen from Table 6, almost all

of the stabilized SFO samples showed the formation

of CD to a significantly lower level than the control,

thus reflecting the antioxidant efficacy of the inves-

tigated Moringa leaf extracts.


CONCLUSION

The present study has shown that aqueous meth-

anol (80%) extract of the powdered leaves of M.

stenopetala is able to scavenge DPPH radicals, re-

duce K3[Fe(CN)6] to K4[Fe (CN)6]

(reducing power

ability) and inhibit oxidation of sunflower oil. Low-

er values of PV and CD contents for sunflower oil

containing M. stenopetala leaf extract relative to the

control indicated effectiveness of the extract in pre-

venting oxidation of the oil. It has also been noted

in the present study that the ability of Moringa leaf

extracts to deter oxidative deterioration of sunflower

oil is comparable to that of the familiar antioxidant

vitamin E. This property of the extract might be

due to the presence of polyphenolic compounds that

can act as natural antioxidants. This prompts further

study to look into the feasibility of using this plant

material as a natural antioxidant to protect, for in-

stance, edible oils from oxidative deterioration. The

antioxidant activity of aqueous methanol extract of

M. stenopetala leaf documented in the current study

may underlie the widespread use of the plant in folk

medicine. However, further work is in order to es-

tablish the medicinal value traditionally associated

with the leaves of this plant.

ACKNOWLEDGMENTS

Tesfaye Tebeka is thankful to Arba Minch Univer-

sity and Bahir Dar University for granting him a

study leave and financial assistance, respectively.

The assistance of Dr. Ali Seid of the Department of

Biology, Bahir Dar University, in the identification

and authentication of the plant species is gratefully

acknowledged.

Table 6: Relative increase in conjugated dienes of SFO stabilized with extracts of MS, MP and vitamin E.

a) Conjugated dienes (1% ε1cm (λ=232 nm) of SFO stored at 650C for six days

St C-SFO Vit ES-SFO MSS-SFO MS*S-SFO MPS-SFO MP*S-SFO

0 0.6±0.07a 0.6±0.07b 0.5±0.06c 0.5±0.04d 0.55±0.04e 0.549±0.40f

2 1.9±0.03a 1.5±0.08b 1.4±0.02c 1.2±0.006d 1.44±0.06e 1.28±0.05f

4 3.4±0.00a 2.5±0.09b 2.3±0.05c 1.98±0.02d 2.35±004e 2.01±0.005f

6 6.4±0.06a 3.9±0.05b 3.5±0.02c 3.17±0.15d 3.6±0.009e 3.32±0.002f

b). Conjugated dienes (1% ε1cm (λ=232 nm) of SFO stored at room temperature for 18 days.

ST C-SFO Vit ES-SFO MSS-SFO MS*S-SFO MPS-SFO MP*S-SFO

0 0.6±0.066a 0.6±0.07b 0.6±0.07c 0.6±0.07d 0.6±0.07e 0.6±0.07f

6 1.6±0.01a 1.3±0.01b 1.2±0.16c 1.0±0.008d 1.2±0.008e 1.0±0.02f

12 2.9±0.133a 2.19±0.011b 1.97±0.021c 1.88±0.004d 2.00±0.01e 1.9±0.01f

18 4.3±0.30a 3.4±0.001b 3.3±0.04c 3.2±0.007d 3.3±0.01e 3.3±0.02f

Values in the same column that are followed by a different letters (a-f) are significantly different p < 0.05 by Tukey’s multiple range tests.

a) Conjugated dienes (1%ε1cm (λ=232 nm) of SFO stored at 650C for six days.b) Conjugated dienes (1%ε1cm (λ=232 nm) of SFO stored at room temperature for 18 days.


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Ethnobotanical study of nedicinal plants in Ankober woreda, central Ethiopia

Daniel Kalu1 and Ali Seid*2

1,2Department of Biology, Bahir Dar [email protected], Cell Phone: (251) 0918767869

ABSTRACTMedicinal plants’ diversity and associated indigenous knowledge in the Ankober district, central Ethiopia was studied from November, 2011 to May, 2012 using Ethnobotanical study approach. A total of 165 randomly selected informants aged 18 to 90 years were randomly selected from nine peasant associations (PAs) or kebeles. Of these, 34 were pur-posively selected as key informants. Data were collected using a semi-structured questionnaire, field observations and Focused Group Discussion (FGD). A total of 109 medicinal plant (MP) species from 56 families were described from the natural vegetation (60.55%) and home gardens (24%). Eighty three (76.15%) medicinal plant species were used only to cure human diseases. Asteraceae and Lamiaceae were the two families containing the most cited species. MPs with high informants’ consensus (HIC) were: Maesa lanceolata, Foniculum vulgare, Croton macrostachyus, Calot-ropis procera and Grewia ferruginea. Scabies, ring-worm and leishmaniasis were the top common diseases treated using the traditional use of medicinal plants. Knowledge of indigenous MP use significantly correlated increase with age. The existing common threats were identified by participatory approach. Awareness creation, motivating tradition-al healers to wisely use medicinal plants and availing their knowledge through proper negotiations are recommended.Key words: Amhara Region, Ethiopia, Ethnobotany, Ethnomedicine, Medicinal PlantsDOI: http://dx.doi.org/10.4314/ejst.v7i2.4

