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Science Spectrum(An Official Journal of Andhra Pradesh Akademi of Sciences)

. Volume 2

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ISSN

Science Spectrum(An Official Journal of Andhra Pradesh Akademi of Sciences)

Issue 1 January

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ISSN - 2455-5053

Science Spectrum

(An Official Journal of Andhra Pradesh Akademi of Sciences)

anuary 2017

www.apas.org.in .

Science Spectrum

_____________________________________________________________________________________________ Volume 2 (1) CONTENTS January 2017

Research Papers

Utilization and Nutritional Evaluation of Babul Bark (Acacia Arabica) for the Development 1-11

of Value Added Product to Implement on the Diabetic Patient

Jaya Sinha and Zeba Naz

Morphological and Biochemical Responses to Heat Stress in Arachis hypogaea. L 12-22

K. Rekha Rani, A. Sujitha, Y.Mrunalini, D. Sujitha and R. Usha

Extraction of Iron (III) with Tri-caprylamine oxide from hydrochloric& sulphuric acid 23-26

solutions

A.V.L.N.S.H. Hariharan

An Environmentally benign synthesis of N-alkyl-2-((benzimidazol-2-yl) thio) acetonitrile 27-30

Sadhu Srinivas Rao

A New Validated RP-HPLC Method for the Quantification of Cabazitaxel - An Anticancer 31-38

Plant product

Mathrusri Annapurna Mukthinuthalapati and Venkatesh Bukkapatnam

Cloud Data Security using hybrid RSA and Cuckoo Search Algorithm 39-42

A Arjuna Rao, K Sujatha, P Praveen Kumar and V Sravani IKC

Structural and Dielectric Properties of Sm3+ Doped SrTiO3 Ceramic Powders 43-48

J. Guravamma, C. Sai Vandana and B.H.Rudramadevi

Construction of Subsets of B and B0 49-53

S.Nagendra and Dr.E.Keshava Reddy

Influence of silver nitrate and different carbon sources on in vitro shoot development in 54-61

Solanum nigrum (Linn)-an important antiulcer medicinal plant

G. Geetha, K. Harathi, D. Giribabu and C.V. Naidu

Synthesis and Spectral characterization and DNA Binding properties of Copper (II) 62-71

complexes with Nicotinoyl and Isonicotinoylhydrazones

S. Chandrasekhar & K. Hussain Reddy

Total Coloring and Chromatic Number of Strong Intuitionistic Fuzzy Graph 72-76

S. Narayanamoorthy and P. Karthick

1

Science Spectrum Vol.2 (1) 1- 11 January 2017, ISSN 2455-5053

Utilization and Nutritional Evaluation of Babul Bark (Acacia Arabica) For the

Development of Value Added Product to Implement On the Diabetic Patient

Jaya Sinha and Zeba Naz University Department of Clinical Nutrition and Dietetics, Vinoba Bhave University, Hazaribag, Jharkhand

*For Correspondence: [email protected]

Abstract

Babul bark is worldwide distributed. In India it is

widely observed from Rajasthan, Gujarat, Uttar

Pradesh, Bihar, Jharkhand, Bengal and Orissa.

Babul tree is of significant importance in

Ayurveda, Unani medicine and African folk

medicine. Babul tree contains Arabinose,

Galactose, Rhamnose and Glucuronic acid. Barks

are rich source of micronutrients such as Vitamin-

C, Iron, Calcium, Carbohydrate (found in babul in

less amount in polysaccharide form). It is a richest

source of Tannins which are responsible for

reducing Blood sugar level. The study was

undertaken with the objectives (i) to process Babul

bark in power form and find out the component

changes after dehydration (ii) To introduce the

babul plant as medicinal uses for reducing the

complication of diabetes by preparing biscuits

enriched with babul bark powder (iii) to assess the

organoleptic quality of biscuits enriched with

babul bark powder (iv) To evaluate the

effectiveness of babul bark biscuits on diabetic

patient after intervention. The present study was

an attempt to utilize the medicinal bark to develop

value added product namely namkin biscuits and

its effect on diabetic patients for reducing the

Blood sugar level (BSL). Babul bark were

dehydrated by tray drying and nutrient estimation

of dehydrated babul bark powder was done for

protein, fat, ash, moisture, crude fibre, iron,

calcium and vitamin-C. Organoleptic test of the

product was done by nine point hedonic scale. For

intervention 100 respondents aged between 27-80

years were randomly selected from different

hospitals of Ramgarh district of Jharkhand and

was dived into two groups of 50 each. One was

control and the other was experimental. The

nutritional status of the patients was recorded

through dietary survey. BSL was estimated with

alkaline citrate method for both the groups before

and after intervention to see the effect of biscuits.

The data collected were analysed statistically by

using mean, standard deviation, standard error,

ANOVA and paired‘t’ test. The nutrient

estimation per 100 gm of dehydrated babul bark

shows that it is a good source of iron (140mg),

fibre (18 g), Vit- C (25.3 mg), and calcium (2939

mg). It is low in fat (2 g) and it contains 58 g

carbohydrate in polysaccharide form. A significant

difference was found between the color, flavor,

texture, taste and over all acceptability of the

biscuits enriched with babul bark powder on

applying ANOVA test. The mean age of the

respondent was 46 years. 87% were non-

vegetarian.

The energy and fibre intake of respondents in both

the groups was less then the RDA. On applying

analysis of variance test it was found that there

was no significant difference in intake of different

nutrients in both the groups. On applying paired‘t’

test a significant difference was found in fasting

and PP glucose level of experimental group which

was intervened with namkin biscuits enriched with

babul bark powder for one month in comparison to

the control group. It was concluded that namkin

biscuits enriched with babul bark powder was

effective in reducing BSL in diabetic patient.

2

Vol.2 (1) 1- 11 January 2017, ISSN 2455-5053 Science Spectrum

Introduction

Babool (Acacia arabica) is commonly known as

kikar and bava in Hindi, Indian gum arabic in

English, Babula in Sanskrit, Acacia arabica Willd

Pennel in Latin. It belongs to the family:

Leguminosae, sub-family: Fabacaea. First of all

described by Linnaeus in 1773. The genus acacia

was first described by philipmiller in 1754 and

until 1842. It is estimated that there are roughly

1380 species of Acacia worldwide, about two-

third of them native to Australia and rest of spread

around tropical and subtropical regions of the

world. Gamble, (1918) have reported more than 40

species of this genus in India in his ‘Flora of

Madras Presidency.’

Perennial shrub or tree, 2.5-10 (-20) mt tall,

variable in many aspects. Branches are spreading,

forming a dense flat or rounded crown with dark

to black colored stems. Bark thin, rough, fissured,

deep red-brown. Spines (thorns) thin, straight,

light-grey in axilliary pairs, usually in 3-12 pairs,

5-7.5 cm long in young trees, mature trees

commonly without thorns. Babul plant is

worldwide distributed native to Africa, Algeria,

Ethiopia and Ghana. In Asia it is also observed

from Iran, Iraq and Israel along with Nepal,

Pakistan and India. In India it is widely observed

from Rajasthan, Gujarat, Uttar Pradesh, Bihar,

Jharkhand, Bengal and Orissa.

Babul tree is of significant importance in

Ayurveda, Unanl medicine and African folk

medicine. Babul tree contains Arabinose,

Galaclose, Rhamnose and Glucuronic acid. Gum

of the tree contains calcium, magnesium and

potassium, malic acid, sugar. Bark and pods

contain a large quantity of tannins and Gallic acid.

(The Satvik Blog and www.dabur.com)

Phytochemical investigations of Acacia arabica

found that phenolic compounds are presents in

Acacia arabica extracts. Acacia arabica contains

flavonoids, sterols, triterpenoids, alkaloids and

phenolics which possess various health benefits.

The isolation and characterization of quercetin,

gallic acid, catechin, epicatechin, dicatechin, and

leucocyanidin gallate from the acetone extract is

reported. The seeds of Acacia arabica contain

5.2% oil. Physico-chemical constants and fatty

acid composition of the refined seed oil were

estimated. The oil was rich in linoleic acid, oleic

acid and trace quantities of epoxy and hydroxy

fatty acids. Acacia arabica bark is reported to

contain catechin, epicatechin, dicatechin,

quercetin, grallic acid leucocyanidin gallate,

sucrose and catechin. (drugandcure.blogspot.in)

The parts of babul such as Leaves, bark and fruit

are used beneficial therapeutically. The doses of

Babul is Bark decoction 30-80 ml; Bark powder 3-

6gm; leaves powder 3-6gm; and fruit powder 3-

6gm. (www.avurveda.com).

It has a slight tan-tike odour, astringent taste and

mucilaginous (botanica.com)

Babul is used as anti-diabetic. Acacia arabica

seeds contains a substance(s) which depressed the

blood glucose level in normoglycemic but not in

alloxan-diabetic rabbits, suggesting that the

mechanism of action involved release of insulin

from pancreatic beta-cells. The bark in the form of

decoction (20 mg/kg) as well as the standard drug

talbutamide produced a significant reduction in

blood glucose levels in mild alloxonised diabetic

rabbits fasted for 18 - 38 hr.

Methanolic extract of the bark decreased the UV-

induced mutagenicity using the Escherichia coli

WP-2 in a dose of 5 mg/plate. This decrease might

be due to some enzymatic action which reverted

the formation of pyrimidine dimmers.

Acacia seeds extracts displayed more pronounced

action on human trypsin and chymotrypsin, it was

more effective in inhibiting the total proteolytic

activity of the bovine system.

3


Studies have shown that babul bark extracts

resulted in lipid peroxidation or cholesterol

control. On the other hand, the antioxidant

properties also showed hepato-protective

properties or liver protection from carbon

tetrachloride by free radical scavenging.

The seeds of babul can control ordinary diarrhoea,

Fresh leaves of the plant can be administered with

same quantity of cumin seeds. This mixture of 12g

each can be taken thrice every day. The decoction

of the gum can be made for the same.

The stem bark extracts of babul or Acacia arabica

showed significant antibacterial properties against

S. viridans, S. aures, E coli, B subtilis, Sonnei and

against fungi such as C albicans, A niger.

The seeds and pod extracts of babul have been

shown to significant control the arterial blood

pressure. Antispasmodic effects were also

observed in the extracts. The antispasmodic effects

were very similar to the control of arterial blood

pressure. Babul demonstrated acetyl cholinesterase

inhibitory effects. Acetyl cholinesterase inhibition

has been named critical in proper functioning of

the nervous system and in the treatment of

Alzheimer’s disease.

Research on babul showed chemopreventive and

antimutagenic properties through the presence of

polyphenals and gallic acid. The extracts of

flowers and gum were observed to be very

effective.

For hundreds of years, babul bark and gum have

been used for Dental problems. Studies on the

babul gum have yielded positive results in

removing plaque, gingivitis and periodontitis. The

gum was found to be effective in inhibiting the

growth of periodontitis bacteria such as

Actinobacillus actinomycetemcomitans,

Capnocytophaga spp., Porrphyrcmonas gingivalis,

Prevotella intermedia and Treponema denticola.

The bark of babul tree is useful in the treatment of

eczema. About 25 grams each of this bark and the

mango bark should be boiled in about 1 litre of

water and the vapours allowed fomenting the

affected part. After the fomentation, the affected

part should be anointed with ghee.

A decoction of the bark, mixed with rock salt,

should be used as a gargle in treating tonsillitis.

(www.dabur.com & www.allayurveda.com)

The Present study was conducted with following

objectives:

(i) To process Babul bark in powder form and

find out the component changes after

dehydration.

(ii) To introduce the babul plant as medicinal

uses for reducing the complication of

diabetes by preparing biscuits enriched with

babul bark powder.

(iii) To assess the organoleptic quality of namkin

biscuit enriched with babul bark powder.

(iv) To see the effectiveness of babul bark

namkin biscuit on diabetic patient by

intervention.

Material & Methods

The present investigation “Utilization and

Nutritional Evaluation of Babul (Acacia Arabica)

For the Development of Value Added Product to

implement on the Diabetic Patient” was carried

out by using the material and methods described in

this chapter.

The details of materials, experimental procedure

and techniques adopted during the course of the

present investigation have been elaborated:

1. Selection of Products: The product namely

babul bark were selected for present study.

2. Procurement of raw materials: Babul (Acacia

arabica) barks were collected from local areas

of Hazaribag in the month of November to

January.

4

Vol.2 (1) 1- 11 January 2017, ISSN 2455

3. Experimental site: The present investigation

was carried out in the laboratory of university

Clinical Nutrition and Dietetics, Vinoba

Bhave University, Hazaribag and Department

of Home Science, Birsa Agriculture

University, Ranchi.

4. Dehydration of Babul (Acacia arabica) barks:

Selected herbal plants namely Babul (Acacia

arabica) barks were subjected to dehydration.

The Babul (Acacia arabica) barks were tray

dried at controlled temperature of 180

for 4-5 hrs.

The Process is described in the following steps:

5. Development of value added food Products:

Dehydrated Babul barks powder were used for

the development of value added locally

familiar food product namely Namkin biscuits

at the ratio of 10:90 in 100 gm.

Final moisture content

Storage

Seiving

Grinding

Tray drying

Sorrting, cutting,

Washing (to remove micro-organisms, dirts

etc.)

Collection of Babul barks (Initial stage moisture

content)

11 January 2017, ISSN 2455-5053

Experimental site: The present investigation

was carried out in the laboratory of university

Clinical Nutrition and Dietetics, Vinoba

Bhave University, Hazaribag and Department

a Agriculture

Dehydration of Babul (Acacia arabica) barks:

Selected herbal plants namely Babul (Acacia

arabica) barks were subjected to dehydration.

The Babul (Acacia arabica) barks were tray

dried at controlled temperature of 180-2000C

The Process is described in the following steps:

Development of value added food Products:

Dehydrated Babul barks powder were used for

the development of value added locally

familiar food product namely Namkin biscuits

Replication of value added food products enriched

with dehydrated Babul barks were done as

follows:

5.1. Chemical Analysis: Chemical analysis of

dehydrated Babul barks powder was done by

standard procedures. For this 3 replications

were done.

Babul barks Dehydrated Babul bark powder

5.2. Organoleptic Test: Organoleptic test was

done by using 9 point hedonic scale namkin

biscuits and it was replicated 5 times.

Product Replication

Namkin Biscuits

6. Organoleptic analysis of the

To analyse the products Namkin Biscuits were

freshly prepared and evaluated

organoleptically by a panel of five judges

selected from the Department of Clinical

Nutrition and Dietetics, Vinoba Bhave

University, Hazaribag. The judges were

requested to score the products with the help

of score card based on the nine point hedonic

scale; mean scores for biscuits were

calculated.

7. Nutrient estimation: After estimation

dehydrated babul bark was analyzed for their

moisture content, total ash,

fibre, crude fat, calcium, iron, carotene. Iron

content was estimated by Atomic Absorption

Spectrophotometer method. Protein, fat, ash,

fibre was estimated by AOAC 1990 method.

Calcium was estimated by Atomic Absorption

Spectrophotometer. Carbohydrate was

estimated by Difference method. Vitamin C

was estimated by Dimethod.

Science Spectrum

Replication of value added food products enriched

with dehydrated Babul barks were done as

Chemical Analysis: Chemical analysis of

dehydrated Babul barks powder was done by

standard procedures. For this 3 replications

Replication Dehydrated Babul bark 3

Organoleptic Test: Organoleptic test was

done by using 9 point hedonic scale namkin

biscuits and it was replicated 5 times.

Replication

5

Organoleptic analysis of the cooked products:

To analyse the products Namkin Biscuits were

freshly prepared and evaluated

organoleptically by a panel of five judges

selected from the Department of Clinical

Nutrition and Dietetics, Vinoba Bhave

University, Hazaribag. The judges were

quested to score the products with the help

of score card based on the nine point hedonic

scale; mean scores for biscuits were

After estimation - The

dehydrated babul bark was analyzed for their

moisture content, total ash, protein, crude

fibre, crude fat, calcium, iron, carotene. Iron

content was estimated by Atomic Absorption

Spectrophotometer method. Protein, fat, ash,

fibre was estimated by AOAC 1990 method.

Calcium was estimated by Atomic Absorption

arbohydrate was

estimated by Difference method. Vitamin C

was estimated by Dimethod.

5


8. Calculation of Nutritive value: The protein,

crude fibre, iron, calcium, carotene,

carbohydrate and vitamin-c of the products

were calculated by using the proximate

analysis values determined of dehydrate babul

bark as well as the Food Composition Table

by Gopalan et. al. (2004).

For Intervention Selection of Sample:

Three stages of sampling were adopted for the

present study.

(i) Selection of District: Ramgarh district was

selected purposively for the present study as

it was convenient, as the researcher had

good access to it, so regular visit could be

made authentic for the collection of data.

(ii) Selection of Area: CCL Hospital of Saunda,

Sayal, Bhurkunda and Ramgarh were

selected purposively for the study as these

are in the centre of the town and were easier

for the researcher to make regular visit and

carry out the survey.

(iii) Selection of Respondent: The total no of

respondents selected were 100, out of whom

50 were in control group & reviewing 50 in

experimental group. Selection was done by

simple random sampling method. All the

respondents of experimental group were

from age group of 27-80 years. Intervened

with the biscuits enriched with babul bark

powder. Each respondents of experimental

group were given 5 biscuits/day (50g) for

one month.

Preparation of Instrument and Tools for the

Data Collection:

For the date collection and structured was

developed. The schedule consisted of the

following information which is elaborated below.

(i) General Profile: The data regarding the

general profile of respondents were

collected using first part of the schedule

which include name, age, sex, religion and

family type no. of family members, total

income of the respondents.

(ii) Dietary Survey: In the present study 24

hours dietary recall method (Swaminathan,

2006) was adopted and nutrient intake per

day was calculated. Calculation of nutrient

intake was done with the help of “Nutritive

value of Indian foods”. Gopalan et al., 2007,

and compared with Recommended Dietary

Allowances given by ICMR. The food

consumption frequency was recorded in

terms of cereals, green leafy vegetables and

other vegetable, roots and tubers, milk &

milk products, meat and fish, egg, fruits, oil

& seeds other food (fast and junk food)

consumption. Information related to dietary

pattern, food habits, food intake and nutrient

intake with references to protein, fat,

carbohydrate, energy, iron, calcium and

fibre were recorded. The respondents were

asked to provide information regarding the

menu as well as ingredients and amounts

used for meal preparation.

(iii) Blood Testing: Blood testing carried out

two times in a month of the respondents in

both the groups i.e. as control group and

experimental group. This data was collected

for the comparison study.

Method: Estimation of BSL of both group i.e.,

experimental and control group were estimated

with alkaline citrate method.

Statistical Analysis: Data was tabulated and

subjected to analysis at the end of the study. The

statistical techniques used were as described by

Prasad. S (2012) and included the following four

tests: Standard deviation, Standard Error, Paired t-

test &Analysis of variance.

Period of Inquiry: The data for the study were

calculated during the period from January - April

2014.

6


Results & Discussion

According to the data obtained from table 1 the

proximate composition in response to protein,

carbohydrate, fat, vitamin C, Fibre, Iron, Calcium,

ash and moisture is in sufficient amount. After

dehydration the moisture content in dehydrated

powder was 8 gm, carbohydrate estimation of

dehydrate bark was 58 gm which was in

polysaccharide form being helpful for diabetic

patients. It contain (140 mg) of Iron which can

treat the iron deficiency diseases. It is high in fibre

content which covers the 18 gm. It is low in fat (2

gm) and high in vitamin-C (25.3 mg). Calcium

content in dehydrated babul bark is (2939.2 mg)

which can reduce the calcium deficiency.

Table 1. Proximate composition of dehydrated

Acacia arabica bark powder per 100 gm.

Nutrient Dehydrated Acacia

Arabic (babul)

Mean

R1 R2 R3

Moisture

(gm)

8.360 8.477 8.470 8.435

0.03

Protein (g) 5.33 5.51 5.25 5.36

0.07

Carbohydr

ate (g)

55.41 56.093 56.30 55.93

0.27

Crude

fibre (g)

18.11 18.01 18.14 18.08

0.03

Ash (g) 10.61 10.00 9.91 10.91

0.57

Calcium

(mg)

2905.8

1

2905.8 3006 2939.2

34.0

Iron (mg) 140.5 141.1 141.5 140.86

0.31

Vitamin-C

(mg)

20.7 27.6 27.6 25.3

2.34

Fat (g) 2.18 1.91 1.93 2.00

0.08

Table 2. Effect of addition of dehydrated Acica

arabica (Babul) bark powder on the colour,

texture, flavor and taste of the Namkin Biscuit.

Results

The table 2 shows average score based on 9 point

hedonic scale for colour, texture, flavor, taste, and

overall acceptability of biscuit enriched with babul

bark powder. Overall acceptability and taste

scored maximum followed by flavor, texture and

colour respectively.

From the ANOVA table it was evident that the

calculated value of biscuit was greater than the

tabulated value at 5% probability level. Therefore

it was found that there was significant difference

between the colour, texture, flavor, taste and

overall acceptability of the biscuit (P 0.05). So,

it was concluded that overall acceptability was

overall like to very much.

For Intervention: All the information about the

hundred respondents collected is presented and

discussed here. Among the subjects studied

majority of the respondents (92%) belonged to

Hindu religion whereas (8%) belonged to Muslim

religion, (65%) lived in nuclear family whereas

(35%) lived in joint family (86%) respondents and

his family were literate, earned between Rs.

20,000/- – 25,000/-, had per capita income

between Rs. 4,000/- - 5,500/- (47%) were working

as employee and has sedentary life style.

Replicat

ion

Sensory scores Mean

S.E R1 R2 R3 R4 R5

Colour 7.3 8.1 7.9 8.4 9 8.1 0.27

Texture 7.1 7.9 8.4 8.4 9 8.1 0.31

Flavor 7.4 8.1 8.3 8.4 9 8.2 0.25

Taste 7.7 8.3 8.7 8.6 9 8.4 0.21

Overall

acceptab

ility

7.9 8.3 8.5 8.6 9 8.4 0.17

7


Table 3. Nutritive value of Namkin biscuit enriched with dehydrated babul bark powder per 100 gm.

S.

No.

Ingredie

nts

Energy

(K cal)

CHO

(gm)

Protein

(gm)

Fat

(gm)

Iron

(mg)

Vit. C

(mg)

Fibre

(gm)

Calcium

(mg)

Moisture

(gm)

1 Flour

(70gm)

238 48.58 8.47 1.19 3.43 - 1.33 33.6 8.54

2 Bark

Powder

(10gm)

22 5.593 0.536 0.2 14.15 2.53 1.808 293.92 0.8435

3 Oil

(10gm)

90 - - 10 - - - - -

4 Milk

Powder

(6gm)

29.76 2.28 1.548 1.60

2

0.036 0.24 - 57 0.21

Table 4. Food consumption frequency of respondents

Food

EXPERIMENT GROUP CONTROL GROUP

Daily 2-4

times a

week

4-6

times a

week

Seldom Never Daily 2-4

times a

week

4-6

times a

week

Seldom Never

N

=

50

%

N

=

50

%

N

=

50

%

N

=

50

%

N

=

50

%

N

=

50

%

N

=

50

%

N

=

50

%

N

=

50

%

N

=

50

%

Cereals

and Cereal

products

50 10

0 50

10

0

Pulses &

Legumes 47 94 3 6 44 88 4 8 2 4

Green leafy

vegetables 43 86 4 8 3 6 38 76 4 8 8 16

Other

vegetables 16 32 13 26 9 18 8 16 4 23 46 11 22 14 28 2 4

Fruits 36 72 5 10 6 12 3 6 42 84 4 8 2 4

Milk & its

products 48 96 2 4 49 98 1 2

Meat &

Fish 16 28 14 28 11 22 9 18 21 46 17 34 8 16 4 8

Eggs 39 78 7 14 4 8 37 74 11 22 2 4

Other

snacks

(chips,

pakoda)

23 58 6 12 8 16 13 26 14 28 10 20 17 34 9 18

Alcohol 23 46 4 8 12 24 10

22

2 20 40 12 24 9 18 9 18

8


Table 4 showed that, most of the respondents

(87%) were Non-vegetarian. Both groups of the

respondents were consuming cereals and its

products daily. The percentage of consumption of

pulses & legumes was higher (94%) in

experimental group while higher percentage of

respondents in both group consume milk and its

product daily. Fruit consumption is very low in

both groups (66%) as the comparison of other food

consumption frequency. According to Table-5

intake of most of the nutrients in the experimental

and control groups were comparatively more than

the recommended dietary allowance (RDA).

Intake of energy and fibre of both groups i.e.,

experimental and control were very less than the

RDA. On applying analysis of variance test

(ANOVA) test, it was found that there were no

significant differences with respect of all nutrients

in different age groups at 5% probability level

Table 5. Average daily Nutrient intake of the respondents in both groups

Group

Energ

y

(Kcal)

CHOs

(gm)

Protei

n

(gm)

Fat

(gm

)

Fibr

e

(gm)

Iron

(mg)

Calciu

m

(mg)

Thia

mine

(mg)

Ribofla

vin

(mg)

Niacin

e (mg)

Experi

mental

Group

Intake 1566

22

196

2.5

67

1.44

55

1.41

6.4

0.14

17

0.27

856

33

1.6

0.07

3.38

0.24

16.2

0.56

RDA 2320 195 60 25 25 17 600 1.2 1.4 16

Differ

ence -754 1 7 30 -18.6 17 256 0.47 1.98 0.2

Contro

l

Group

Intake 1564

17.1

194

2.73

68

1.21

57

0.98

5.8

0.16

17

0.26

8.26

10.22

1.53

0.07

3.41

0.24

19.1

0.58

RDA 2320 195 60 25 -25 17 600 1.2 1.4 16

Differ

ence -756 1 8 37 19.2 0 220 0.33 2.01 3.1

F. Cal. : 18.51 5.12

F. Tab. : 19.00 3.63

P 0.05

Result NS NS

9


Fig.1. Showed that F.BSL of Control and experimental groups before intervention.

Fig.2. Showed that PP of Control and experimental groups before intervention.

Effect of intervention of Biscuits incorporated

with dehydrated babul barks powder before

incorporation

Fasting and PP glucose level of control and

experimental group was taken before

incorporation of biscuits enriched with babul bark

powder. Significant differences (Appendix ‘E’) of

F.BSL was found in both the groups on applying

paired‘t’ test (t.cal of control 2.9 and experimental

4.02) (t. tab. 2.02, d.f. 49, p 0.05) and pp was

also found that significant differences on

applying‘t’ test (t.cal. of control group 3.8 and

experimental 2.3) (t.tab. 2.02, d.f. 49, p 0.05).

0

50

100

150

200

250

300

350

FASTI…

EXPERIMENTAL

CONTROL

0

50

100

150

200

250

300

350

PPEXPERIMENTAL CONTROL

10


Fig.3. Showed that F.BSL of Control and experimental groups after intervention.

Fig.4. Showed that PP of Control and experimental groups after intervention.

Effect of intervention of Biscuits incorporated

with dehydrated babul bark powder after

incorporation

Fasting and PP glucose level of control and

experimental group was taken after incorporation

of biscuits enriched with babul bark powder.

Significant differences of F.BSL was found in

both the groups on applying paired‘t’ test (t.cal of

control 3.9 and experimental 5.3) (t. tab. 2.02, d.f.

49, p 0.05) and pp was also found that

significant differences on applying‘t’ test (t.cal. of

control group 2.7 and experimental 5.2) (t.tab.

2.02, d.f. 49, p 0.05).

0

50

100

150

200

250

FASTINGEXPERIMENTAL

CONTROL

0

50

100

150

200

250

FASTINGEXPERIMENTAL

CONTROL

11


Conclusions

It is concluded that babul bark, powder mainly 5

g/day given in the form of namkin biscuit reduces

the blood glucose level in diabetic subjects and it

may be beneficial for diabetes. This is the primary

study, for a short period of time. It may be more

effective when more human studies require finding

out the effective doses and effect of doses.

However nutrition education should also be given

for the management and control of disease.

References

1. A. Banso, Photochemical and antibacterial

investigation of bark extract of Acacia

nilotica, Journal of medicinal plant research,

2009, 3(2), 082-085.

2. A.R. Pradeep, E.P. Agrwal, S.B. Bajaj, N.

Naik, Shanbhag, and S.R. Uma, Clinical and

microbiolgic effects of commercially available

gel and powder containing Acacia arabica on

gingivitis, Australian Dental Journal, 2012, 57,

312-318.

3. A.S. Andreson, An overview of diet survey

methodology, British Food Journal, 1995, 97,

ISS 7, 22-26.

4. C.D. Jensen, G.A. Spiller, and J.E. Gates, The

effect of acacia gum and a water soluble

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5. FAO 1992: Gum arabic (Published in FAO

food and nutrition paper 49, 1990), In

compendium of food additive specification.

FAO food & Nutrition paper 52 (Joint

FAO/WHO) Expert committee on food

Additive, Food & Agriculture organization,

735-737.

6. H.C. Durry, Ayurvedic useful plants of India,

with their medicinal properties and use in

medicine, Article published by Colonel Herber

Durry, 2006.

7. M. Chellapandian, and M. Murugan, Chemical

composition and nutritive value of Acacia

nilotic pods for sheep, Indian J. small rumin,

2003, 9(2), 130-132.

8. M.A. Khan, T. Khan, and Z. Ahmed, Barks

used as source of medicine in Madhya

Pradesh, India, Fitoterpia, 1994, 65, 444-446.

9. Masuellie & A. Martin, Hydrodynamic

properties of whole Arabic gum, American

Journal of Food Science and Technology,

2013, 1.3, 6.

10. P. Priyanka, and C. Maya, Management of

type 2 DM by Indian gum Arabic pods

powder, International Journals of Food &

Nutritional Search, e-ISSN: 2320-7876 Col.-2

ISS – 2, April – June, 2013.

11. S. Goels, and T. Kaur, Impact of

Hypoglycemic herbal mixture based biscuits

intervention on blood glucose level and lipid

profile of Type 2 DM, 2012, IJFANS: E ISSN

2320-7876.

12. S. Rajvaidhya, B.P. Nagori, G.K. Singh, B.K.

Dubey, P. Desai, and S. Jain, A review on

Acacia Arabica – An Indian Medicinal Plant,

International Journals of Pharma and Science

Research, 2012, 3(7), 1995-2005.

13. U.B. Cheema, J.I. Sultan, A. Javaid, P. Akhtar,

and Mr. Shahid, Chemical composition,

mineral profile and in situ digestion kinetics of

fodder leaves of four native etrees, Pakistan J.

Bot., 2011, 42(1), 397-704.

12

Vol.2 (1) 12 – 22 January 2017, ISSN 2455-5053 Science Spectrum

Morphological and Biochemical Responses to Heat Stress in

Arachis hypogaea L.

K. Rekha Rani, A. Sujitha, Y. Mrunalini, D. Sujitha and R. Usha* Department of Biotechnology, Sri Padmavati Mahila Visvavidyalayam, Tirupathi-517502, Andhra Pradesh, India


Abstract

Globally peanut (Arachis hypogaea L.) is

economically one of the important oil and food

crop and it ranks third and fourth as a source of

protein and edible-oil respectively. The world

population is increasing rapidly and may reach 6

to 9.3 billion by the year 2050, whereas the crop

production is decreasing rapidly because of the

negative impact of various environmental stresses;

therefore, it is now very important to develop

stress tolerant varieties to cope up with this

upcoming problem of food security. Abiotic

stresses are major constraints for many crop plants

in specific areas over the globe which limits the

crop production. Heat stress due to high ambient

temperatures is a serious threat to crop production

worldwide. Screening of ground nut genotypes for

high temperature stresses in natural conditions

which are highly variable is very difficult. The

adverse effects of heat stress can be mitigated by

developing crop plants with improved thermo-

tolerance using various genetic approaches.

Temperature Induction Response (TIR) technique

is the best alternative to evaluate ground nut

genotypes for thermo-tolerance. The physiological

and biochemical responses to heat stress are active

research areas, and the molecular approaches are

being adopted for developing heat stress tolerance

in plants. Most recently, biotechnology has

contributed significantly to a better understanding

of the genetic basis of heat tolerance. Molecular

knowledge of response and tolerance mechanisms

will pave the way for engineering plants that can

tolerate heat stress and could be the basis for

production of crops which can produce economic

yield under heat-stress conditions.

Keywords: Arachis hypogaea L., Heat stress,

Temperature induction response, Osmoprotectants,

Membrane thermostability.

Introduction

Globally peanut (Arachis hypogaea L.) is

economically one of the important oil and food

crop and it ranks third and fourth as a source of

protein and edible-oil respectively, with over two-

thirds of global production coming from

seasonally rainfed areas of tropical, sub-tropical

and warm regions of the world. Therefore it

constitutes an important portion of food nutrition

for people in these regions. Cultivated groundnut

or peanut (A. hypogaea L.) is a self pollinated,

allotetraploid (2n=4x=40) with a genome size of

2891 Mbp (Fig. 1) Though a native of South

America, peanut is presently cultivated mainly in

Asian (11.82 m ha), African (7.6 m ha) and

American (1.1 m ha) countries in semi- arid

regions and also in India, China, Nigeria, USA etc.

(USDA, 2013).

13

Science Spectrum Vol.2 (1) 12-22 January 2017, ISSN 2455-5053

Fig 1. Groundnut (Arachis hypogaea L.)

Impact of Heat Stress

Among the ever-changing components of the

environment, the constantly rising ambient

temperature is considered one of the most

detrimental stresses. Heat stress due to high

ambient temperatures is a serious threat to crop

production worldwide (Hall, 2001). According to a

report of the Intergovernmental Panel on Climatic

Change (IPCC, 2007), global mean temperature

will rise 0.30C per decade (Jones et al., 1999)

reaching to approximately 1 to 30C above the

present value by years 2025 and 2100,

respectively, and leading to global warming.