In Ethiopia, about 70% of human and 90% of live-stock population depend on traditional medicine (En-dalew Amenu, 2007).In many developed countries, increased use of com-plementary and alternative medicine (CAM) indi-cates that factors other than tradition and cost are at work (WHO, 2002). The progress made in medicinal plant use has identified plants serving as drug precur-sors, and/or pharmacological probes (Balick and Cox, 1996). About half the world’s medicinal compounds are still derived or obtained from plants and they may give the chemical blueprints for the development of related synthetic drugs (Hamann, 1991). It is there-fore, important to conserve the genetic material for future drug development programs (Agrawal, 2009). Many endemic medicinal plant species restricted to Ethiopia are of great concern. Medicinal plants and associated indigenous knowledge are under risk, mainly because of agricultural expansion, deforesta-tion, fuel wood harvesting, overgrazing and urban-ization (Getu Alemayehu, 2010; Moa Mergasa, 2010;

INTRODUCTIONMedicinal plants have been globally used for mil-lennia by indigenous people to get relief from illness beauty care as well as spiritual aspects (Hawkins, 2008). This is true in East African countries where medicinal and aromatic plant resources with high potential are used in the production of diverse herb-al products at industrial scale (Ermias Dagne, 2003). Ten to twelve percent of the flora of Ethiopia is es-timated to be endemic, many of them with aromatic and medicinal value (Endalew Amenu, 2007; IBC, 2007) providing traditional uses to a multiplicity of ethnic groupings with complex cultural diversity (Azene Bekele, 2007; IBC, 2007).

Since, indigenous medicines are relatively inexpen-sive, locally available, and are usually readily accept-ed by the local people, several African and Asian na-tions are increasingly introducing traditional medicine to their public health care programs (Agrawal, 2009).


106 Daniel Kalu and Ali Seid

Nurya Abdurhman, 2010; Mohammed Adefa and Berhanu Abraha, 2011). Besides, cultural transforma-tion occurring before the documentation of medicinal plant use knowledge is an important challenge in the conservation of medicinal plants that ethnobotanical research is concerned about (Balick and Cox, 1996). In Ethiopia, a number of ethnobotanical researches have documented the declining indigenous knowl-edge held by traditional societies living in different parts of the (WHO, 2002; Haile Yineger and Delena-saw Yewhalaw, 2007; Endalew Amenu, 2008; Ermias Lulekal et al., 2008) country. The people of Ankober lead a traditional way of life, but no ethnobotani-cal research has been so far made in the area. Thus, this investigation of medicine plants and associated knowledge is believed to fill a gap in knowledge and hence, contribute to biodiversity conservation. The major objective of this research was documenting me-dicinal plants’ diversity and the associated indigenous knowledge and identifying the major threats to MPs.

MATERIALS AND METHODS

Description of the Study Area

The Ankober Woreda (District) is found in the North Shoa zone of the Amhara Regional State, Ethiopia

partly forming the western escarpment of the Great Rift Valley. It is located at the eastern edge of the Ethiopian central highlands (Figure 1). The popula-tion of Ankober Wereda is about 76,510 (CSA, 2007). The Woreda has two major ethnic groups, namely Amhara (92.77%), and Argoba (7.04%).

Data Collection and Analysis

A reconnaissance survey was carried out from Oc-tober 2-9, 2011, in order to have an overview of the plant assemblages (‘emic’ or cultural vegeta-tion classification) and determine the data collection methods. Ethnobotanical data were collected using a semi-structured questionnaire, key informants in-terview, field observation, guided field walk and Fo-cused Group Discussion made in local language as recommended in (Balick and Cox, 1996; Cunning-ham, 2001; Alexiades, 1996; Martin, 1995). Informa-tion gathered includes: medicinal value, plant parts used, methods of preparation, route of administration, human and /or animal diseases treated, growth habit, and causes of threats to medicinal plants. Appropriate herbarium specimens were collected and identification was made with the help of field guides and floras, comparison and experts determination

Figure1. Map of the study area, Ankober District (Woreda), and Sampled Kebeles (Source: http://www.maplandia.com/ethiopia/amhara/north-shewa/ankober)


in the National Herbarium, and taxonomic key us-ing flora books of Ethiopia and Eritrea (Hedberg and Edwards, 1989; Phillips, 1995; Edwards et al., 1995, 1997, 2000; Hedberg et al., 2003, 2006; Mesfin Ta-desse, 2004)

A total of 165 informants, twenty seven from the six target PAs (kebeles) and three informants pref-erentially selected from Alyuamba kebele were the study subjects. The later were selected, based on the recommendation of local elders, health workers, de-velopment agents, local administrators and villagers’ recommendation. The 34 Key informants were purpo-sively elected from those informants selected before.

Data Anaylsis Descriptive statistics including preference ranking, paired comparison, fidelity level index, informant consensus, and diversity indices were computed using Microsoft Excel (2007) spread sheet. Pearson cor-relation coefficient was calculated to determine the relationship between informants and medicinal plant knowledge. Fidelity level (FL) for the most frequent-ly reported diseases or ailments was calculated as:

FL=

Where: Np is the number of informants that claim a use of a plant species to treat a partic-ular disease, and N is the number of infor-mants that use the plants as a medicine for any given disease.

RESULTS AND DISCUSSION

Emic and Etic Categorization of Vegetation

The study discovered that medicinal plants were named emically based on different criteria including habitat such as Rubes steudneri ‘Yedega-enjori’ (high-land berry), Rubus apetalus ‘Yekola-enjori’ (lowland

berry); plant color; healers perceptions of plants spe-cies (local verity) medicinal values and after their disease name that it heals. Such names are ‘Ymich Medanit’ (febrile healer) for Ocimum lamifolium, ‘Yedengetagna Medanit’ (Acute Healer) for Cucum-is ficifolius and ‘Entil Betis’ (Tonsil Healer) for Aju-ga integrifolia. Each medicinal plant recorded in this study has a well known vernacular name indicating its popularity as a medicinal plant.