Rising temperatures may lead to altered

geographical distribution and growing season of

agricultural crops by allowing the threshold

temperature for the start of the season and crop

maturity to reach earlier (Porter, 2005).

Heat stress causes alterations in plant growth,

development, physiological processes, and yield

(Fig.1). One of the major consequences of heat

stress is the excess generation of reactive oxygen

species (ROS), which leads to oxidative stress

(Hasanuzzaman et al., 2013). A plant is able, to

some extent, to tolerate heat stress by physical

changes within the plant body and frequently by

creating signals for changing metabolism. Plants

alter their metabolism in various ways in response

to high temperature, particularly by producing

compatible solutes that are able to organize

proteins and cellular structures, maintain cell

turgor by osmotic adjustment, and modify the

antioxidant system to re-establish the cellular

redox balance and homeostasis (Janska et al.,

2010). At the molecular level, heat stress causes

alterations in expression of genes involved in

direct protection from high temperature

(Chinnusamy et al., 2007; Shinozaki and

Yamaguchi-Shinozaki, 2007). These include genes

responsible for the expression of osmoprotectants,

detoxifying enzymes, transporters, and regulatory

proteins (Krasensky and Jonak, 2012; Semenov

and Halford, 2009). Due to high temeparatures,

modification of physiological and biochemical

processes by gene expression changes gradually

leads to the development of heat tolerance in the

form of acclimation, or in the ideal case, to

adaptation (Moreno and Orellana, 2011;

Hasanuzzaman et al., 2010).

Direct injuries due to high temperatures include

protein denaturation and aggregation, and

increased fluidity of membrane lipids. Indirect or

slower heat injuries include inactivation of

enzymes in chloroplast and mitochondria,

inhibition of protein synthesis, protein degradation

and loss of membrane integrity (Howarth, 2005).

Fig. 2. Major effects of high temperature in Plants.

14


Heat stress also affects the organization of

microtubules by splitting and/or elongation of

spindles, formation of microtubule asters in

mitotic cells, and elongation of phragmoplast

microtubules. These injuries eventually lead to

starvation, inhibition of growth, reduced ion flux,

production of toxic compounds and reactive

oxygen species (ROS) (Schoffl et al., 1999). Plants

overcome high-temperature stress by several

physiological and biochemical mechanisms

(Rampino et al., 2009).

Morpho-Physiological Responses

When plants are exposed to high temperatures

they exhibit various mechanisms for surviving

which include long-term evolutionary,

phenological and morphological adaptations or

short-term avoidance or acclimation mechanisms

involving leaf orientation, transpiration cooling

(Wahid, 2007). Physiological role of leaf rolling

was the maintenance of adaptation potential by

increasing the efficiency of water metabolism in

the flag leaves of wheat under high temperature

(Sarieva et al., 2010). High temperature also

greatly affects starch and sucrose synthesis, by

reduced activity of sucrose phosphate synthase,

ADP-glucose pyrophosphorylase, and invertase

(Rodríguez et al., 2005). Heat imposes negative

impacts on leaf of plant like reduced leaf water

potential, reduced leaf area and pre-mature leaf

senescence which have negative impacts on total

photosynthesis performance of plant (Greer and

Weedon 2012; Young et al., 2004). Brief exposure

of plants to high temperatures during seed filling

can accelerate senescence, diminish seed set and

seed weight, and reduce yield (Siddique et al.,

1999). In temperate and tropical lowlands, heat

susceptibility is a cause of yield loss in groundnut,

Arachis hypogaea L. (Vara Prasad et al., 1999).

Under prolonged heat stress depletion of

carbohydrate reserves and plant starvation are also

observed (Djanaguiraman et al., 2009). High

temperatures can cause considerable pre- and post-

harvest damages, including scorching of leaves

and twigs, sunburns on leaves, branches and

stems, leaf senescence and abscission, shoot and

root growth inhibition, fruit discoloration and

damage, and reduced yield (Guilioni et al., 1997;

Ismail and Hall, 1999; Vollenweider and

Gunthardt- Goerg, 2005).

Similarly, in temperate regions, heat stress has

been reported as one of the most important causes

of reduction in yield and dry matter production in

many crops, including maize (Giaveno and

Ferrero, 2003). Heat stress affects the process of

photosynthesis through various means, i.e., ROS-

mediated membrane damage and reduced

chlorophyll production (Chu et al., 1974; Dhindsa

et al., 1981; Nishizawa et al., 2008).

Growth chamber and greenhouse studies suggest

that high temperature is most deleterious when

flowers are first visible and sensitivity continues

for 10–15 days. Reproductive stages of peanut are

sensitive to temperatures over 35°C and yield

losses were reported when temperatures exceeded

this threshold value (Ketring, 1984; Vara Prasad et

al., 2000). Heat stress may alter membrane lipid

bilayer structure and cause membrane protein

displacement, which together with solute leakage

is believed to contribute to the loss of membrane

selectivity (Du et al., 2011). A membrane injury

test such as electrolyte conductivity test has been

widely used to differentiate stress tolerant and

susceptible cultivars in peanut (Celikkol Akcay et

al., 2010; Ketring, 1985; Lauriano et al., 2000).

The relationship between membrane injury and

other physiological traits such as specific leaf area

and chlorophyll content was also demonstrated in

peanut (Nautiyal et al., 2008). Thus, membrane

injury tests offer a promising tool for assessing the

stress tolerance ability in peanut. In peanut,

genotypic differences in heat tolerance/

susceptibility have been reported for partitioning

of dry matter to pods and kernels (Craufurd et al.,

15


2002), fruit set, membrane thermostability

(Srinivasan et al., 1996) and chlorophyll

fluorescence (Chauhan and Senboku, 1997). In a

controlled growth chamber study (Vara prasad et

al., 2003; Talwar et al., 1999) reported that

stomatal conductance and transpiration rates

significantly increased with the increase in

temperature and higher net photosynthetic rate.

Biochemical Responses

Various analytical techniques are being used for

identification of metabolites involved in plant

adaptation to environmental changes (Fiehn, 2002;

Kaplan et al., 2004; Guy et al., 2008). Analysis of

stress-induced metabolic profiles can provide new

insights into the mechanisms of stress tolerance in

crops and the resulting information can further

assist in developing stress-tolerant cultivars

through conventional breeding and metabolic

engineering techniques (Fernie and Schauer, 2009;

Valliyodan and Nguyen, 2006).

In response to abiotic stresses, plants produce

metabolites that are involved in primary and

secondary metabolism (Rizhsky et al., 2004;

Shulaev et al., 2008). In plants, these metabolites

act as osmoprotectants, compatible solutes,

reactive oxygen species scavengers, and signal

transduction molecules (Farooq et al., 2009).

During stress, lipid peroxidation can cause severe

membrane injury and as such, can be measured to

assess the degree of heat stress in crops including

peanuts (Bajji et al., 2002; Blum and Ebercon,

1981). A General increases of unsaturated fatty

acid levels during heat stress have been reported

previously (Upchurch, 2008; Zhang et al., 2005).

Several studies reported increased amino acid

levels in response to various stresses in sensitive

cultivars (Vasquez-Robinet et al., 2008; Zuther et

al., 2007).

Their increase is sometimes attributed to stress-

induced protein degradation due to tissue damage

and senescence (Diaz et al., 2005; Widodo et al.,

2009). Leaf chlorophyll fluorescence, measured as

the ratio of variable (Fv) to maximum (Fm)

fluorescence (Fv/Fm), is extensively used in heat

stress studies by physiologists and

ecophysiologists (Burke, 2007; Maxwell, 2000).

The relationship between chlorophyll fluorescence

parameters (including Fv/Fm) and pod yield in

field-grown peanuts was also reported (Clavel et

al., 2006).

A very important adaptive mechanism under high

temperatures is the accumulation of compatible

osmoprotectants like proline, glycine betaine,

sugars, alcohols, polyamines (Hare et al., 1998).

Activities of different antioxidant enzymes such

as super oxide dismutase (SOD), catalase (CAT),

peroxidase (POX), glutathione reductase (GR) and

ascorbate peroxidase (APX) are increased with

increase in temperature. One of the primary

responses of plants exposed to high temperature is

enchanced synthesis of reactive oxygen species

(ROS) (Larkindale et al., 2005; Sung et al., 2003).

Some of the major biochemical responses include

changes in protein content, ion transporters,

signalling molecules, free radical scavengers etc.

To generate response in specific cellular

compartments or tissues against a certain stimuli,

interaction of cofactors and signaling molecules

are required. Signaling molecules are involved in

activation of stress responsive genes. Once the

stress responsive genes activate, they help to

detoxify the ROS (by activating detoxifying

enzymes, free radical scavengers); reactivate the

essential enzymes and structural proteins

(Ciarmiello et al., 2011) and all the above stated

processes help to maintain the cellular homeostasis

(Figure 3).

16


Fig.3. Schematic illustration of heat induced signal

transduction mechanism and development of heat

tolerance in plants.

Screening for Thermotolerance

Screening of ground nut genotypes for high

temperature stresses in natural conditions which

are highly variable is very difficult. The adverse

effects of heat stress can be mitigated by

developing crop plants with improved

thermotolerance using various genetic approaches.

Temperature Induction Response (TIR) technique

is the best alternative to evaluate ground nut

genotypes for thermo tolerance (Gangappa et al.,

2006). The relevance of a physiological or

biochemical trait for thermotolerance can best be

studied by pre-exposure of seedlings/plants to a

sub-lethal acclimation temperature before they are

challenged with severe temperature and

subsequently recovery growth is measured. The

seedling survival and recovery growth is

considered as criteria to arrive at optimum

acclimation stress levels. Thermoprotection on exposure to acclimation

treatment was also observed not only in seedlings

but also at mature plant level (Attaluri, 1998;

Srikanthbabu, 1999). Thermotolerant lines

developed in sunflower, pea, groundnut, pigeon

pea and few vegetable crops adopting TIR

approach substantiate the efficiency and

usefulness of this protocol (Senthil-Kumar et al.,

2003). The advantage of the TIR-based screening

method is that large number of seedlings can be

screened in a short time. It also provides an option

to screen for different temperature regimes. But

the limitation of this method is that screening is

amenable only at seedling stage. Viewed in this

context, it is important to ensure that the

genotypes selected as thermotolerant based on TIR

at seedling level also exhibit tolerance at plant

level. Only then the application of this screening

method is justified.

The best characterized aspect of acquired

thermotolerance is production of heat shock

proteins (HSPs) (Vierling, 1991; Burke, 2001; Iba,

2002). Several other studies in different species

demonstrated that upon acclimation in seedlings as

well as plants, significant increase in HSPs (HSP

70, HSP 104, HSP 90 and HSP 18.1) occurred

(Uma et al., 1995; Kumar et al., 1999;

Srikanthbabu et al., 2002). Along with different

physiological and biochemical mechanisms,

molecular approaches are boosting to understand

the concept of heat stress tolerance very clearly.

Plants tolerate such stresses by modulating

multiple genes and by coordinating the expression

of genes in different pathways (Vinocur and

Altman, 2005; Bohnert et al., 2006).

Conclusion and Future Prospects

The world population is increasing rapidly and

may reach 6 to 9.3 billion by the year 2050,

whereas the crop production is decreasing rapidly

because of the negative impact of various

environmental stresses; therefore, it is now very

important to develop stress tolerant varieties to

cope up with this upcoming problem of food

security. Abiotic stresses are major constraints for

many crop plants in specific areas over the globe

which limits the crop production. Heat stress due

to high ambient temperatures is a serious threat to

crop production worldwide. The global air

temperature is predicted to rise by 0.2°C per

decade, which will lead to temperatures 1.8–4.0°C

higher than the current level by 2100.

17


This prediction is creating apprehension among

scientists, as heat stress has known effects on the

life processes of organisms, acting directly or

through the modification of surrounding

environmental components. Screening of ground

nut genotypes for high temperature stresses in

natural conditions which are highly variable is

very difficult. The adverse effects of heat stress

can be mitigated by developing crop plants with

improved thermotolerance using various genetic

approaches. Temperature Induction Response

(TIR) technique is the best alternative to evaluate

ground nut genotypes for thermo tolerance.

TIR is a potential screening method not only to

identify contrasting genotypes differing in

thermotolerance but also to identify highly tolerant

lines from segregating progenies or from a

population. This screening protocol is robust and a

large number of seedlings can be screened in a

short period.

The physiological and biochemical responses of

peanut to heat stress are active research areas, and

most recently, biotechnology has contributed

significantly to a better understanding of the

genetic basis of heat tolerance.

Molecular knowledge of response and tolerance

mechanisms will pave the way for engineering

plants that can tolerate heat stress and could be the

basis for production of crops which can produce

economic yield under heat-stress conditions.

Functional characterization of stress inducible

genes is important not only for understanding the

molecular mechanisms of stress tolerance, but also

for practical application in improving stress

tolerance of crops by gene manipulation.

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23


A Novel and Simple Method Developed For Analysis of Iron (III) In Food

Samples

A.V.L.N.S.H. Hariharan* Department of Chemistry, GIT, GITAM University, Visakhapatnam – 530 045, India


Abstract

Extraction of iron (III) was carried out with

5.0X10-2 M of Tri capryl amine oxide [TCAO] in

benzene from aqueous hydrochloric and sulphuric

acid solutions have been done. Stripping of iron

(III) from the organic phase was attempted with

1.0M NaOH. The extractions were nearly

quantitative with the acid solutions employed in

the study. Based on the results obtained in this

study, estimation of iron in food samples as well

alloys was attempted successfully.

Key words: Extraction -iron (III) - Tri capryl

amine oxide – Food samples – Iron alloys

Introduction

Iron is one of the most essential elements in the

human body. Iron deficiency [anemia] is one of

the world’s most common nutritional deficiency

diseases (Ghadamali et al., 2009). Because of the

different biological roles of iron in humans,

animals, plants, and oceans, the need for

analysis of iron in environmental and biomedical

materials have been receiving attention. The

extraction of iron in its trivalent state from

aqueous hydrochloric (Sahu, 2000; Lee and Lee,

2005; Staszak et al., 2011; Gupta et al., 2003) and

sulphuric acid (Cattrall and west, 1966; Alguacil,

and Amer, 1986) solutions by various extractants

has been studied. As there were no reports

available on the extraction of iron (III) with Tri

capryl amine oxide [TCAO], an attempt was made

on its extraction in hydrochloric as well as

sulphuric acid solutions. The results obtained are

discussed in the present communication. The

applicability of the method was extended for

separation of Iron (III) in food samples and iron

alloys.

Experimental

Apparatus and Reagents: A stock solution of

0.3M Iron (III) was prepared by dissolving

appropriate amount of ammonium iron (III) sulfate

(E. Merck) in 500 ml of double distilled water.

The solution was standardized volumetrically with

potassium dichromate using diphenyl amine as the

indicator. Iron (III) solutions of required

concentration were prepared from the stock

solution. TCAO was synthesized (Kennedy, 1964)

by N- oxidation of Tricapryl amine using

hydrogen peroxide as oxidant. A sock solution of

5.0X10-2 M TCAO in benzene was used

throughout the course of investigations.

Determination of iron content has been done with

ELICO SL 191 UV-Visible Double beam

Spectrophotometer.

Procedure for Iron (III) Extraction: An aliquot

(10ml) of solution containing iron (III) was added

with appropriate concentration of the acid in a

separating funnel and 10 ml of 5X 10-2 M of

TCAO was added to it. The solution was

vigorously shaken for ten minutes and the two

phases were allowed to s separate. Iron (III) from

the organic phase was stripped with 10 ml of 1M

NaOH and was determined spectrophotometrically

(Vogel, 1962) at 480 nm as its colored complex

with thiocyanate. The concentration of Iron (III)

was computed from the calibration curve.

24

Vol.2 (1) 23-26 January 2017, ISSN 2455-5053 Science Spectrum

Results and Discussion

Variation of Acidity: Iron (III) was extracted from

different concentrations of the acids with 5.0X10-2

M TCAO in benzene and the results are presented

in Table 1. It was observed that the distribution

ratio (Kd) increased with increasing concentration

of the acid up to 8.0 M (98.18%) and remained

constant up to 9.0 M acidity and 10.0-11.0M

(96.89%) from sulphuric acid media respectively.

The extractions are nearly quantitative from both

the acid solutions.

Table 1. % Extraction as a function of varying

Acidity

[Fe(III) ]= 1.2 x 10-3 M [TIOA] = 5.0 x 10-2 M

Molarity

(M)

%Extraction

(HCl)

%Extraction

(H2SO4)

1.0 59.99 47.79

2.0 82.58 52.92

3.0 91.20 60.50

4.0 94.08 64.26

5.0 95.17 66.42

6.0 96.62 69.16

7.0 97.25 73.28

8.0 98.18 78.02

9.0 98.18 86.27

10.0 96.45 96.89

11.0 92.46 96.89

12.0 83.37 90.25

Composition of the Extracted species: The

extraction isotherm method (Coleman et al., 1958)

and distribution ratio method (Hesford and Mckay,

1958) were employed to determine the

composition of the extracted species. In the

extraction isotherm method the limiting ratio of

the metal to TCAO was found unity under the

experimental conditions. Representative data from

hydrochloric acid solutions has been provided in

Table 1.