Based on the researchers ideas about what people know about plants (Etic Categorization or ecological communities), the vegetation types of the study area are of two types. The highland vegetation zones char-acterized by Erica arborea and Chloris sp., is found at altitude above 3000m a.s.l., forming the afro-al-pine vegetation. The second vegetation is Eucalyp-tus globules and mixed vegetation where a number of medicinal plants are located. The medicinal plants knowledge distribution is found to have significant correlation (r = 0.86) with age of healers.

Medicinal Plants Diversity

In this study a total of 109 medicinal plant species, belonging to 56 families are documented. Astereace-ae and Lamiaceae were the most dominant plant fam-ilies, represented by 10 species each, followed by Solanaceae and Euphorbiaceae each represented by 7 species. The top two plant families are also have higher contribution as reported by different authors in medicinal plant studies carried out elsewhere (Haw-kins, 2008; Nurya Abdurhman, 2010; Tesfaye Awas and Sebsebe Demissew, 2009; Ermias Lulekal et al., 2008).

In terms of growth habit 47 (43.12%) of the medici-nal plants were shrubs (Figure 2) and all species with local names indicated the popularity and importance of the plants in the study area. Some of them are en-demics, and include Inula confertiflora, Laggera to-mentosa, Lippia adoensis, Lobelia rhynchopetalum,


Solenecio gigas, Thymus schimperi and Urtica simen-sis accounting 6.42% of the total number of medicinal plants recorded in the study. Of the endemics Lobe-lia rhynchopetalum is reported as a threatened species (Vivero et al., 2005).

2010; Nurya Abdurhman, 2010) who also reported more number of medicinal plant use for human ail-ments than for livestock.

Medicinal Pant Utilization

Pant Parts Used and Remedy Preparations

The very common medicinal plants are used by self collection and preparation. However, MPs knowl-edge in Ankober showed a significant correlation (R2 = 0.858) with age of informants. Plant used by elderly members of the community was higher than youngsters. Many informants in age category between (51- 90) were able to list more than 48 medicin inde-pendently but the other two lower age categories were not able to cite more than few plants. This could be related to a higher degree of cultural contact and ex-perience of the elderly members and/or the effects of both cultural transformation, access to modern drags and lack of awareness.

Leaves were the most frequently used plant part used in 63 (45.65%) cases, followed by roots in 23 (16.67 %) cases and fruits used in 15 (10.87%) cases. Oth-er studies (Tesfaye Hailemariam et al., 2009; Behailu Etana, 2010; Moa Mergasa, 2010; Nurya Abdurhman, 2010) had reported similar findings, contrary to Ermi-as Lulekal et al. (2008). As revealed in this study, col-lecting leaves for remedies could have lesser impact than collecting roots and stems.

The method of processing 49 (34.51%) include chop-ping and harmonizing in water and/or other solvents; 27 (19.01%), crushing and squeezing extraction 22 (15.49 %), powdering and 10 (7.04%) boiling. Sol-vents like water, local alcoholic beverages such as, ‘Tella’ (local beer) and/or ‘Teji’ (fermented honey), and milk were used to make dissolved and wet prepa-rations. In most cases water was used as a solvent, but semi solid preparations were also made with butter and honey. Moreover, sugar, honey, tea, coffee were

Figure 2. Proportions of Medicinal Plants by Growth Habits

Analysis of growth form revealed that shrub consti-tute the largest category with 47 (43.12%) species followed by herbs with 38 (34.86%) species (Figure 2) which is in agreement with the resulst of other re-searchers (Fisseha Mesfin, 2007; Ermias Lulekal et al.,2008; Getu Alemayehu, 2010). However the re-sults were contrary to Endalew Amenu (2007) and Tesfaye Awas and Sebsebe Demissew, 2009) who documented larger proportion of herbaceous medici-nal plants.

The majority 67 (60.55%) of medicinal plant species were collected from natural habitats 27 (24.77%) from home-garden and the remaining from both indi-cating natural habitats are the main source of medici-nal plants like in other studies (Fisseha Mesfin, 2007; Ermias Lulekal et al., 2008; Tesfaye Hailemariam et al., 2009; Getu Alemayehu, 2010; Moa Mergasa, 2010). In terms of ailments, 83 (76.15%) species were used for human, 15 (13.76) species for both livestock and human ailments and the remaining 11 (10.09%) species for livestock and consolidates the works of (Endalew Amenu. 2007; Ermias Lulekal et al., 2008; Tesfaye Hailemariam et al., 2009; Moa Mergasa,


added for taste, especially when remedies are ad-ministered orally, beside some animal products such as better, chicken fat and essential organs of selected species.

Diseases Treated, Route of Administration and Dosage of Medicinal Plants

Out of the 109 medicinal plant species, 76.15% are used to treat about 42 different types of human ail-ments (Table 1). There were a total of 142 types of treatment preparations. The routes of administration were oral 66 (46.48%), dermal 59 (41.55%), nasal 6 (4.23%), ocular 5 (3.52%), through ears and both dermal and oral each 2 (1.41%). However, only sin-gle preparation administerd through anal and applied on tooth. As reported by others, oral administration is the major root of delivery (Dawit Abebe and Aha-du Ayehu, 993; Behailu Etana, 2010; Moa Mergasa, 2010; Gidey Yirga and Samuel Zeraburk, 2011).

Depending on the age and health condition of the pa-tients, the dosage measurements were spoon, Kuntit (pinch), Chibit (Fist) for powdered preparation, and ‘Sêni’ (Coffee Cup), ‘Tassa’ (Can), and ‘Birchiko’ (Glass) for liquid preparation. Children are given less doses than adults, as less as one fourth of a coffee cup compared to an adult that may be given up to one Tassa depending on the type of illness and treatment. The dosage is however, greater for animals than for human beings.

The informants in the study area reported that some of the MPs including Hagenia abyssinica, Phytolac-ca dodecandra, Verbasicum siniaticum, Euphorbia candelabrum and Croton macrostachyus are poison-ous to human if not handled with proper care. Though these MPs do have side effects, they are also effective against human and animal ailments, hence, the em-phases must shift to how much the local people are aware of side effects of using MPs.

The study revealed that though measurement units lack precision, healers routinely measure the plant

Table 1. List of human and livestock diseases that are treated using plants

Disease treated

Local name ofThe disease

No. of species

used

Percent of plants

Acute disease Dingetegna 14 12.84Body swelling Ebach 12 11.01

Diarrhea Tekimat 10 9.17

Herpes zoster Almaz balechira, 10 9.17

Rabies Yewush beshita 10 9.17

Sore throat Entilmewured 8 7.33

Eczema Chife 8 7.34Jaundice Wof (gubet) 8 7.34Hemorrhoid Kintarot 8 7.34Bleeding Yedemabinet 7 6.42

Belly blotting Entako (hod yeminefa)

7 6.42

Common cold Gunfan 6 5.50

Dandruff Forefor 5 4.59

Skin infection Megagna 5 4.59

Hypertension Demgfit 5 4.59

Abdominal colic

Kurtet 5 4.59

Fibril illness Michi 5 4.59

Evil spirit Buda 5 4.59

part and the amount of water that will be added to prepare the plant remedy. The most widely used mea-suring unit reported in this study was the tip part of forefinger (‘atiq)’. As reported by others, the amount and rates of remedy prescribed by a healer depends on age, physical variation and level of sickness (Er-mias Lulekal et al., 2008, Haile Yineger and Delena-saw Yewhalaw (2007). Even though, almost all infor-mants have developed awareness of the toxic nature of some plants, especially when administered in large doses, they prefer MPs than modern drugs for some diseases pointing that the dose should be refined for those preferred MPs. To control the harmful effects of the doses it was also found that homogenous solution of ‘Beso’ (tossed barley preparation) and in few cases tella’ (local beer) has been given as antidotes in some cases.


Important Medicinal Plants in Ankober

Informant consensus values were made to identi-fy important medicinal plants (Table 2). The method is used to indicate that particular species that can be used to solve particular health problems and specific MPs used for several health problems (Martin, 1995). Some of MPs recorded in this study have also been used in other parts of Ethiopia as reported in (Fisse-ha Mesfin, 2007; Tilahun Teklehaymanot and Mirutse Giday, 2007; Ermias Lulekal et al., 2008; Getu Ale-mayehu, 2010) indicating their pharmacological sig-nificance. Higher informant consensus warrants fur-

Table 2. Medicinal plants of higher Informant Consensus Factor (ICF) for treatment

Scientific Name No of species N cited Disease treated ICF Value

Maese lanceolata 1 45 Scabies 1.00Foniculum vulgare 1 7 Retention of urine 1.00Croton macrostachyus 2 55 Tinea corporis 0.98Calotropis procera 3 43 Leishmaniasis 0.95Leucas abyssinicum 3 43 To expel leech 0.95Grewia ferruginea 2 19 Retained placenta 0.94Croton macrostachyus 4 56 Gonorrhea 0.94Inula confertiflora 4 35 Tinea nigra 0.91Achyranthes aspera 4 27 Traumatic Wound 0.88Justicia schiperiana 8 52 Jaundice 0.86

Table 3. Direct matrix ranking of six selected multi use MPs with use diversity

Main use

Medicinal plants

C. macrosttachus D. angustifolia O. europea H. abyssinica J. procera M. salicifolia

Construction 8 9 14 14 15 11Fire wood 9 14 14 8 13 11House utensils 6 6 11 14 14 13Forage 6 13 14 3 5 13Tooth brush 0 10 15 7 0 9Medicine 15 12 14 15 9 14Farming tools 8 9 14 11 8 10Grand total 52 73 96 72 64 78Rank 6th 3rd 1st 4th 5th 2nd

Key: 5 = Excellent, 4 = Very good, 3 = Good, 2 = Less, 1 = least and 0 = Not used

ther ethnopharmacological studies of medicinal plants for identifying active ingredients of the plants.

Direct Matrix Ranking For Multipurpose Medicinal Plants

The ranking exercise showed that Olea europea is the top multipurpose MP, followed by Myrica salicifolia and Dodonia angustifolia respectably. Table 3 shows the direct matrix ranking of six plant species by three key informants with seven criteria for each plant spe-cies.


ble 5). Agricultural expansion was the most serious threat in the highlands and followed by deforestation and overgrazing, eucalyptus plantation and fire wood and house hold material harvesting in the mid-altitude mixed vegetation. While drought is mentioned as a big threat with highly negative impact on plant diver-sity, there was little interest from youngsters to learn about traditional healing and retain the MPs’ knowl-edge. This is due to lack of awareness, education and thinking westernization as civilization. The expansion of clinics and health facilities could have intensified cultural transformation. Moreover, adulteration and acculturation are emerging challenges.

Table 4. List of nine MPs with highest Fidelity level value

Plant species Local name Therapeutically use Fidelity Level (FL)Kanahia laniflora Tifrindo Acute disease 100%Euphorbia tirucalli Kinchib Leishmania 100%Xanthium strumarium Fikrutena Tinea nigra 100%Thalictrum rhynchocarpum Sirebizu Pest entered ear 100%Endostemen tereticaulia Ena Tinea nigra 100%Dichrostachys cinerea Ader Scorpion bite 100%Hagenia abyssinica Kosso Tinea saginata 100%Vernonia amygdalina Girawa Skin disease 100%Grewia ferruginea Lenkuata Retained placenta 100%

Fidelity Level Value Ranking of Medicinal Plants

Computation of the fidelity level values of medicinal plants showed that ten MPs with highest fidelity level (Table 4).