Effect of TCAO concentration: With all other

factors being kept constant, iron (III) was

extracted with 10 ml of TCAO with concentration,

varying from 1.0 X 10-2 M to 4.85 X 10-2 M. The

log-log plots of Kd Vs TCAO from both the acid

solutions gave straight lines of with unit slope in

hydrochloric acid (Fig.1) and two from sulphuric

acid media respectively.

Effect of diluent: Several solvents with varying

dielectric constants were tested as the diluents

(Table 2). Quantitative extractions were achieved

with benzene as diluent. More than 80% efficiency

was obtained with carbon tetrachloride, hexane,

toluene, cyclohexane and xylene. Nitrobenzene

and n-heptane were found to be poor in extraction.

Hence benzene was preferred as diluent

throughout the study.

Effect of various stripping agents: After

extraction, iron (III) was stripped with 20ml

reagents of various concentrations (0.1 – 1 .0 M)

of HCl, H2SO4, HNO3, ACOH, and NaOH

solutions. It was observed that 1.0 M NaOH alone

is a good stripping agent. However in no case the

acid strips out all the iron (III) in a single

extraction. 99.7% iron (III) could be recovered

from organic phase by making contact four times

with equal volumes of 1.0 M NaOH.

0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

Fig.1. Extractant variation

Slope-1.0

[Fe(III)] = 1.2x10-3M

[HCl] = 8.5M

LogK

d

1+Log[TCAO]g/lt

25


Table 2. Effect of various diluents on extraction

(HCl medium)

[Fe (III)] = 1.2 x 10-3 M [TIOA] = 5.0 x 10-2 M

Diluent Dielectric

constant

%

extraction

Benzene 4.81 98.25

CHCl3 2.28 90.04

CCl4 2.23 88.81

Cyclo hexane 2.00 80.12

n-Hexane 1.89 82.17

n-heptane 1.92 73.72

Nitrobenzene 34.82 68.25

Toulene 2.43 82.34

Xylene 2.56 85.23

The observed iron: TCAO molar ratio of two

from sulphuric acid media and unity from

hydrochloric acid solutions (by distribution ratio

method) could be explained as arising from the

extraction of iron (III) by the following solvation

mechanism.:

From hydrochloric acid solutions:

(TCAO) org + H+aq

+ CrO3Cl-aq

HCrO3Cl.(TCAO) org.

From sulphuric acid solutions:

2(TCAO) org + H+aq

+ HCrO4-aq

H2CrO4.2(TCAO) org.

On the basis of the proposed mechanism for the

extraction of iron (III), the dependence of the

distribution ratio on the nature of the mineral acid

was well under stood.

Analysis of iron in various samples: The validity

of the method of extraction for recovery of iron

has been tested by analyzing food samples (Naidu

et al., 1993) and iron alloys. A known weight (1.0

gm) of the finely powdered sample was dissolved

in aquaregia. The solution was evaporated and

extracted with dilute hydrochloric acid solution.

The mixture was shaken well for about 15 min.

Then the mixture was diluted by 0.01 M HCl

solution to the mark and then filtered by

Whatmann filter paper No. 40. The first portion of

filtrate was discarded. The clear solution so

obtained was made up to 100 ml and used as stock

solution. 10 ml of this iron solution was shaken for

five minutes with an equal volume of 5.0X 10-2 M

of TCAO. After separation of two phases, Iron

(III) from the organic phase was stripped with 10

ml of 1.0M NaOH and was determined

spectrophotometrically as described earlier.

Results are presented in Tables 3& 4.

Table 3. Analysis of Iron in Food Samples

Sample % of Fe

(III)

Present

% of Fe

(III)

Found

%

Recovery

Ragi 3.00 2.86 95.34

Green

gram

4.05 3.96 97.78

Dextrin 100.22 99.65 99.45

Conclusion

The proposed method is simple, rapid and

selective. It takes less than half an hour to extract

and determine iron content in natural food samples

as well as alloys.

Acknowledgements

Thanks are due to Dr. V. Muralidhara Rao, Retd.

Professor, School of Chemistry, Andhra

University, Visakhapatnam for his valuable

suggestions. Thanks are also due to Principal, GIT

and Management of GITAM University for

providing necessary facilities.

26


Table 4. Determination of Iron in Alloys

Material Carbon Manganese Sulfur Phosphorus Silicon Iron Amount

of

Iron(III)

taken

(ppm)

Amount

of

Iron(III)

found

(ppm)

%

Recovery

Cast

Iron

3.430 0.880 0.041 ---- 2.120 91 -

91.2

91.1 90.7 99.56

Carbon

steel

0.007-

1.3

0.3-1.0 0.02-

0.06

0.002-0.1 0.005-

0.5

98.1-

99.5

99.2 98.8 99.30

Wrought

iron

0.05-

0.25

0.01-0.1 0.02-

0.1

0.05-0.2 0.02-

0.2

99-

99.8

99.5 99.6 99.59

References

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K.A.Allen, Proc.2nd Intl. Conf., Peaceful Uses

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G.R.K. Naidu, J. Ind. council chemists, chem.

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27


An Environmentally benign synthesis of N-alkyl-2-((benzimidazol-2-yl) thio)

acetonitrile

Sadhu Srinivas Rao* Department of Chemistry, Vidya Jyothi Institute of Technology (Autonomous), Himayatnagar (Vill.), C.B.Post,

Hyderabad, India – 500 075


Abstract

An environmentally benign synthesis of

N-substituted-2- ((benzimidazol-2-yl) thio)

acetonitrile (2) under different conditions has been

developed under green conditions. In this method,

2 - (( 1H-benzimidazol-2-yl) thio) acetonitrile (1)

was treated with an alkylating agent such as

DMS/DES/PhCH2Cl under green conditions i.e.,

by physical grinding in the presence of K2CO3 at

RT or by heating in PEG-600 as a green solvent at

100 0C or by irradiation with micro-waves at RT to

obtain N-alkyl-2- ((benzimidazol-2-yl) thio)

acetonitrile (2).

Key Words: 2-mercaptobenzimidazole, thiourea,

green synthesis, physical grinding.

Introduction

Benzimidazoles are very important class of

compounds due to their wide spectrum of

biological activity (Gravatt et al., 1994; Kim et al.,

1996). Benzimidazole derivatives play an

important role with diverse types of

pharmacological actions (Roth et al., 1997;

Hasegawa et al., 1975; Rovnyak et al., 1974; Bell

et al., 1976; Graber et al., 1987; Korotkikh et al.,

1995; Korotkikh et al., 1997). In continuation of

our earlier studies (Rao et al., 2013; Rao & Dubey

et al., 2016) on alkylation of 2-mercapto

benzimidazole, we now wish to report our studies

on alkylation of 2-((benzimidazol-2-yl) thio)

acetonitrile using Green methods.


Treatment of 2-mercaptobenzimidazole with

chloroacetonitrile in dimethylformamide

containing K2CO3 as base and tetra-n-butyl

ammonium bromide (TBAB) as phase transfer

catalyst for 3 h gave previously reported (S.S.Rao

et al., 2015) 1H-benzimidazol-2-ylsulfanyl)

acetonitrile (1). Reaction of 1, independently, with

each of dimethyl sulphate (DMS), diethyl sulphate

(DES) and benzyl chloride (PhCH2Cl) in the

presence of K2CO3 as a mild base, by a simple

physical grinding of the reaction mixture in a

mortar with a pestle under solvent-free conditions

for 10-15 min at RT, followed by processing, gave

respectively 1-methyl-2-chlorobenzimidazole (2a,

i.e., R=CH3) , 1-ethyl-2-chlorobenzimidazole (2b,

i.e., R=C2H5), 1-benzyl-2-chlorobenzimidazole

(2c, i.e., R=PhCH2), as the products identical with

the ones reported in the earlier methods10 in all

respects (m.p. m.m.p. and co-tlc analysis).

The reaction was also carried out in PEG-600 as a

solvent. Thus, heating a mixture of 1,

independently, with each of dimethylsulphate

(DMS), diethylsulphate (DES) and benzyl chloride

(PhCH2Cl) in PEG-600 at 100oC for 3hrs without

the use of any added base, followed by simple

processing, gave respectively 2a (i.e., 2, R=CH3),

2b (i.e., 2, R=CH2CH3) and 2c (i.e., 2, R=PhCH2)

identical with the same products obtained above. 2

could also be prepared by an alternative method.

28


Thus, 1 on treating, independently, with each of

dimethyl sulphate (DMS), diethyl sulphate (DES)

and benzyl chloride (PhCH2Cl) under microwave

irradiation conditions for 5 min and subsequent

processing, gave respectively 2a ( i.e., 2, R=CH3)

2b (i.e., 2, R=CH2CH3) , 2c ( i.e., 2, R=PhCH2)

identical with the products obtained earlier above.

Thus, the above four methods have different yields

with one suffering from relatively poor yields.

Among these, in solvent system ethanol giving

high yields where as PEG-600 giving lower yields.

Due to high molecular weight and viscous nature

of PEG-600 (135 cp at 250C) may lower reaction

rates, reduce product yields and cause the reaction

to be mass-transfer limited. Because of this, three

of the four methodologies result in the preparation

of the compounds giving high yields whereas

PEG-600 gives lower yields. Among these four

methodologies, the microwave irradiation is

superior than that of three methods because of

microwave dielectric heating is more energy

efficient than classical conductive heat transfer

methods.

Experimental Section

Preparation of 4 from 3:

Physical grinding method: A mixture of 1

(10mM), K2CO3 (20mM) and alkylating

agent(10mM) was ground together for about 10-15

min in a mortar with a pestle at RT to obtain a

homogeneous mixture.

The completion of the reaction was monitored by

TLC on prepared silica gel-G Plates using

authentic samples of the starting material and the

target compounds as references. The mixture was

then treated with ice-cold water (≈30-40ml). The

separated solid was filtered, washed with water

(2x10ml) and dried to obtain crude 2a-c. For

yields please see Table-1. Recrystallization of the

crude product from a suitable solvent gave pure

2a-c. IR, 1H-NMR and LC-MS spectra for the

compounds 2a-c were found to be in agreement

with the structures assigned to them.

In PEG-600: A mixture of 1 (10 mM), alkylating

agent (10mM) and PEG-600 (20 ml) was heated

on a steam-bath at 100oC for 3hrs. At the end of

this period, the mixture was cooled to RT and

poured into ice-cold water (≈50ml).The separated

solid was filtered, washed with water (2x10ml)

and dried. The crude products were recrystallized

from a suitable solvent to obtain pure 2a-c,

identical with the same products obtained above.

For yields please see Table-1.

Under microwave condition: A mixture of 1 (10

mM) and alkylating agent (10mM) was taken in a

10 mL CEM-reaction tube sealed by rubber

stopper and subjected to microwave irradiation for

2 min in the commercial micro-wave reactor.

After that, the tube was cooled and the completion

of reaction was checked by TLC. Then, the

reaction mixture was poured into ice-cold water

(50 mL). The separated solid was filtered, washed

with water (2x10ml) and dried. The crude products

were recrystallized from a suitable solvent to

obtain pure 3a-c, identical with the same products

obtained above. For yields, please see Table-1.

Conclusion

In conclusion, we have developed a green

methodology for the synthesis of N-substituted-2-

thiobenzimidazole under different conditions.

Acknowledgement

The author is highly indebted to UGC New Delhi

for sanctioned Minor Research Project and also

thankful to Principal, Vidya Jyothi Institute of

Technology, Hyderabad.

29


SCHEME: 1

(2a, R = CH3; 2b, R = C2H5; 2c, R = CH2Ph)

Table.1. Preparation of 2a-c from 1 under different green conditions

M.P. of 2a: 108-111 0C (Lit. (10) m.p. 110-112 0C)

M.P. of 2b: 99-101 0C (Lit. (10) m.p. 98-100 0C)

M.P. of 2c: 85-88 0C (Lit. (10) m.p. 86-88 0C )

*Yield refers to isolated crude product only.

References:

1. D. R. Graber, R. A. Morge and Raenko,

Reaction of 2-(alkylsulfinyl)-, 2-

(arylsulfinyl)-, and (aralkylsulfinyl)

benzimidazoles with thiols: a convenient

synthesis of unsymmetrical disulfides,

Journal of Org. Chem, 1987, 52, 4620.

S.

No.

S

M

Reagent

Prod

uct

Physical grinding

Green solvent Microwave

irradiation

PEG-600

Time

(Min)

Tem

p

(0C)

Yield*

(%)

Time

(Min)

Tem

p

(0C)

Yield

*

(%)

Time

(Min)

Temp

(0C)

Yield

*

(%)

1.

1

DMS 2a 10-15 RT 89 180 100 69 2 RT /

450 W

87

DES 2b 10-15 RT 87 180 100 72 2 RT /

450 W

89

PhCH2Cl 2c 10-15 RT 85 180 100 63 2 RT /

450 W

83

30


2. G. Rovnyak, V. L. Narayana, R. D.

Haugwitz and C. M. Cimarusti, US Pat,

014, 1973, Chem. Abstr, 1974, 105596.

3. G.L. Gravatt, B.C. Baugley , W. R. Wilson

and W. A. Denny, DNA-Directed Alkylating

Agents, Synthesis and Antitumor Activity of

DNA Minor Groove-Targeted Aniline

Mustard Analogs of Pibenzimol (Hoechst

33258), J. Med. Chem., 1994, 37,4338.

4. H. Hasegawa, N. Tsuda and M. Hasoya,

Japanese Pat, 1974, 198; Chem Abstr, 1975,

156308.

5. J. S. Kim, B. Gatto and L. F. Liu,

Substituted 2,5‘-Bi-1H-benzimidazoles:

Topoisomerase I Inhibition and

Cytotoxicity, Eur J Med Chem, 1996, 39,

992.

6. N. I. Korotkikh, G. F. Raenko and O. P.

Shavaika, Reaction of some azoles

and azolinthiones with 2-chlorobenzoxazole,

Chem. Heterocycl. compd, 1995, 31, 359.

7. N. I. Korotkikh, A. F. Aslanov and G. F.

Raenko, Heterocyclizations of 2-

allylthiobenzimidazoles under the action

of bromine, Russ. J. Org. Chem, 1995, 31,

721; Chem. Abstr, 1997, 18833.

8. S. C. Bell and P. H. Wei, Syntheses of

heterocyclic fused thiazole acetic acids, J.

Med. Chem., 1976, 19, 524.

9. S. S. Rao, P. K. Dubey & Y. B. Kumari, A

green and simple synthesis of 2-mercapto

benzimidazoles , Indian J. Chem, 2013, 52B,

1210.

10. S. S. Rao, Ch. V. R. Reddy & P. K. Dubey,

Highly efficient tandem syntheses of

unsymmetrically substituted isomeric S, N–

disubstituted-2-mercaptobenzimidazoles,

Indian J. Chem, 2015, 54, 829-832.

11. S. S. Rao, Ch. V. R.Reddy & P. K. Dubey,

Synthesis of α-Benzylthiobenzimidazole

acetonitriles and Their Chemoselective

Reduction of the Double Bond with NaBH4,

J. Heterocyclic. Chem, 2016, 53 (74). DOI

10.1002/jhet.2282.

12. T. Roth, M. L. Morningstar, P. L. Boyer, S.

H. Hughes, R. W. Buckheit and C. J.

Michejda, Synthesis and Biological Activity

of Novel Nonnucleoside Inhibitors of HIV-1

Reverse Transcriptase. 2-Aryl-Substituted

Benzimidazoles, J. Med. Chem., 1997, 40,

4199.

31


A New Validated RP-HPLC Method for the Quantification of Cabazitaxel -

An Anticancer Plant product

Mathrusri Annapurna Mukthinuthalapati* and Venkatesh Bukkapatnam Department of Pharmaceutical Analysis & Quality Assurance, GITAM Institute of Pharmacy, GITAM University,

Visakhapatnam, India


Abstract

Cabazitaxel, a semi-synthetic derivative of a

natural taxoid extracted from the yew tree needles.

Cabazitaxel is recommended for the treatment of

metastatic castration-resistant prostate cancer. A

new stability-indicating high performance liquid

chromatographic technique was developed for

Cabazitaxel in the presence of the degradation.

Chromatographic elution was performed on

Shimadzu Model CBM-20A/20 Alite, equipped

with Zorbax SB-C18 column (150 mm × 4.6 mm

i.d., 3.5 µm particle size) using mobile phase

consisting of sodium acetate buffer and methanol

(flow rate of 1.0 ml/min). Forced/Stress

degradation studies were performed with various

conditions (acidic, alkaline, oxidation, thermal and

photolytic degradations) and the method was

validated (ICH guidelines). The method follows

the Beer-Lambert’s law over a linearity range 0.1–

200 µg/ml with linear regression equation, y =

21316x - 3152 (r2 = 0.9999). It was observed that

Cabazitaxel is more sensitive towards basic

environment in comparison to other stress

conditions. The projected method is accurate,

precise and economical and it is practically useful

in quality control department for the analysis of

Cabazitaxel marketed formulations as well as

biological fluids.

Keywords: Cabazitaxel, Taxoid, Anti-cancer, RP-

HPLC, Stability-indicating, Validation.

Introduction

Cabazitaxel is a new generation taxane used for

the management of hormone-refractory prostate

cancer (Neil, 2006). It is a dimethyloxy derivative

of docetaxel. The methyl group moieties provide

Cabazitaxel an uncommon capability among

chemotherapy agents i.e. the capability to cross the

blood–brain barrier. It is effective against

docetaxel-refractory prostate cancer. Cabazitaxel

is used for the treatment of prostate cancer. The

anti-tumour activity was proven by blocking

tumour cell division (Jordan, 2004) and also

stabilizes the microtubules. In the US Cabazitaxel

has been accepted by the Food and Drug

Administration (Oudard, 2011) in June 2010 and

in Europe by the European Medicines Agency in

January 2011 for the patients with castration

resistant metastatic prostate cancer whose disease

progresses after docetaxel treatment (Assessment

Report, 2011) along with prednisone.

Current research is going on metastatic breast

cancer (Pivot, 2008) (Villanueva, 2011).

Cabazitaxel (Figure 1) is chemically known as

(2aR, 4S, 4aS, 6R, 9S, 11S, 12S, 12aR, 12bS)-

12b- acetoxy- 9-(((2R, 3S)- 3-((tert-

butoxycarbonyl) amino)- 2- hydroxy- 3- phenyl

propanoyl) oxy)- 11-hydroxy- 4, 6- dimethoxy- 4a,

8, 13, 13- tetramethyl- 5- oxo- 2a, 3, 4, 4a, 5, 6, 9,

10, 11, 12, 12a, 12b- dodecahydro- 1H- 7, 11-

methanocyclodeca benzo [1, 2-b] oxet-12-yl

benzoate (C45H57NO14) with molecular weight

835.93 g/mol. (Sanofi-Aventis, 2006), (Cheetham,

2013).

32


Fig.1. Structure of Cabazitaxel

The analytical methods so far developed are based

on LC-MS/MS (Kort, 2013), (Jagannath, 2012),

(Peter, 2012), spectrophotometry (Kishore, 2012)

and RP-HPLC (Mathrusri, 2013) (Mathrusri,

2014a) (Mathrusri, 2014b) techniques. A present

the authors have proposed a robust, and selective

(stability indicating RP-HPLC method for the

estimation of Cabazitaxel in presence of its

degradants.

Materials and Methods

Chemicals and reagents: Cabazitaxel was

obtained from Dr. Reddy’s Laboratories Ltd.,

(India) and all other chemicals used were of AR

grade. Methanol (HPLC grade), Glacial acetic acid

(HPLC grade), Hydrogen peroxide, Hydrochloric

acid, and Sodium hydroxide were purchased from

Merck (India) and used as received. Cabazitaxel is

available as infusion with brand name Jevtana®

(Sanofi-Aventis, Malaysia) with label claim of 60

mg of drug.

Instrumentation and Chromatographic

conditions: Shimadzu Model CBM-20A/20 Alite

HPLC system with PDA detector and Zorbax SB-

C18 column (150 mm × 4.6 mm i.d., 3.5 µm) was

used for chromatographic separation. Isocratic

mode of elution was selected with sodium acetate

buffer and methanol (20:80, v/v) mixture as

mobile phase at a flow rate of 1.0 ml/min. The

overall run time was 10 min (UV detection at 234

nm).

Preparation of stock solution: The stock solution

(1000 µg/ml) was prepared by transferring about

25 mg of Cabazitaxel in to a 25 ml volumetric

flask and dissolved in methanol. Working standard

solutions were prepared from stock on dilution as

per the requirement and filtered through 0.45 µm

membrane filter.

Preparation of sodium acetate buffer solution

(pH 4.0): The buffer solution (pH 4.0) was

prepared by thorough mixing of 28.6ml of glacial

acetic acid with 10 ml of 50% w/v NaOH in a

1000 ml volumetric flask with the help of HPLC

grade water.

Method Validation

The proposed method was validated according to

ICH prescribed validation parameters such as

linearity, precision, accuracy, limit of quantitation

(LOQ), limit of detection (LOD), selectivity and

robustness (ICH, 2005). Linearity was conducted

by using a series of solutions (0.1–200 µg/ml)

prepared from the stock solution by dilution and

introduced in to the HPLC system (n=3). The

average peak area of the chromatograms was

plotted against concentration to construct the

calibration curve. The limit of quantification and

limit of detection were also determined from the

calibration curve.

The intra-day and inter-day precision studies were

conducted (20, 50 and 100 µg/ml) and the %

relative standard deviation was calculated. The

accuracy study was performed by standard

addition and recovery experiments (80, 100 and

120%) and the percentage recoveries were

calculated. Robustness studies were performed by

incorporating small changes in the parameters

such as wavelength (232 and 236 nm),

composition of mobile phase (78 and 82%) and

flow rate (0.9 and 1.1 ml/min). Forced degradation

studies (ICH, 2003) were evaluated by refluxing

the drug solution (1 mg/ml) to different treatments

for 30 min at 80 ºC in a thermostat.

33


Acidic degradation was conducted by exposing

Cabazitaxel solution to 1 N hydrochloric acid for

30 min at 80 ºC. The stressed sample was then

cooled and neutralized prior to dilution with

mobile phase. Alkaline stress degradation study

was conducted with 1ml 0.01 N NaOH and

neutralized prior to dilution with mobile phase.

Oxidative degradation was studied using 1 ml of

30 % hydrogen peroxide and thermal degradation

studies were conducted in thermostat at 80 ºC for

30 min. 20 µL solution of these solutions were

injected in to the HPLC system and the percentage

recoveries calculated from the recorded

chromatograms.

As the Cabazitaxel was not available as

formulations the drug was quantitatively mixed

with some of the commonly available excipients in

the laboratory, extracted with mobile phase and

percentage recovery was calculated.


Method optimization: Initially mobile phases of

various reagents and compositions with the

available columns in the laboratory were tried to

achieve better resolution and separation. Among

those trials, the stressed samples analyzed using

sodium acetate: methanol with a ratio 45:55, v/v

have shown unsymmetrical peaks. Therefore, the

mobile phase composition was modified as 20:80,

v/v by which a sharp peak was eluted at 4.175 ±

0.02 min without tailing (UV detection 234 nm)

and therefore taken as the greatest

chromatographic response for the study.

The representative chromatograms of the blank as

well as the drug solution was shown in Figure 3A

and 3B respectively. The present developed liquid

chromatographic method (stability indicating) was

compared with the previously published methods

in Table 1.

Table 1. Comparison of the present proposed method with the previously published liquid

chromatographic methods.

Mobile phase/Reagent λ

(nm)

Linearity

(g/ml) Remarks Reference

Phosphate buffer: Acetonitrile

(30:70, v/v) 230 0.1-150 HPLC (Mathrusri, 2013)

Sodium acetate buffer: Acetonitrile

(30:70, v/v) 234 0.1-250 HPLC

(Mathrusri,

2014a)

0.1% ortho phosphoric acid and methanol

(20:80, v/v) 210 0.1-200 HPLC

(Mathrusri,

2014b)

Ammonium hydroxide and acetonitrile

(83:17, v/v) (pH 3 ± 0.1) 275 2-20 LC-MS (Kort, 2013)

Acetonitrile: Ammonium acetate (80:20, v/v) 236 2.49-99.60 (LC-MS/MS)

(dried blood spots) (Jagannath, 2012)

Acetonitrile: Ammonium formate (gradient) 362 (10-100) 103 (LC-MS)

Human plasma (Peter, 2012)

Sodium acetate buffer: Methanol (20: 80, v/v) 234 0.1-200 Stability indicating

HPLC (PDA detector) Present work

34


Method Validation: Cabazitaxel has shown

linearity over a concentration range of 0.1–200

μg/ml (Table 2) (% RSD 0.11-0.54) with linear

regression equation y = 21316x - 3152

(r2 = 0.9999) (Figure 2). The LOQ was found to

be 0.0967 μg/ml and the LOD was found to be

0.0319 μg/ml.

Table 2. Linearity of Cabazitaxel

Conc. (g/ml) *Mean peak area ± SD * RSD (%)

0.1 1942 ± 8.74 0.35

0.5 9968 ± 44.86 0.45

1 20354 ± 50.89 0.25

5 104251 ± 229.35 0.22

10 214033 ± 813.33 0.38

20 415242 ± 415.24 0.10

50 1064255 ± 4789.15 0.55

100 2100511 ± 6091.48 0.29

150 3212545 ± 20881.54 0.65

200 4260567 ± 15764.10 0.37

*Mean of three replicates

Fig. 2. Calibration curve of Cabazitaxel

For the precision studies the % RSD range was

found to be 0.19-0.25 (Intra-day) and 0.15-0.20

(Inter-day) which was within the limit (<2%)

whereas in accuracy studies the % RSD was 0.26-

0.49 with a recovery of 99.35-99.51%. Table 3

indicates the results representing that the method

is precise and accurate. The results obtained in the

robustness study were shown in Table 4 in which

the % RSD was found to be less than 2.0% (0.22-

1.34) indicating that the method is robust.

35


Table 3. Precision and accuracy studies of Cabazitaxel

Conc.

(µg/ml)

Intra-day precision Inter-day precision

* Mean peak area ± SD

(% RSD)

*Mean peak area ± SD

(% RSD)

20 415070.00±784.28 (0.19) 414654.67±624.89 (0.15)

50 1064426.33±5328.44 (0.22) 1062260.00±2108.43 (0.20)

100 2104855.00±5328.44 (0.25) 2096391.33±3730.38 (0.18)

Accuracy

Spiked conc.

(µg/ml)

Total conc.

(µg/ml)

* Mean peak area ± SD (%

RSD)

Drug

Found

(µg/ml)

% Recovery*

8 (80%) 18 384345.33±989.57 (0.26) 17.88 99.35

10 (100%) 20 427383.67±1466.92 (0.34) 19.90 99.51

12 (120%) 22 469499.00±2300.15 (0.49) 21.88 99.44


Table 4. Robustness study of Cabazitaxel

Parameter Condition *Mean peak area ± SD

(% RSD)

*Assay

(%)

Flow rate

(± 0.1 mL/min)

0.9 2095489.33 ± 4678.33

(0.22) 99.76 1.0

1.1

Detection wavelength

(± 2 nm)

232 2103571.33 ± 10710.59

(0.51) 100.15 234

236

Mobile phase composition

(TBAHS: methanol)

(± 2 %, v/v)

18:82 2098611.00 ± 17866.93

(0.85) 99.91 20:80

22:78

pH (± 0.1 unit)

3.9 2086517.67 ± 2803.89

(1.34) 99.33 4.0

4.1


36

Vol.2 (1) 31-38 January 2017, ISSN 2455

Forced degradation studies:

indicating capability of the method was

established from the separation of Cabazitaxel

peak from the degraded samples to judge the

specificity of the developed method. The typical

chromatograms obtained for the for

degradation studies were shown in Figure 3C

Cabazitaxel during basic stress indicating has

undergone 9.60 % degradation with degradant at

2.306 min and 2.569 min showing that the drug is

sensitive towards basic environment. In the drug

structure the amino group may be responsible for

this degradation. Cabazitaxel solution was seen

decomposing on exposure to acidic (0.99 %),

alkaline (9.60 %), oxidative (0.41 %) thermal

(0.77 %) and photolytic (0.31 %) conditions

Table 5. Forced degradation studies of Cabazitaxel

Stress Condition *Drug recovered

(%)

Standard (Untreated)

100

Acidic degradation

99.01

Alkaline degradation

90.40

Oxidative degradation 99.59

Thermal degradation 99.23

Photolytic degradation 99.69


Fig. 3. Typical chromatograms of blank [A], Cabazitaxel (100 μg/ml) oxidative [E],

January 2017, ISSN 2455-5053

Forced degradation studies: The stability

indicating capability of the method was

established from the separation of Cabazitaxel

peak from the degraded samples to judge the

specificity of the developed method. The typical

chromatograms obtained for the forced

degradation studies were shown in Figure 3C-3G.

Cabazitaxel during basic stress indicating has

undergone 9.60 % degradation with degradant at

2.306 min and 2.569 min showing that the drug is

sensitive towards basic environment. In the drug

e amino group may be responsible for

this degradation. Cabazitaxel solution was seen

decomposing on exposure to acidic (0.99 %),

alkaline (9.60 %), oxidative (0.41 %) thermal

(0.77 %) and photolytic (0.31 %) conditions

(Table 5). From these studiesl it is

drug is much sensitive towards alkaline

conditions. The system suitability parameters for

the Cabazitaxel peak shows that the theoretical

plates were more than 2000 and the tailing factor

was less than 2 (or <1.5-2.0) (Table 5).The 3D

chromatograms obtained during the forced

degradation studies were shown in Figure 4.

The present proposed stability-

quantification of Cabazitaxel is specific because

the drug peak was well separated even in presence

of degradation products. The proposed method

was applied to the laboratory prepared formulation

and the percentage recovery was calculated as

99.52.

degradation studies of Cabazitaxel

*Drug recovered *Drug decomposed

(%)

Theoretical

Plates

100 - 2854.152

99.01 0.99 2732.556

90.40 9.60 2810.816

99.59 0.41 2719.496

99.23 0.77 2818.388

99.69 0.31 2861.906

Typical chromatograms of blank [A], Cabazitaxel (100 μg/ml) [B], acidic [C], alkaline [D],

oxidative [E], thermal [F] and photolytic [G] degradations

Science Spectrum

it is concluded that

is much sensitive towards alkaline

The system suitability parameters for

the Cabazitaxel peak shows that the theoretical

plates were more than 2000 and the tailing factor

2.0) (Table 5).The 3D

tograms obtained during the forced

degradation studies were shown in Figure 4.

-indicating for the

is specific because

the drug peak was well separated even in presence

The proposed method

was applied to the laboratory prepared formulation

and the percentage recovery was calculated as

Tailing factor

1.056

1.039

1.046

1.046

1.046

1.050

acidic [C], alkaline [D],

37


Fig.4. A) Cabazitaxel standard B) acid degradation C) base degradation D) Oxidation degradation E)

Thermal degradation F) Photolytic degradation

38


Conclusion The developed stability-indicating liquid chromatographic method was validated as per ICH guidelines and can be applied for the pharmaceutical dosage forms and also for the accelerated stability studies as well as for the determination of drug in biological fluids. Acknowledgements The authors are grateful to University Grants Commission, New Delhi, India for their financial support and M/s GITAM University, Visakhapatnam for providing the research facilities. The authors have no conflict of interest. References 1. "Jevtana (Cabazitaxel) Injection approved by

U.S. FDA after priority review" (Press release), Sanofi-Aventis, 2010-06-17. Retrieved June 17, 2010.

2. A. Kort., M.J.X. Hillebrand, G. A. Cirkel, E. E. Voest, A. H. Schinkel, H. Rosing, J. H. M. Schellens, J.H. Beijnen, J.Chromatogr. B. 2013, 925, 117-123.

3. Assessment Report for Jevtana (Cabazitaxel), Procedure No.: EMEA/H/C/002018, European Medicines Agency, London, 2011.

4. C. Villanueva, A. Awada, M. Campone, J. P. Machiels, T. Besse, E. Magherini, F. Dubin, D. Semiond, X. Pivot, European Journal of Cancer 2011, 47, 1037-1045.

5. G. Kishore, Int. J. Res. Rev. Pharm. Appl. Sci. 2012, 2, 950-958.

6. ICH stability testing of new drug substances and products Q1A (R2), International Conference on Harmonization, 2003.

7. ICH validation of analytical procedures: text

and methodology Q2 (R1), International Conference on Harmonization, 2005.

8. M. A. Jordan, L. Wilson, Nat Rev Cancer 2004, 4, 253–265.

9. M. J. O’Neil, (Ed.) The Merck Index, Merck Research Laboratories, Whitehouse Station, NJ, 2006.

10. M. Mathrusri Annapurna, B. Venkatesh, G. Naga Supriya, J. Bioeq. Bioavai 2014, 6, 134-138.

11. M.Mathrusri Annapurna, B. Venkatesh, K. Pramadvara, S. Hemchand, Chem. Sci. Trans. 2014, 3, 854-860.

12. M. Mathrusri Annapurna, K. Pramadvara, B. Venkatesh, G. Sowjanya, Indo American J. Pharm. Res. 2013, 3, 9262-9269.

13. P. Cheetham, and D.P. Petrylak, Cancer Journal 2013, 19, 59-65.

14. Peter de Bruijn, Anne-Joy M de Graan, Annemieke Nieuweboer, Ron HJ Mathijssen, Mei-Ho Lam, Ronald de Wit, Erik, AC Wiemer, Walter J Loos, J. Pharm. Biomed. Analy. 2012, 59, 117–122.