Threat to Medicinal Plants

The causes of threats to MPs can be grouped in to natural or anthropogenic factors. The threats to me-dicinal plants were scaled 1-5 and have been found that agricultural expansion, deforestation, overgraz-ing, charcoal and fire wood collection were major threats to medicinal plants in descending order (Ta-

Table 5. Priority ranking of perceived threats to MPs

Major ThreatRespondents (R1-R9)

Total % RankR1 R2 R3 R4 R5 R6 R7 R8 R9

Agriculture Expansion 5 4 5 5 4 4 5 5 5 42 17.87 1st

Over grazing 3 4 5 4 4 5 3 3 2 33 14.04 2nd

Construction 5 5 3 4 4 2 4 2 4 33 14.04 2nd

Eucalyptus plantation 4 5 2 3 3 3 4 5 2 31 13.19 3rd

Fire wood 2 1 1 2 5 4 3 4 5 27 11.49 4th

Drought 4 3 4 2 2 1 2 1 4 23 9.79 6th

Medicinal plant Trade 3 2 3 2 1 2 2 3 3 21 8.94 7th

Household Equipment 4 3 2 4 2 3 1 2 4 25 10.64 5th


CONCLUSIONSOne hundred and nine medicinal plant species in 56 families for Ankober Woreda indicates a relative-ly high MPs diversity and existence of indigenous knowledge relative to its small size. As reported from other places in Ethiopia, Asteraceae and Lamiaceae are the two plant families containing the most cited medicinal plant species. The local people and tradi-tional healers still knew distribution, medicinal use and conservation status of MPs. In addition to their medicinal values, MPs have been used for different purposes. This together with the shortage of farm-lands is major threat to both plants and indigenous knowledge in the woreda. There is little interest from youngsters to learn about traditional healing. Thus, facilitating the sustainable utilization of MPs and in-digenous knowledge of the Ankober people need a concerted efforts and timely actions.

ACKNOWLEDGEMENTSWe greatly acknowledge the local people of Ankober, particularly the traditional healers who shared their knowledge on medicinal plants and Dakon Abiy Ti-lahun for giving pertinent advice during field works. We are grateful for the Ministry of Education and Ba-hir Dar University for partly funding the cost of the research; and the National Herbarium for their coop-eration during herbarium work.

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Second degree generalized gauss-Seidel iteration method for solving linear system of equations

Tesfaye Kebede Bahir Dar University, College of Science, Department of Mathematics

[email protected]

ABSTRACTIn this paper, a second degree generalized Gauss –Seidel iteration (SDGGS) method for solving linear system of equa-tions whose iterative matrix has real and complex eigenvalues are less than unity in magnitude is presented. Few numerical examples are considered to show the efficiency of the new method compared to first degree Gauss-Seidel (GS), first degree Generalized Gauss-Seidel (GGS) and Second degree Gauss-Seidel (SDGS) methods. It is observed that the spectral radius of the new Second degree Generalized Gauss-Seidel (SDGGS) method is less than the spectral radius of the methods GS, GGS and SDGS. By use of second degree iteration (SD) method, it is possible to accelerate the convergence of any iterative method.

Key words: Gauss-Seidel method (GS); Generalized Gauss-Seidel method (GGS); Strictly Diagonally Dominant Matrix. DOI: http://dx.doi.org/10.4314/ejst.v7i2.5

INTRODUCTIONConsider a class of linear stationary second degree methods for solving linear system

A x =b (1)where A is a given real non singular n x n matrix and b is a given vector or nx1 (column) matrix (Saad ,1995). It-erative methods, based on splitting A into A = D-L-U, compute successive approximations to obtain more accurate solutions to a linear sys-tem at each iteration step n. This process can be written in the form of the general iteration matrix equation as

( 1) ( )1

n nx G x C+ = + In numerical linear algebra the Gauss–Seidel method, also known as the Liebmann method or the method of successive displacement, is an iterative method used to solve a linear system of equations (Kahan, 1958). It is named after the German mathematicians Carl Friedrich Gauss and Philipp Ludwig Von Seidel, and is similar to the Jacobi method. Though it can be applied to any matrix with non-zero elements on the diagonals, convergence is only guaranteed if the matrix is either diagonally dominant, or symmetric and positive definite. The computed Gauss-Seidel iterate successively for each component. It has been proved that, if A is strictly diagonally dominant (SDD) or irreducibly diagonally dominant, then the associated Jacobi and Gauss-Seidel iterations converge for any initial guess X(0) (Li,2005). If A is symmetric positive definite (SPD) matrix, then the Gauss-Seidel method also converges for any initial guess X(0)

(David, 2007).

The Gauss-Seidel iteration (GS) method for first degree is ( 1) 1 ( ) 1( ) ( )n nx D L Ux D L b+ − −= − + − (2)

© This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/CC BY4.0)

116 Tesfaye Kebede

The Generalized Gauss –Seidel (GGS) iterative method for first degree stated by (David, 2007) is

( 1) 1 ( ) 1( ) ( )n nm m m m mx D L U x D L b+ − −= − + − (3)

where, 1( )m m m mG D L U−= − is the iteration matrix of the GGS method.

1( )m mC D L b−= − is a column vector. In the next section, a review of Second degree generalized Gauss-Seidel iterative method (SDGGS) is presented. Following this, the relationship between spectral radius of first degree Gauss-Seidel (GS), first degree gener-alized Gauss-Seidel (GGS) and Second degree Gauss-Seidel (SDGS) methods and Second degree generalized Gauss-Seidel iteration methods is given. Finally, based on the results on the numerical examples considered, dis-cussion and conclusion made.