15. S. Oudard, Future Oncology 2011, 7, 497-506.

16. V Jagannath Patro, R. Nageshwara Rao, N. K. Tripathy, Open Access Scientific Reports 2012, 1, 1-4.

17. X. Pivot, P. Koralewski, J. L. Hidalgo, A. Chan, A. Goncalves, G. Schwartsmann, S. Assadourian, J. P. Lotz, Annals of Oncology, 2008, 19, 1547-1552.

39


Cloud Data Security using hybrid RSA and Cuckoo Search Algorithm

A. Arjuna Rao, K. Sujatha*, P. Praveen Kumar and V. Sravani I K C

Department of CSE, Miracle Educational Society Group of Institutions, Bhogapuram, Vizianagram, India


Abstract

Cloud provides the means to instantly use new

services and expand the infrastructure. However

lack of physical control, bring a whole host of

Cloud Security Issues and arises problems like

data deletion, leakage etc. Encryption of data

stored in cloud allows solving these issues. RSA

Algorithm is public key encryption algorithm

proposed by Rivest-Shamir-Adleman. This has

been used for years due to its simplicity and

security. Hybrid RSA and Cuckoo Search

Algorithm is proposed here to enhance the security

of RSA algorithm. Cuckoo Search Algorithm is

the optimization algorithm that is used to select the

random values used in RSA algorithm. This is

based on biological facts where Cuckoo Birds

selects the nests of other birds to lay the eggs.

Cloud Data Security is provided by using this

hybrid algorithm. This algorithm provides

enhanced security and effectively uses the

algorithm. Test Results are simulated which

proves that the proposed algorithm provides

reliable confidentiality.

Keywords: Cloud Security Issues, Authentication,

data deletion, leakage, Encryption, RSA

Algorithm, Rivest-Shamir-Adleman, Hybrid RSA

and Cuckoo Search Algorithm, Cloud Data

Security.

Introduction

Cloud computing security refers to the set of

procedures, processes and standards designed to

provide information security assurance in a cloud

computing environment. Cloud computing

security addresses both physical and logical

security issues across all the different service

models of software, platform and infrastructure.

This also addresses how these services are

delivered. Cloud security encompasses a broad

range of security constraints from an end-user and

cloud provider's perspective, where the end-user

will primarily will be concerned with the

provider's security policy, how and where their

data is stored and who has access to that data. For

a cloud provider, on the other hand, cloud

computer security issues can range from the

physical security of the infrastructure and the

access control mechanism of cloud assets, to the

execution and maintenance of security policy.

Cloud security is important because it is probably

the biggest reason why organizations fear the

cloud. Cloud Encryption can be used within the

AppProtex Cloud Security Gateway which acts to

protect data – both at rest and in the cloud – from

unauthorized access. One can learn more

about cloud data encryption(Samiksha et.al.,

2011).

Cloud knowledge Security may be achieved

through watching and reportage on cloud use via

management console that permits users to outline

and maintain knowledge discovery, analysis and

protection policies. The Cloud Security Alliance

(CSA), a noncommercial organization of trade

specialists, has developed a pool of pointers and

frameworks for implementing and imposing

security among a cloud in operation surroundings.

40


RSA serves as solution to these problems (Yang

et.al., 2009).

Problems identified in Cloud Storage

Data can be stolen

Information is misused

Unauthorised access

Modification of content

Secured data is attacked

Cloud Data Storage using Hybrid RSA and CS

Algorithm

Security can be provided to the data stored in

Cloud by using Hybrid RSA and CS Algorithm.

This maintains the confidentiality and integrity in

data that is stored in cloud. Unauthorized users

cannot access the contents that are placed in cloud.

The data stored cannot be altered or modified as

this is not understood by them.

Cuckoo Search Algorithm: Cuckoo search (CS)

is associate degree optimisation algorithmic rule

developed by Xin-she principle and Suash woman

in 2009. It had been galvanized by the obligate

brood mutuality of some cuckoo species by

parturition their eggs within the nests of different

host birds (of different species). Some host birds

will have interaction direct conflict with the

intrusive cuckoos. As an example, if a bird

discovers the eggs don't seem to be here own, it'll

either throw these alien eggs away or just abandon

its nest and build a brand new nest elsewhere.

Some cuckoo species like the New World brood-

parasitic Tapera have evolved in such some way

that feminine parasitic cuckoos square measure

typically terribly specialised within the mimicry in

colours and pattern of the eggs of many chosen

host species (Samiksha et.al., 2011).

Cuckoo search perfect such breeding behavior,

and therefore is applied for varied optimisation

issues. It looks that it will surmount different

metaheuristic algorithms in applications. Cuckoo

search (CS) uses the subsequent representations.

Each egg during a nest represents an answer, and a

cuckoo egg represents a brand new resolution. The

aim is to use the new and doubtless higher

resolutions (cuckoos) to exchange a not-so-good

solution within the nests. Within the simplest type,

every nest has one egg. The algorithmic rule is

extended to additional difficult cases during which

every nest has multiple eggs representing a group

of solutions (Yang et.al., 2009).CS is predicated on

3 idealized rules:

1. Every cuckoo lays one egg at a time, and

dumps its egg in an exceedingly arbitrarily

chosen nest;

2. The simplest nests with prime quality of

eggs can carry over to ensuing generation;

3. The quantity of accessible hosts nests is

fastened, and also the egg set by a cuckoo is

discovered by the host bird with a likelihood

electronic device in (0,1). Discovering treat

some set of worst nests, and discovered

solutions drop from farther calculations.

Modified RSA: RSA technique is one of the most

popular public-key techniques and is predicted on

the problem of factoring large numbers. RSA is a

cryptosystem that supports public-key encryption.

This is widely used for securing sensitive data

particularly when being sent over an insecure

network such as the Internet (Zhang et al., 2011).

The public and therefore the private key-generation

algorithmic design is that the most advanced a part

of RSA cryptography and requires choosing

optimal parameter. Two massive prime numbers, p

and q, area unit generated exploitation the Rabin-

Miller property take a look at algorithmic program.

A modulus n is calculated by multiplying p and

alphabetic character (Chhabra et.al, 2011). This

range is employed by each the general public and

personal keys and provides the link between them.

Its length, typically expressed in bits, is named the

key length. the general public key consists of the

modulus n, and a public exponent, e, that is

ordinarily set at 65537, as it is a prime that's not

large. The e figure ought not to be a on the Q.T.

41


designated prime because the public secret's shared

with everybody. The personal key consists of the

modulus n and therefore the personal exponent d,

that is calculated exploitation the Extended

geometer algorithmic program to search out the

opposite with relevance the totient of n(Taher,

1985).

The changed RSA algorithmic program with

cuckoo search is as follows.

1. First, a few of enormous primes p and letter

of the alphabet square measure elite by

victimization cuckoo search algorithmic

program.

2. victimization p and letter of the alphabet, n

and Ø square measure calculated.

n= p * q

Ø = (p-1)*(q-1)

3. Exponent e is chosen basing on n and

personal exponent d from e, p and q. Here,

(n, e) is treated because the public key and

(n, d) because the non-public key.

4. The RSA encoding shown in equation (7) is

that the mathematical operation to the eth

power modulo n

C = Me mod n - (7)

5. The decoding shown in equation (8) is

performed as mathematical operation to the

dth power modulo n

M = Cd mod n - (8)

Encryption of knowledge that's hold on in cloud is

handled by MRSA that is encrypted by

victimization the general public key and may be

decrypted solely by the user UN agency possesses

the non-public key. therefore any user UN agency

has the shared public key of given user will

encode the info however solely the desired user

will decode.

Result

RSA implements construct of public-key

cryptography. This could produce smaller, quicker

and a lot of economical scientific discipline keys.

RSA authors specify that the cryptography time

per block will increase no quicker than the cube of

the quantity of digits in n. The secured algorithmic

rule is developed and tested victimization

numerous sample sets of information and is found

secure. Then this is often tested on cloud

application to supply unified security to cloud. The

RSA and Modified RSA(MRSA) are compared

and shown in table 1.

Table 1. Accuracy Percentage comparing RSA

and MRSA

Sample Set RSA(%) MRSA(%)

Set 1 82 98

Set 2 88 97

Set 3 85 100

Set 4 88 98

Set 5 90 97

Set 6 84 99

From table 1 it can be analysed that MRSA is

more accurate compared to RSA as this generates

results accurately. The encryption and decryption

values are considered from each sample set to find

the accuracy count. Hence MRSA is preferable

technique for performing encryption.

Conclusion

The issue of security was handled by enforcing

encryption to cloud data security. All the issues in

manual system are solved by using this automated

encryption system. This can be used in any

system that requires identifying the user securely

and with reliability. Security is never limited to an

application and hence this algorithm can be used

in many related problems which are having such

issues. Cloud is becoming popular along with

Internet of things and people worldwide started

using cloud to store their images, photographs and

data. Hence Security is always a issue in cloud.

This is resolved by using Modified RSA that

provides both authentication and privacy. This is

basically popular public key cryptographic

algorithm with optimally choosing parameters by

using Cuckoo Search. The future scope includes

42


applying other optimization algorithms to choose

the parameters of RSA and compare them to

identify the best technique.

References

1. A.Chhabra, S.Mathur, Modified RSA

Algorithm: A Secure Approach,

Computational Intelligence and

Communication Networks (CICN), IEEE,

2011 International Conference, 7-9 Oct.

2011, 545 – 548.

2. Sameera Abdulrahman Almulla and Chan

Yeob Yeun, Cloud computing security

management, Engineering Systems

Management and Its Applications

(ICESMA), 2010 Second International

Conference, 2010, 1-7.

3. Samiksha Goel, Arpita Sharma, Punam

Bedi, Cuckoo Search Clustering Algorithm:

A novel strategy of biomimicry, Information

and Communication Technologies (WICT),

2011 World Congress, 2011, 916 – 921.

4. Taher El Gamal, A public key cryptosystem

and a signature scheme based on discrete

logarithms, in Proceedings of CRYPTO 84

on Advances in cryptology, Springer-Verlag

New York, Inc., 1985, 10–18.

5. Xin Zhou and Xiaofei Tang, Research and

implementation of RSA algorithm for

encryption and decryption, Strategic

Technology (IFOST), 2011, 6th

International Forum, Aug. 2011, Vol 2, 1118

– 1121, IEEE.

6. Xin-She Yang, Suash Deb, Cuckoo Search

via Lévy flights, Nature & Biologically

Inspired Computing, 2009, NaBIC 2009,

World Congress, 2009, 210 – 214.

7. Zhang Qing, Hu Zhihua, The Large Prime

Numbers Generation of RSA Algorithm

Based on Genetic Algorithm, Intelligence

Science and Information Engineering (ISIE),

2011 International Conference on 20-21,

Aug. 2011, 434 – 437.

43


Structural and Dielectric Properties of Sm3+ Doped SrTiO3 Ceramic Powders

J.Guravamma, C.Sai Vandana and B.H.Rudramadevi*

Department of Physics, Sri Venkateswara University, Tirupati-517 502 *For Correspondence: [email protected]

Abstract

The structural and dielectric properties of SrTiO3

and Sm3+:SrTiO3 ceramic powders were prepared

by using conventional solid-state reaction method.

The samples were characterized by using the

XRD, FTIR, and SEM with EDS for structural,

functional groups, morphological and elemental

analysis respectively. For Sm3+:SrTiO3 ceramic

powders, the dielectric properties such as

dielectric constant (εr), dielectric loss (tan δ) as

well as ac conductivity in the frequency range

(100Hz – 1MHz) were studied for their use of

technological applications such as capacitors,

transducers, actuators, and nonvolatile random

access memory devices etc.

Keywords: SrTiO3, XRD, FTIR, SEM &

Dielectric measurements

Introduction

Ceramic compounds exhibiting the high dielectric

constant are of enormous importance to the

electronic industry due to their wide range of

applications such as capacitors, sensors, actuators,

power transmission devices, memory devices and

high energy storage devices (Saifi and Cross,

1970; Sakudo and Unoki, 1971; Ueno et al., 2008;

Roth, 1957; Song et al., 1996). Most of the high

dielectric constant materials contain the lead

which causes environmentally pollution and are

harmful to human beings due to the toxicity of

lead oxide. Therefore compulsory to search

alternative lead free compounds for such

applications which should take the comparable and

superior dielectric properties of SrTiO 3 is one of

the ferroelectric materials having ABO3 type

perovskite structure (Dietz et al., 1995;

Schumacher et al., 1995). TiO2 posses the good

mechanical resistance and stability and therefore it

is widely used for development of the stable host

matrices like as SrTiO3, BaTiO3 CaTiO3. Solid

solutions of SrTiO3 recently studied for

microwave devices and gas sensors. Among all the

lanthanide ions, the samarium (Sm3+) ion has been

known as the most efficient and could be

converting in to ultraviolet light to visible light

emissions (Longo et al., 2008; Tagantsev et al.,

2003; Kanemitsu and Yamada, 2011), because the

doping of rare earth ion. Samarium (Sm3+) ion has

been known as the most efficient down–converting

material and it could convert the ultraviolet light to

visible emissions which can be reabsorbed by dyes

in Dye-Sensitized Solar Cells (Kan et al., 2005;

Takashima et al., 2009).In this paper we

demonstrated a systematic study of structural and

dielectric properties of samarium doped strontium

titanate ceramic powders. Samarium ions doped

normally occupy the Sr+2 sites in a perovskite

structure. The Sm3+ doped SrTiO3 ceramics for the

applications of capacitors are used in high voltage

conditions. For these high voltage applications we

need a high storage dielectric ceramics. For this

purpose samarium doped strontium titanate can be

used in energy storage applications.

Experimental Procedure

Materials: Sm2O3 (99.9% purity), SrCO3 and TiO2

(Annular grade) were used as the raw chemicals to

prepare the SrTiO3 and (0.2 mol %) Sm3+:SrTiO3

ceramic powders by a solid state reaction method.

44


Preparation: Suitably weighed chemicals were

mixed with acetone for homogeneous mixing in an

agate mortar for 1hr and later it was collected into

a porcelain crucible for its appropriate heating.

The temperature was gradually raised from room

temperature to 1150°C, at this temperature the

chemical mixture was kept for about 4h in an

ambient atmosphere with intermediate grinding.

After that, the resultant powders were ground and

pelletized at 50 MPa pressure into disks of 10 mm

in diameter and1 mm in thickness. The final

products obtained were used for further

characterizations.

Characterizations: The structure of the ceramic

powders is examined by powder X-ray diffraction

technique using X-ray diffractometer with nickel

filtered Cu Kα radiation (Model Philips Expert

Pro). The infrared (IR) spectra of the samples were

recorded in the range 400 – 4000 cm−1 on a

Thermo Nicolet Avatar 370 Fourier Transform

Infrared (FTIR) Spectrometer using KBr pellet

method. SEM and EDAX studies were carried out

using Philips XL30 ESEM and JEOL JSM 840

electron microscope. The dielectric constant (ε')

and dielectric loss factor (tan δ) and ac

conductivity of the sintered pellets were measured

using an impedance analyzer (Model PSM 1700

RS232).


The prepared samples were analyzed by powder X-

ray diffraction using Cu-Kα radiation (λ=1.5406 Ǻ)

with 2θ ranging from 20˚ to 80˚ with a scan rate of

0.02 steps per second. Fig.1shows the XRD pattern

of the SrTiO3 and Sm3+:SrTiO3 ceramic powders

and they are indexed on the basis of the reflections

from the JCPDS file no. 84- 0443. The major

phase was identified to be cubic SrTiO3 with,

(110), (111), (200), (210), (211), (220) and (310)

reflections appearing at 32.0˚, 39.7˚, 46.1˚, 52.0˚,

57.5˚, 67.6˚, and 77.1˚ 2θ positions respectively.

The phase transition is important role in the

Powder diffraction and is mainly classified in to

two types.

Fig.1. XRD pattern of host SrTiO3 and Sm3+:

SrTiO3 ceramic powders

The first transition is reconstructive nature in the

crystal structure and second transition is displacive

nature. The powder diffraction is very useful in

first order transition. The main difference between

the crystal structure and phases means it is

difficult to use the structural information obtained

from the one phases to second one. The unit cell of

the both the phases often related to the space

group symmetries.

The symmetries changes from the one crystal

system to another one that is cubic to tetragonal or

orthorhombic or monoclinic. For cubic →

tetragonal system is presented in the Sm3+:SrTiO3

powder diffraction. In this system the (100) cubic

peak splits in to two peaks with indices (100) and

(001) in that case a ≠ c. Likewise the (110) cubic

peak will splits in to two peaks with indices (110)

and (101). However, the (111) cubic peak will not

split under this symmetry transformations. The

(200) cubic peak will be split into two peaks with

indices (2 0 0) and (0 0 2).The interplanar spacing

for (011); i.e., d110 was calculated from the

corresponding 2θ position using Bragg law as

described in the experimental methodology section

and found to be 2.7939 Ǻ and then theSrTiO3

lattice constant, crystalline size will be calculated

is about 3.898 Ǻ and 54 nm (Dietz et al., 1995;

Souza et al., 2012; Schumacher et al., 1995).

45


Fig.2. FTIR spectrum of prepared SrTiO3 ceramic

powder

The Fig. 2 shows the FTIR spectrum of the SrTiO3

ceramic powder pellet. The bands at 1466 cm−1 are

due to the stretching and bending vibrations of

water molecule. The band at 852 cm−1 is assigned

to stretching vibrations of Sr-O (Komatsu et al.,

1998).

The band at 558 cm−1 corresponds to stretching

vibrations of TiO2 and the band at 1466 cm-1 is

related to asymmetric stretch of COO- (Sulaeman

et al., 2011).

Fig. 3 shows the SEM & EDS images of the pure

SrTiO3 and Sm3+: SrTiO3 Ceramic powders. The

SEM images of the samples had obvious pores or

displayed homogeneous microstructure. The grain

size of SrTiO3 and Sm3+:SrTiO3 samples are about

184.3 and 241.4 nm respectively. The grain size of

the samarium doped strontium titanate is increased

compared to the pure strontium titanate. We

believe that these fine particles belong to the

second phase and that their sizes are on the

nanometer scale. These nanosize particles increase

the scattering centers as well as decrease the

thermal conductivity and electrical conductivity.

The energy dispersive x-ray spectroscopy (EDS)

spectrum shows the elements that are present in

SrTiO3 and Sm3+:SrTiO3 ceramic powders.

Fig.3. The SEM&EDS images of the host SrTiO3 and Sm3+:SrTiO3 ceramic powders

46


Fig.4. Variation of dielectric constant ( ) and loss factor (tan) of SrTiO3 ceramic powder pellet

and inset figure shows the ac conductivity of the SrTiO3 ceramic powder pellet

The Fig. 4 shows the frequency dependence of the

real part 'of the dielectric constant and loss

factor (tan) values as a function of log (w) for the

host SrTiO3 pellet and was studied in the

frequency range 100Hz – 1MHz at room

temperature. The dielectric constant was

calculated using this formula '= cd/OA where c is

the capacitance of the sample, d is the thickness,

O is the vacuum dielectric constant (8.85x10-12

farad/m) and A is the cross-sectional area of the

sample. The dielectric constant of SrTiO3 is found

to be 1880 (by using formula) Dielectric loss can

be calculated using this formula tan = ''/' where

'' is the imaginary part of the dielectric constant

and ' is the real part of the dielectric constant. The

value of dielectric loss is found to be 1625 x10-4.

From the figures it is observed that dielectric

constant (') and dielectric loss (tan) values are

decreasing with an increase in frequency of

strontium titanate ceramic powder. When the

frequency of the applied electric field increased to

a certain value that is more than the frequency of

dielectric relaxation polarization, the contribution

of dielectric relaxation polarization to dielectric

constant decreased, which is attributed to

Maxwell-Wagner relaxation behavior (Zhang et

al., 2010; Kristina Zagar et al., 2013; Wu et al.,

2012). Hence the dielectric constant decreased as

the frequency increased, and dielectric loss was

evident in the dielectric constant. Also, the

dielectric constant and loss increased with

increasing due to less contribution of ions in the

direction of applied electric field.The inset figure

shows that the ac conductivity of the SrTiO3

ceramic powder pellet as a function of log (w) at

room temperature. The ac conductivity was

calculated using this formula σ ac = Or w tan

where O is the vacuum dielectric constant

(8.85x10-12 farad/m), r is the relative dielectric

constant, w is the angular frequency (w=2πυ) and

tan is the loss factor. The ac conductivity of the

SrTiO3 is found to be 0.56x102 S/cm. From the

figure it is observed that the ac conductivity is

increasing with increasing the frequency. At high

frequency, the conductivity is increased. Where it

obeys the power law relation that is σ (w) = Aws;

w is the angular frequency of ac signal.

47


Fig.5. Variation of dielectric constant ( ) and loss factor (tan) of Sm3+:SrTiO3ceramic powder pellet

and inset figure shows the ac conductivity of the Sm3+:SrTiO3ceramic powder pellet

The Fig. 5 shows that the dielectric constant and

loss factor tan values as a function of log (w) for

the Sm3+:SrTiO3 pellet and was studied in the

frequency range 100Hz – 1MHz at room

temperature. From the figures it is observed that

dielectric constant () and dielectric loss (tan)

values are decreasing with an increase in

frequency of samarium doped strontium titanate

ceramic powder. The high dielectric constant is

also related to the substitution of Sm3+ to Sr2+ It is

reported that the rare earth doped SrTiO3 ceramics

with dielectric constant is increased compared to

that of pure SrTiO3 value is 1.24x1011, dielectric

loss lower than 0.87 and the ac conductivity is

1.32x105 S/cm were obtained. Sr vacancies and Ti

vacancies charge compensation mechanism is

reasonable for high dielectric constant trivalent

rare-earth doped SrTiO3 ceramics. In this work,

the trivalent ions Sm3+ is replaced the divalent ions

Sr2+ at the A-sites of the perovskite lattice would

result in Sr-site vacancies to maintain the charge

equilibrium. It is believed that a small quantity of

Sr-site vacancies will be helpful to improve the

dielectric constant. It is clear that all doped

ceramic powders show higher values than the un

doped ceramic powder due to the introduction of

localized states (Jung, and Lee, 2013).

Conclusions

SrTiO3 and Sm3+:SrTiO3 ceramics powders were

successfully synthesized by solid-state reaction

method. The strontium titanate powder X-ray

diffraction analysis confirms the phase transition

occurred.The strontium titanate can be changed in

to cubic to tetragonal nature. The crystalline size

of the powder has been calculated by using the

Scherrer’s equation. The SEM&EDS image shows

the morphological and elemental analysis. By

using the SEM we calculated the grain size of

SrTiO3 and Sm3+:SrTiO3 powders. The samarium

doped strontium titanate grain size was increased

compared to the host strontium titanate ceramic

powder. The functional groups and structural

phase transition have been evaluated from the

FTIR analysis. The dielectric properties of SrTiO3

and Sm3+:SrTiO3 ions also studied. Sm3+doped

SrTiO3 can be considered as a promising dielectric

material due to the increased dielectric constant,

48


dielectric loss and increasing ac conductivity

values compared to the undoped SrTiO3. Therefore

Sm3+:SrTiO3 is good dielectric material and it can

be used high voltage capacitors in energy storage

applications.

References

1. A.E. Souza, G.T.A. Santos, B.C. Barra, W.D. Macedo Jr., S.R. Teixeira, C.M. Santos, A.M.O.R. Senos, L. Amaral, and E. Longo, Cryst. Growth Des., 2012, 12, 5671–5679.

2. A.K.Tagantsev, V.O. Sherman, K.F. Astafiev, J. Venkatesh, N. Setter, J.Electroceram., 2003, 11, 5–66.

3. D. Kan, T. Terashima, R. Kanda, A. Masuno, K. Tanaka, S. Chu, H. Kan, A. Ishizumi, Y. Kanemitsu, Y. Shimakawa, and M. Takano, Nat. Mater., 2005, 4, 816–819.

4. G.W. Dietz, W. Antpohler, M. Klee, and R. Waser, J. Appl. Phys., 1995, 78, 6113-6121.

5. H. Takashima, K. Shimada, N. Miura, T. Katsumata, Y. Inaguma, and K. Ueda, Adv. Mater., 2009, 21, 3699–3702.

6. H.S. Jung, and J.K. Lee, J. Phys. Chem. Lett. 2013, 4, 1682-1693.

7. J. Zhang, C. Tang, and J.H. Bang, Electrochem. Commun., 2010, 12, 1124-1128.

8. K. Ueno, S. Nakamura, H. Shimotani, A. Ohtomo, N. Kimura, T. Nojima, H. Aoki, Y. Iwasa, and M. Kawasaki, Nature Mater, 2008, 7, 855-858.

9. Kristina Zagar, Cristian Fàbrega, Francisco Hernandez-Ramirez, Joan Daniel Prades, Joan Ramon Morante, Aleksander Recnik, and Miran Ceh, Mater.Chem.Phys., 2013,141, 9-13.

10. M. A. Saifi and L. E. Cross, Phys. Rev. B2, 1970, 677-684.

11. M. Schumacher, G.W. Dietz, R. Waser, Integrated Ferroelectrics, 1995, 10, 231-245.

12. M.X. Wu, X. Lin, Y.D. Wang, L. Wang, W. Guo, D.D. Qi, X.J. Peng, A. Hagfeldt, M. Gratzel, and T. Ma, J. Am. Chem. Soc., 2012, 134, 3419-3421.

13. R.S. Roth, J. Research NBS, RP.1957, 58, 75-88.

14. S. Komatsu, K. Abe, and N. Fukushima, Japanese, J. Appl. Phys., 1998, 37, 5651-5654.

15. T. Sakudo and H. Unoki, Phys. Rev. Lett., 1971, 26, 851-854.

16. T.K. Song, J. Kim, S.I. Kwun, Solid State Commun., 1996, 97, 143-147.

17. U. Sulaeman, S. Yin and T. Sato, Applied catalysis B: Environmental, 2011, 102, 286-290.

18. V.M. Longo, A.T. de Figueiredo, S. de Lázaro, M.F. Gurgel, M.G.S. Costa, C.O. Paiva Santos, J.A. Varela, E. Longo, V.R. Mastelaro, F.S. de Vicente, A.C. Hernandes, R.W.A. Franco, J. Appl. Phys., 2008,104, 023515 (1-11).

19. Y. Kanemitsu, Y. Yamada, Phys. Status Solidi B2, 2011, 48 (2), 416–421.

49


Construction of Subsets of B and B0

S.Nagendra1 and Dr. E. Keshava Reddy2 1Department of Mathematics, Govt. Degree College, Porumamilla, Kadapa- 516193

2Department of Mathematics, JNTUA, Anantapur * For Correspondence: [email protected]

Abstract

In continuation to the study of subsets of the Bloch space B and the Little Bloch space B0 (Nagendra and

Keshava Reddy, 2015), we construct new subsets of B and B0 based on the results obtained in Bloch

Multipliers by Nagendra and Keshava Reddy (2015).

Keywords: Bloch space, Little Bloch space, Hp spaces, Minkowski’s inequality

Introduction

Let be the unit disc in the Complex plane� . An analytic function :f � is said to be a Bloch

function if f 2'sup 1z

f z z

. The function space of Bloch functions is called Bloch Space and

denoted by B. It is known that B is a Banach Space with respect to the norm defined as

2'0 sup 1B

z

f f f z z

.

The subspace of B denoted by B0 known as the little Bloch Space consists of members of B for which

lim|�|→��′(�)�(1 − |�|�)= 0.

Simple examples of Bloch functions are polynomials which are also in B0. For detailed discussion and

interesting theorems related to B and B0, an interested reader can refer to Dinakar ().

In the Study of subsets of the Bloch space and the Little Bloch space (Nagendra and Keshava Reddy,

2015), we made a detailed study of function spaces such as��, BMOA, VMOA, A, D in view of subsets

of B or B0. In the next sections, we construct new subsets of B and B0.

Subset of Bloch Multipliers

In this section, we prepare new subsets of B based upon the results obtained in Bloch Multipliers (2015).

In this, the concept of Bloch Multiplier was defined and the collection of Bloch multipliers was denoted

by ‘M’ which was proved to be a normed linear space. Theorem-1 and 2 give necessary and sufficient

conditions for Bloch multipliers. While doing this, we came across a new function space denoted by ‘L’

which was also a normed linear space. We brief about these concepts below.

� = {∅: ∆→ ℂ:∅� ∈ ��ℎ�� ∈ �}

� = {∅: ∆→ ℂ ∶ ∅��(∅)< ∞}

Where �(∅)= (1 − |�|�)|∅�(�)|�∈∆��

�1 + log�

��|�|�

For details of these concepts, we refer reader to (Nagendra and Keshava Reddy, 2015).

50


Theorems-1 and 2 of Bloch Multipliers by Nagendra and Keshava Reddy (2015) imply that

a) � ∩ �� = �

b) From a) M L and M H

c) From definition of M, once ∅ ∈ � ⇒ ∅ ∈ � which implies that M B

d) L B

For ∅ ∈ �, |∅�(�)|(1 − |�|�)< �(∅)

2'sup 1

z

z z L

B

L B

M L B

In the next section, we prepare new subsets which are similar to �� spaces.

A. �� space:

With the inspiration obtained from �� spaces (Nagendra and Keshava Reddy, 2015), we started

constructing �� type space which we are calling �� space.

Definition

Let �: ∆→ ℂ be an analytic function and � ∈ [0,1[, � > 0 then � ∈ �� if f

��(�, �)< +∞��

where

��(�, �)= � �

��∫ ��

��

��

� ��

Clearly polynomials are members of ��.

Now let us do some propositions about ��.

Proposition-1

lim�→��(�, �)= �� where �� = max|�|��|��(�)|

Proof: For � > 0,�� − � is not maximum of |��(�)| on |�| = �. So there exists � > 0 such that

��|� − ��| <�2� ⇒ �� > �� − �

For given� ∈ [0,1[, �� = ��, then

��

2��

��

(�� − �)< ��

2��

��

�� ≤ ��

2��

��

�� = ��

��

��

��

��

��

��

�

��

≤ ��(�, �)≤ ��

taking � → ∞ in the above inequality, we get

�

2�(�� − �)≤ ��(�, �)≤ ��, ∀� ∈ [0,1]

∴ ��(�, �)= �� = |��(�)|.�∈∆��

51


Using above proposition, we define

�� = ��: ∆→ ℂ ∶ �� |��(�)| < +∞�∈∆��

�

It is to note that �(�)= log(1 − �), � ∈ ∆ is not a member of �� as

|��(�)| = �1

� − 1�

For� = � ∈ [0,1[, |��(�)| = ��

�� → ∞�� → 1�

Proposition-2

For 0 < � < �, �� ⊆ �� and hence �� ⊆ ��,∀� > 0

Proof: For this, we use

0 < � < � ⇒ �� < �� + 1, ∀� ≥ 0

Now for � ∈ ��,

��(�, �)=

�

��

��

�

��

��

��

�

��

�

< ��(�, �)+ 1

<+∞

∴ � ∈ ��

Therefore, �� ⊆ ��0 < � < �

It is to note that �� ⊆ ��,∀� > 0.

In the next section, we discuss the normed linear structure of �� and it’s containment in B0.

B. Linear structure of ��:

For �, � ∈ ��,

��(� + �, �)= ��

�� (��)��

��

��

�

�

��

≤ � �

��∫ ��

��

��

��

Using Minkowski’s inequality, we have

��(� + �, �)≤ ��(�, �)+ ��(�, �)

< +∞

⇒ � + � ∈ ��

For � ∈ ℂ�� ∈ ��,��(�, �)= � �

��∫ �(��)��

��

��

��

52


= |�|��(�, �)< +∞

⇒ �� ∈ ��.

∴ (��,+, . )/ℂ�� a linear space.

�� can also be made a normed linear space equipped with norm

‖�‖�� = |�(0)| + ��(�, �)��

Where ��(�, �) is defined as in this section 6.2.1

Clearly ‖�‖�� ≥ 0 and if ‖�‖�� = 0

�(0)= 0�� = 0, ∀�&∀�

�(0)= 0��

⇒ � ≡ 0

For �, � ∈ K�,

‖� + �‖�� = |(� + �)(0)| + ��(� + �, �)��

Using Minkowski’s inequality

≤ ‖�‖�� + ‖�‖��

For � ∈ ℂ, � ∈ K�, ‖αf‖�� = |(αf)(0)| + K�(αf, r)��

= |�|‖�‖��

Therefore (��,+, . , ‖ ‖��)��.

Next we prove a theorem about containment of �� in B.

Theorem: �� ⊆ ��

Proof: If� ∈ ��, then by definition

��(�, �)< +∞��

⇒ ��(�, �)≤ ‖�‖��, ∀0 ≤ � < 1

⇒ ��(�, �)≤ ‖�‖��, ∀0 ≤ � < 1

⇒�

��

��

�

�� ≤ ‖�‖��, ∀0 ≤ � < 1…… . . (∗)

The above inequality implies that

�� is bounded ∀0 ≤ � < 1��0 ≤ � < 2�………(∗∗)

53


Otherwise, let ��

� is unbounded for 0 ≤ � < 2� and for some �� ∈ [0,2�].From the theory of

Riemann integration

�

��∫ ��

��

�� would be unbounded which contradicts equ.(*).

Therefore assertion (**) is true and thus �� is bounded ∀0 ≤ � < 1��0 ≤ � < 2�

Hence |��(�)| ≤ �, ∀� ∈ ∆ and for some � > 0

⇒ lim|�|→��(1 − |�|�)|��(�)| = 0

⇒ � ∈ ��

Hence � ∈ �.