SECOND DEGREE GENERALIZED GAUSS-SEIDEL ITERATIVE METHOD The linear stationary second degree method is given by (David, 1970) is

( 1) ( ) ( ) ( 1) ( 1) ( )1 1( ) ( )n n n n n nx x a x x b x x+ − += + − + − (4)

Here, X (n+1) appearing in the right hand side as given in (2) is completely consistent for any constant a1 and b1such that b1≠0.

( 1) ( ) ( ) ( 1) ( ) ( )1 1 1

( 1) ( ) ( ) ( 1) ( ) ( )1 1 1 1 1 1

( 1) ( ) ( 1)1 1 1 1 1 1

( 1) ( ) ( 1)

( ) ( )

[(1 ) ]

,

n n n n n n

n n n n n n

n n n

n n n

x x a x x b G x C xx x a x a x b G x b C b xx a b I b G x a x b CTherefore x Gx Hx K

+ −

+ −

+ −

+ −

= + − + + −

= + − + + −

= + − + − +

= + +

(5)

Where 1 1 1 1(1 )G a b I b G= + − + (6)

1H a I= − (7)

1K b C= (8)Theorem1.1:- If matrix A is strictly diagonally dominant, then the associated

generalized Gauss-Seidel iteration method converges for any initial approximation, x (0).

Proof: Since matrix A is strictly diagonally dominant, we have the iteration matrix 1( )m m m mG D L U−= − . Taking the norm at infinity of both sides we have

( ) ( ) ( ) 11 1 . 1.mm m m m m m m m m m

m m

UG D L U D L U D L U

D L−− − ∞

∞ ∞ ∞∞∞ ∞∞

= − < − = − = <−

That is 1<∞mG .

Thus, the generalized Gauss-Seidel iterative method converges for any initial approximation (0)x .The second degree generalized Gauss - Seidel (GGS) method is defined as


( 1) ( ) ( 1)n n nx Gx Hx K+ −= + + (9)

Where 1 1 1 1(1 )G a b I b G= + − + (10)

1H a I= − (11)

1K b C= (12)

1( )m m m mG D L U−= − be the iteration matrix of the GGS methods.

1( )m mC D L b−= − is a column vector. Using the idea of (Golub and Varga, 1961), (9) can be written in the form.

( ) ( 1)

( 1) ( )

0 0n n

n n

Ix xH G Kx x

−

+

= +

The necessary and sufficient condition for convergence of the method is that spectral radius of G must be less than unity in magnitude for any (0) (1)x and x .

Using (10) and (11), for 0ˆ I

GH G

=

, ˆ( ) 1Gσ < ,if and only if all roots λ of

2det( ) 0I G Hλ λ− − = (13) are less than unity in modulus.

Substituting G and H of (10) and (11) in (13), we have

21 1 1 1det( [(1 ) ] ) 0mI a b I b G a Iλ λ− + − + + =

After collecting and rearranging, we have2

1 1 1 1det( [(1 ) ] ) 0mI a b I b G a Iλ λ− + − + + =

21 1 1

11 1

(1 ) ( )det [ 0m

a b ab G I I

b bλ

λλ

+ − +− + − =

21 1 1

11 1

(1 ) ( )det [ 0m

a b ab G I I

b bλ

λλ

+ − +− + − =

, Since 1det( ) 0b λ− = (14)

Thus, the eigenvalues mof Gλ are related to the eigenvalues mof G byµ

2

1 1 1

1 1

(1 ) ( )a b ab b

λµ

λ+ − +

+ = (15)

Let ive θλ = (16)

Substituting (16) in (15), we have

2

1 1 1

1 1

(1 ) ( )i

i

a b ve ab b ve

θ

θµ+ − +

+ = , then 2

1 1 1

1 1

(1 ) ( cos sin )(cos sin )

a b v iv ab b v i

θ θµ

θ θ+ − + +

+ =+

118 Tesfaye Kebede

After collecting and simplifying, we get

2 2

1 1 1 1

1 1 1

( 1 )cos sin

b a v a v ai

b b v b vµ θ θ

− − + −= + +

(17)

From (17) real part of µ is 2

1 1 1

1 1

( 1 )Re cos

b a v ab b v

µ θ − − +

= +

Now add both sides the term 1 1

1

(1 )a bb

+ − , we get

2

1 1 1 1 1 1 1

1 1 1 1

(1 ) ( 1 ) (1 )Re cos

a b b a a b v ab b b b v

µ θ + − − − + − +

+ = + +

.

So the result is2

1 1 1

1 1

(1 )Re cos

a b v ab b v

µ θ + − +

+ =

.

From this we get,

1 1

12

1

1

(1 )Re

cos

a bb

v ab v

µθ

+ −+

= +

Squaring both sides we have

2

1 1

2 12

1

1

(1 )Re

cos

a bb

v ab v

µθ

+ −+

= +

. (18)

From (17) imaginary part of µ is 2

1

1

Im sinv a

b vµ θ

−=

, then we get

2

222

1

1

Imsinv a

b v

µθ = −

(19)


Add (18) and (19), we get

2

1 12

12 22 2

1 1

1 1

(1 )Re

Im 1

a bb

v a v ab v b v

µµ

+ −+

+ = + −

(20)

a b

Real case Complex case

Figure1. General regions for eigenvalues mof Gµ From (20), we have the following

1 1 1 1

1 1 1

11 1 (( ) , ( ) , ( )2

a a b av a v b

b v b v ba b− − +

+ = − = =

If the eigenvalues mof Gµ are real and lie in the interval 1a µ b≤ ≤ < , then the choice of a1 and b1 must satisfy the following conditions

Im 0µ = (since µ is a real number), we have 1

1

1 ( )sin 0a

vb v

θ− = ,

we get v2=a (21)

and from figure (1) above the distance from β to α is 2cb a ′− = , where 2 2c a b′ = −

Squaring both sides we get 2 2 21 ( )4

a bb a− = − . (22)

Now substituting 1 1

1 1

1 1( ) ( )a a

v and vb v b v

+ − in (22) for a and b respectively, we have

2 2 21 1

1 1

1 1 1( ) [ ( )] [ ( )]4

a av v

b v b vb a− = + − − , then we get

22

1

1 4( )4

vb

b a− = , taking square root of

both sides,

a b

a b

120 Tesfaye Kebede

we get 1

22

vb

b a−= (23)

Thus we have from (15), (16) and (17), we get

2(1 ) 22 ( )

v vb ab a−

+ =− +

(24)

If we let 2 ( )b aσb a−

=− +

, then we get

2 2

2

2 ˆ(1 ) 2 11 1

bv v and let vσ ωσ

+ = + = =+ −

(25)

From (16), we have

2

1 2 2 2

2ˆ 1 11 1 [1 1 ]

a σωσ σ

= − = − =+ − + −

(26)

and 1 2

ˆ ˆ2 2 4( ) 2 ( ) [1 1 ][2 ( )]

b bbσω ωb a b a σ b a

= = =− − + + − − +

(27)

Therefore the spectral radius of1 12 2

1 1ˆ ˆ ˆ( . . ( ) 1)bG is a i e G aσ ω= = − (28)

Thus with this choice of a1 and b1, the second degree method for any iteration is given by (David, 2007)

( 1) ( ) ( 1) ˆ2 2ˆ ˆ( ) (1 )2 ( ) 2 ( ) 2 ( )

n n nm bb b

Gx I x x C

ωb aω ωb a b a b a

+ −+= − + − +

− + − + − + (29)

Where2

2ˆ1 1

bωσ

=+ −

,

mG is the generalized iteration matrix and C is column vector.

If A is positive definite matrix and if is a generalized Gauss-Seidel iterative matrix and hence

22

2, 0 ,2 ( ) 2b a µb µ a σb a µ−

= = = =− + −

, whereµ is spectral radius of generalized Jacobi matrix

2

2ˆ1 1

bωσ

=+ −

= 2 2

2 2 2 2 22

2

2 4 2 2(2 )

2 1 (1 1 )1 1

2

µ µ

µ µ µµµ

− −= =

− + − + − + − −

(30)


4

1 2 2 42

2

2ˆ 1 1(1 1 )

1 12

ba µωµµ

µ

= − = − =+ −

+ − −

(31)

and 2

1 2 2 2 2 2

ˆ ˆ2 2 2 2(2 ) 4( )( ) 2 ( ) 2 (1 1 ) (1 1 )

b bbσω ω µb a b a µ µ µ

−= = = =

− − + − + − + − (32)

1 22

1 2 2ˆ ˆ( ) 1

(1 1 )bG a µσ ω

µ= = − =

+ − (33)

Therefore, the second degree generalized Gauss-Seidel method is given by

(1) (0)1x G x C= + (34)

( 1) ( ) ( ) ( ) ( 1)2 2

ˆ ˆ2 2 ˆ( ) (1 ) ( 1)( )2 2

n n n n nb bm m bx G x C x x x

ω ωω

µ µ+ −= + + − + − −

− −

Where2

1 1

2 2

4 2ˆ , ( ) ( )2 1

b m m m m m m m mG D L U and C D L bµωµ µ

− −−= = − = −

− + −

Relationship between Spectral Radiuses Based on the results on the spectral radius, the following relations are observed

First degree Jacobi method (FDJ) is µ .

Second degree Jacobi method (SDJ) is 1 21 1a µ

µ=

+ −.

Second degree generalized Jacobi method (SDGJ) is 1 21 1m

m

aµ

µ=

+ −

Second degree Gauss-Seidel method (SDGS) is 2

1 2 2(1 1 )a µ

µ=

+ −

Second degree generalized Gauss-Seidel method (SDGGS) is 2

1 2 2(1 1 )m

m

aµ

µ=

+ −

We know 2

2 2 2 2(1 1 ) 1 1 1 1m m

m m

µ µ µ µµ µ µ

≤ ≤ ≤+ − + − + −

Since 21 1 0µ+ − > and

also 2 , 0,1, 2,3....m m m nµ µ µ≤ ≤ ∀ = ,

122 Tesfaye Kebede

If m=0, we have 0µ µ= .

Numerical Examples

Example 1: Solve the following strictly diagonal dominant (SDD) linear system of equations:

−=−+=++−

=+−

1525272

1126

321

321

321

xxxxxx

xxx

Using a) GS b) GGS c) SDGGS

Solution:- Let us choose x(0)=(0,0,0)t is an initial approximation value and tolerance number is 10-5. Now the spectral radius of Gauss-Seidel and generalized Gauss-Seidel iteration methods are r(GS)=0.1396 and r(GGS)=0.050459. i.e r(GGS) < r(GS).

Example 2: Solve the following positive definite (PD) linear system of equation using GS,

GGS, SDGGS methods.

=+−−−=−+−−=−+−

=−−

947434

54

432

431

421

321

xxxxxxxxxxxx

Solution: Let us take the initial approximation x(0) =(0,0,0,0)t with an accuracy of 10-5.

The spectral radius of Gauss-Seidel (GS) and generalized Gauss-Seidel iteration methods (GGS) are r (GS)=0.3334, r(GGS)=0.1112 . i.e r(GGS) < r(GS).

RESULTS AND DISCUSSION

As presented in Table 1, the exact solution for the given linear system of equation is (2,1,1). It is observed from the table that the same solution is obtained at the 8th iteration by Gauss-Seidel method (GS), at the 6th iteration by Generalized Gauss-Seidel method(GGS) and by Second degree Generalized Gauss-Seidel (SDGGS) method by considering only m=1. If m=2 one can see that at the first iteration exact solution is obtained.


As presented in Table 2, the exact solution for the given linear system of equation is (1,0,-1,2). It is observed from the table that the same solution is obtained, using Gauss-Seidel method (GS) at the 12th iteration, using Generalized Gauss-Seidel method(GGS) at the 9th iteration and using Second degree Generalized Gauss-Seidel (SDGGS) method at the 6th iteration by considering only m=1. If m=2,3,4 one can get almost equal to the exact solution with small number of iteration. Hence for Positive definite (PD) matrix SDGGS method is faster than first degree GS, GGS and Second degree GS method.

Table 1: Solution of GS, GGS and SDGGS for SDD Matrix

nGS GGS(m=1) SDGGS(m=1)

x1 x2 x3 x1 x2 x3 x1 x2 x3

0 0.00000 0.00000 0.00000 0.00000 0.00000 0.00000 0.00000 0.00000 0.00000

1 1.83333 1.23806 1.06191 2.17890 1.03670 1.05046 2.17890 1.03670 1.05046

2 2.06905 1.00204 1.01463 1.99097 0.99815 0.99745 1.98805 0.99759 0.99666

3 1.99824 0.99531 0.99988 2.00044 1.00009 1.00013 2.00082 1.00016 1.00022

4 1.99881 1.00030 0.99988 1.99998 1.00000 0.99999 1.99995 0.99999 0.99999

5 2.00012 1.00007 1.00005 2.00000 1.00000 1.00000 2.00000 1.00000 1.00000

6 2.00001 1.00000 1.00000 2.00000 1.00000 1.00000 2.00000 1.00000 1.000007 2.00000 1.00000 1.00000

8 2.00000 1.00000 1.00000

Table 2: Solution GS, GGS and SDGGS Iterative Methods

GS GGS (m=1) SDGGS (m=1)

n x1 x2 x3 x4 x1 x2 x3 x4 x1 x2 x3 x4

00.00000 0.00000 0.00000 0.00000 0.00000 0.00000 0.00000 0.00000 0.00000 0.00000 0.00000 0.00000

11.25000 0.43750 -1.4375 1.78125 1.13333 0.46667 -0.99556 1.88444 1.13333 0.46667 -0.9955 1.88444

20.78125 0.10938 -1.9453 1.94531 0.99348 0.03052 -1.00377 1.99143 0.98610 0.00485 -1.0051 1.99946

3. . . . . . . . 0.99921 0.00018 -1.0001 1.99990

4. . . . . . . . 1.00001 0.00005 -1.0000 1.99999

5. . . . . . . . 1.00000 0.00001 -1.0000 2.00000

6. . . . . . . . 1.00000 0.00000 -1.0000 2.00000

7. . . . . . . .

8. . . . 1.00000 0.000000. -1.00000 1.999999.

10 . . . .11 1.00000 0.00000 -1.0000 2.0000012 1.00000 0.00000 -1.0000 2.00000

124 Tesfaye Kebede

CONCLUSIONIf matrix A is strictly diagonal dominate (SDD) and positive definite (PD) matrix, then by the use of second degree generalized Gauss-Seidel iterative method(SDGGS), it is possible to accelerate the convergence rate of the solution of linear system of equations which has real and complex eigenvalues that are less than unity in magnitude . The numerical results shows that the SDGGS method is more effective than first degree Gauss-Seidel (GS), first degree generalized Gauss-Seidel (GGS) and second degree Gauss-Seidel (GS) methods. Moreover, from the relationship of spectral radius, the spectral radius of SDGGS is less than SDGS. In general, the results of numerical examples and spectral radius comparison considered clearly demonstrate the accuracy of the methods developed in this article. It is conjectured that the rate of convergence of some methods developed in this paper can be further enhanced by using extrapolating techniques.

ACKNOWLEDGEMENTSI would like to express my sincere appreciation to Professor Vatti, Bassava Kumar, Department of Engineering mathematics, College of Engineering, Andhra University and Gashaye Dessalew for their comments and sugges-tion on the article.

REFERENCES

David, M. Y. (1970). Second-degree iterative methods for the solution of large linear systems. Journal of Approximation Theory 5:137-148.David, K. S. (2007). Generalized Jacobi and Gauss-Seidel methods for solving linear system of equations. Numerical Mathematics, A Journal of Chinese Universities. (English Series) 16: 164-170.Golub, G.H and Varga, R.S. (1961). Chebyshev semi-iterative methods, Successive Over-Relaxation iterative methods and second-order Richardson iterative Methods. Numerical Mathematics.Kahan, W. (1958). Gauss-Seidel methods for solving large systems of linear equations. PhD Thesis, University of Toronto.Li, W. (2005). A note on the preconditioned (GS) method for Large system of equation. Journal of Computational Applied Mathematics 182:81-90.Saad, Y. (1995). Iterative methods for sparse linear systems. PWS Press, New York.

Ethiop. J. Sci. & Technol. 7(1), 2014

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CONTENTS


Effect of ultrasound on protein metabolism in the silkworm

Murali Mohan, P., Siva Prasad, S. Sahitya Chetan, P.

67


Eyasu Shumbulo Shuba, Dominique Adriaensand, Peter Bossier

85

Assessment of antioxidant potential of Moringa stenopetala leaf extract

Tesfaye Tebeka, Solomon Libsu

93

Ethnobotanical study of medicinal plants in Ankober woreda, central Ethiopia

Daniel Kalu, Ali Seid

105

Second degree generalized gauss-seidel iteration method for solving linear sys-tem of equations

Tesfaye Kebede

115

Author Guidelines

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