Hence the theorem.

Conclusions

In this paper, we constructed two subsets ‘M’ and ‘L’ of B. Also defined and discussed about �� spaces

and proved these spaces as subsets of��.

References

1. S. Nagendra and E. Keshava Reddy, Study of subsets of the Bloch space and the Little Bloch space

published in IJSIMR, 2015, 3(1), pp. 345-348, ISSN 2347-307X.

2. S. Nagendra and E. Keshava Reddy, Bloch Multipliers by published in IJPAMS, 2015, 8(1), pp.55-60,

ISSN 0972-9828.

3. N. Danikar, Some Banach spaces of Analytic functions, Aristotle Univ. of Thessaloniki.

54


Influence of silver nitrate and different carbon sources on in vitro shoot

development in Solanum nigrum (Linn)-an important antiulcer medicinal

plant

G. Geetha, K. Harathi, D. Giribabu and C.V. Naidu*

Department of Biotechnology, Dravidian University, Kuppam-517426, A.P, India


Abstract

In the present study the influence of different

carbon sources such as sucrose, fructose, glucose

and maltose (1-6%) and ethylene inhibitor silver

nitrate (0.4 mg/l) along with 1.0 mg/l Kn and 0.1

mg/l NAA was investigated for the development

of multiple shoots from axillary bud or nodal

explants of Solanum nigrum. The regeneration

frequency, growth and multiplication rate were

highly influenced by the type and concentration of

carbohydrates and AgNO3 used. The highest

number of shoots (34.5) was obtained on MS

medium augmented with 4% fructose and 0.4mg/l

AgNO3 when compared to media devoid of

AgNO3. In the absence of carbohydrates there is

no regeneration was found. Observations of the

shoot cultures developed on media containing one

of these carbohydrates indicated that 4% fructose

was the preferential carbohydrate for the

proliferation of multiple shoots followed by

sucrose, maltose and glucose from nodal explants

of S. nigrum.

Key words Solanum nigrum, Axillary bud

explants, Micropropagation, Silver nitrate, Carbon

sources.

Abbreviations

BAP 6 – benzyl amino purine

AgNO3 Silver nitrate

NAA α – naphthalene acetic acid

IAA Indole – 3 – acetic acid

IBA Indole – 3 – butyric acid

MS Murashige and Skoog

Introduction

Solanum nigrum is an important herbaceous

medicinal plant belongs to solanaceae family.

Solanacae family comprises a number of plants

widely known for the presence of natural products

of medical significance mainly steroidal lactones,

glycosides, alkaloids and flavonoids. The herb is

antiseptic, antidysentric and diuretic used in the

treatment of cardiac, skin disease, psoriasis,

herpivirus and inflammation of kidney. The fruits

and leaves have been traditionally used against

various nerve disorders (Perez et al., 1998). It has

very important gastric ulcerogenic activities

(Aktar and Munir, 1989), and is recommended in

ayurveda for the management of gastric ulcers.

Most prominent medical properties are the

presence of alkaloids solamargin and solosonin

which yield solasodine as glycone. Solasodine has

embryogenic, teratonic and antimicrobial activities

(Kim et al., 1996). The growth and multiplication

of shoots in vitro are affected by many factors

(Anwar et al., 2005), one of which was the

concentration and type of exogenous carbon

sources added to medium to serve as energy and

also to maintain the osmotic potential (Lipavska

and Konradova, 2004). In general sucrose is the

carbohydrate of choice as a carbon source for in

vitro plant culture probably, because it is the most

common carbohydrate in the phloem sap of many

plants (Murashige and Skoog, 1962; Lemos and

Baker, 1998; Fuents et al., 2000). Although

sucrose has been the carbohydrate of choice in

vast majority of work on in vitro shoot induction

55


and shoot development, it is not always the most

effective carbon source for these purpose

(Thomson and Thrope, 1987).

The involvement of ethylene in plant tissue growth

and differentiation has been widely investigated.

Application of ethylene precursors and/or

inhibitors has shown that ethylene may often have

diverse effects in similar tissue culture systems.

Although it has been reported that ethylene may

promote callus growth (Songstad et al., 1991), it

generally appears to inhibit shoot regeneration

(Biddington, 1992). Silver nitrate (AgNO3), a

potent inhibitor of ethylene action (Beyer, 1976),

was shown to promote regeneration in Brassica

campestris (Palmer, 1992) and Helianthus annuus

(Chraibi et al., 1991).

Therefore, the aim of the present study was to

determine the effect of silver nitrate and different

carbon sources such as sucrose, glucose, fructose

and maltose on in vitro shoot regeneration from

nodal explants of Solanum nigrum.

Materials and methods

Source of plant material: Healthy axillary bud

and leaf explants of Solanum nigrum (L.) were

collected from two-month-old seed germinated

field grown plants growing in the Herbal garden of

Dravidian University, Kuppam, Andhra Pradesh,

India.

Surface sterilization: Explants were washed

thoroughly under running tap water to remove

traces of dust etc. followed by treatment with 10%

teeepol or tween -20 for 5 minutes and 0.4%

bavistine (fungicide) for 10-15 minutes. Then the

explants were sterilized in 70% alcohol for a

minute, and finally with 0.01% mercuric chloride

for1-2 minutes and washed 3-4 times with sterile

double distilled water.

Culture Medium: The explants were inoculated

on MS medium (Murashige and Skoog, 1962)

containing different carbohydrates and gelled with

0.8% agar, supplemented with BAP (2.0mg/l) in

combination with NAA (0.5mg/l) and AgNO3

(0.4mg/l). The pH of the medium was adjusted to

5.8 before gelling with agar and autoclaved for 20

minutes at 121°C and 15 lbs pressure.

Sub culturing: The cultures were maintained by

regular subculture at 4 week intervals on fresh MS

medium.

Culture conditions: All cultures of Solanum

nigrum were maintained in a culture room at

temperature of 24 ± 2°C and 55-65% RH with 16

h/8 h photoperiod at a photon flux density of 3000

lux or 50-70 Em-2 s-1 provided by cool white

fluorescent tubes.

Data collection and statistical analysis: Visual

observations were recorded on the frequency in

terms of number of cultures responding for

axillary shoot proliferation, shoot development,

number of shoots per explant, average length of

the regenerated shoots, and number of roots per

shoot and average root length.

Despite scarcity and limitations encountered with

the plant material, for most of the treatment a

minimum of 10 replicates were used. All the

experiments were repeated at least twice/thrice and

the cultures were observed at regular intervals.

The qualitative data were subjected to statistical

analysis by using standard error (SE±) for shoot

length, rate of shoot multiplication and then

number of roots per shoot.


Influence of silver nitrate and various carbon

sources on shoot regeneration from axillary

bud explants: Experiments were conducted with

axillary bud explants at 0.4 mg/l AgNO3 to find

more effective energy source for in vitro

propagation of Solanum nigrum. After pursuing

the observations depicted in the table-1 the type

and amount of carbohydrates on multiple shoot

proliferation are significantly influenced by silver

nitrate in comparison with normal control. The

regeneration frequency which indicates the

56


survival rate of explants has varied depending on

the presence of silver nitrate and energy sources

supplemented to the media. Silver nitrate supplied

media had shown maximum survival rate

compared to control. The media free from energy

source had not shown any regeneration where as

media supplemented with 0.4 mg/l AgNO3 along

with 3% sucrose and 4% fructose had shown

maximum regeneration frequency as high as 95%

and 92% respectively. Among all the

carbohydrates, fructose supplemented media

grown explants had shown better regeneration

frequency ranging from 65% to 92%. The fructose

at 4% in the presence of silver nitrate gave

significantly higher mean number of shoots (34.5

± 0.36) followed by 4% sucrose (27.6 ± 0.25). The

results obtained obviously indicate (Fig-1)

supplementation of silver nitrate along with

various concentrations and types of carbohydrates

in the media had increased the shoots proliferation

over three folds compared to control.

Similar to the multiple shoot regeneration, mean

shoot length was also influenced by silver nitrate

on various types and concentrations of

carbohydrates. The maximum mean shoot length

was given by AgNO3 supplemented media along

with 4% sucrose (11.4 ± 0.30 cm) followed by 4%

fructose (10.4 ± 0.25 cm).

In plant tissue culture continuous supply of

carbohydrate is essential, since the photosynthetic

activity of in vitro plant tissue is reduced due to

low light intensity, high humidity and limited

gaseous exchange (Kozai, 1991a). The type and

levels of exogenous carbohydrate supplements

play a dynamic role in plant growth and

multiplication (Hossain et al., 2005). Surpassing of

glucose over sucrose in the investigation could be

explained, as the presence of sucrose might cause

hypoxia and ethanol accumulation in cells due to

quick metabolisation. But when it comes to

longevity parameter under investigation, 4%

sucrose besides showing 95% regeneration

percentage proved to be promising in repeated sub

culturing. Whereas 4% glucose, though

appreciably extended with 80% withering,

yellowish lean shoots development in next sub

culturing on the same media. Hence there is a

prospect of using 3% sucrose which had promoted

more healthy shoots and high survival frequency

(95%), instead of 4% glucose for further

investigations. However, it is also reported by

many other researchers that the different carbon

sources like glucose, fructose and maltose are also

proved to better carbohydrate sources for in vitro

propagation. Fructose have been reported to be

effective in preventing hyperhydricity and helps in

production of adventitious shoots in Almonds

(Rugini et al., 1987), also achieved maximum

number of shoots in mulberry (Vijay chitra and

Padmaja, 2002), in Mentha piperita (Sujana and

Naidu, 2011). Another major carbon source in

culture media proved to be glucose used in Prunus

mume (Hisashi and Yasuhiro, 1996).

Presence of silver nitrate in in vitro propagation

showed distinct significance in almost all

parameters under present investigation. When

media was devoid of energy source there was no

growth at all and media without silver nitrate

showed lesser response. This clearly explains that

the presence of optimal AgNO3 concentration at

particular carbohydrate concentration had

increased almost three folds in shoot proliferation.

This may be due to presence of silver nitrate,

which might have suppressed the activity of

AgNO3 might have promoted production of poly

amines (Roustan et al., 1990), which implicate

easy translocation and assimilation of these energy

sources available in the media by the explants

resulting in cell division and leading to vigorous

growth. In addition, sugar sensitive plant gene

which plays an important role in cellular

adjustment to critical nutrient availability might

show carbohydrate modulated gene expression at

various levels (Koch, 1996).

57


Table 1. Effect of AgNO3 on different carbohydrates on multiple shoot regeneration from axillary bud

explants of Solanum nigrum supplemented with BAP 2.0 mg/l and NAA 0.5 mg/l. Observation: After 8

weeks, values are mean ± S.E. of 10 independent determinants.

Carbohydrat

e Source

Concentrati

on (%)

Regeneration

frequency (%)

Mean number of

shoots/explants

Mean shoot length

(cm)

Without

AgNO3

0.4mg/l

AgNO3

Without

AgNO3

0.4mg/l

AgNO3

Without

AgNO3

0.4mg/l

AgNO3

Control No

carbohydr-

ate

- - - - - -

Glucose 1.0 55 65 4.5 ± 0.35 10.8 ± 0.25 2.8 ± 0.35 4.4 ± 0.26

2.0 60 72 6.4 ± 0.19 14.6 ± 0.30 4.5 ± 0.39 5.8 ± 0.50

3.0 68 75 7.9 ± 0.47 18.5 ± 0.36 6.8 ± 0.47 9.5 ± 0.28

4.0 75 80 11.5 ± 0.2 21.9 ± 0.20 4.53 ± 0.32 7.56 ± 0.32

5.0 70 75 5.4 ± 0.41 16.9 ± 0.15 3.96 ± 0.40 6.4 ± 0.43

6.0 60 70 3.6 ± 0.25 12.7 ± 0.2 2.9 ±0.17 4.5 ± 0.2

Sucrose 1.0 58 80 3.5 ± 0.18 16.9 ± 0.3 3.1± 0.32 4.53 ± 0.15

2.0 70 85 5.4 ± 0.29 20.5 ± 0.36 3.39 ±0.28 6.3 ± 0.20

3.0 85 95 9.4 ± 0.40 24.3 ± 0.37 5.5 ± 0.15 8.9 ± 0.3

4.0 80 92 12.5 ± 0.28 27.6 ± 0.25 8.0 ± 0.41 11.4 ± 0.30

5.0 70 85 6.9 ± 0.3 17.4 ± 0.41 5.35 ± 0.16 7.5 ± 0.18

6.0 65 70 4.0 ± 0.29 9.9 ± 0.36 3.4 ± 0.28 4.2 ± 0.15

Fructose 1.0 62 70 7.3 ± 0.32 13.5 ± 0.18 2.5 ±0.35 3.9 ± 0.32

2.0 65 75 9.4 ± 0.40 17.8 ± 0.47 3.6 ± 0.30 5.7 ± 0.17

3.0 75 85 11.7 ± 0.15 23.4 ± 0.3 4.41 ± 0.42 7.56 ± 0.41

4.0 85 92 14.3 ± 0.30 34.5 ± 0.36 7.63 ± 0.37 10.4 ± 0.25

5.0 70 75 8.5 ± 0.17 19.0 ± 0.34 5.3 ± 0.26 6.5 ± 0.35

6.0 60 65 4.4 ± 0.25 10.4 ± 0.45 2.2 ± 0.07 4.46 ± 0.03

Maltose 1.0 50 58 2.9 ± 0.30 9.5 ± 0.35 1.76 ± 0.15 2.53 ± 0.37

2.0 55 60 6.4 ± 0.25 11.4 ± 0.40 3.4 ± 0.1 3.9 ± 0.50

3.0 62 65 7.7 ± 0.1 15.7 ± 0.36 3.9 ± 0.35 6.6 ± 0.23

4.0 65 70 9.4 ± 0.41 19.6 ± 0.23 5.3 ± 0.37 7.5 ±0.15

5.0 55 65 4.5 ± 0.16 12.5 ± 0.28 3.43 ± 0.32 5.5 ± 0.14

6.0 50 60 2.4 ± 0.40 8.2 ± 0.15 1.4 ± 0.29 2.6 ± 0.15

58

Vol.2 (1) 54-61 January 2017, ISSN 2455

Fig.1. Effect of different carbon sources at different concentrations and silver nitrate concentration on

shoot morphogenesis from axillary bud

mg/l AgNO3. A-B) Glucose (2.0 %)

Fructose (3.0 %) H) Fructose (3.0 %) I) Fructose (4.0 %).

Effect of silver nitrate on in vitro

vitro derived shoots at a length of 3

separated from shoot clumps and transferred to

half strength MS rooting medium supplemented

with different auxins such as NAA, IAA or IBA

(1.0-3.0mg/l) along with AgNO

fortified with 3% sucrose. Among the different

auxins tried with half strength MS medium, IBA at

(2.0mg/l) resulted in inducing maximum number

of in vitro roots per shoot (39.4) with a maximum

shoot length of (5.4cm), (Table 2; Fig. 2).

Presence of silver nitrate in in vitro

showed marked significance in almost all

parameters under present investigation. Media

without silver nitrate showed lesser response. This

may be due to presence of silver nitrate, which

might have suppressed the activity of excess of

2017, ISSN 2455-5053

Effect of different carbon sources at different concentrations and silver nitrate concentration on

shoot morphogenesis from axillary bud explants cultured on MS + 2.0 mg/l BAP + 0.5 mg/l NAA and 0.4

Glucose (2.0 %) C) Glucose (4.0 %) D-E) Sucrose (2.0 %) F) Sucrose (3.0 %)

Fructose (3.0 %) H) Fructose (3.0 %) I) Fructose (4.0 %).

in vitro Rooting: In

derived shoots at a length of 3-5cm were

separated from shoot clumps and transferred to

half strength MS rooting medium supplemented

with different auxins such as NAA, IAA or IBA

th AgNO3 (0.4mg/l)

fortified with 3% sucrose. Among the different

auxins tried with half strength MS medium, IBA at

(2.0mg/l) resulted in inducing maximum number

roots per shoot (39.4) with a maximum

shoot length of (5.4cm), (Table 2; Fig. 2).

in vitro propagation

showed marked significance in almost all

parameters under present investigation. Media

without silver nitrate showed lesser response. This

may be due to presence of silver nitrate, which

have suppressed the activity of excess of

ethylene present in the in vitro

further promoted for easy translocation and

assimilation of these energy sources available in

the media by the explants resulting in cell division

and leading to vigorous growth. Similar type of

results was documented in Decalepis hamiltonii

(Bais et al., 2000) Vanilla planifolia

al., 2001).

Acclimatization and hardening:

shoots were removed from the culture tubes and

washed thoroughly to remove the traces of agar.

The plantlets of in vitro grown

with well developed roots and shoots were

transplanted to plastic cups containing autoclaved

vermiculite and soil (1:1). About 90% of the

transplanted plantlets surv

Science Spectrum

Effect of different carbon sources at different concentrations and silver nitrate concentration on

explants cultured on MS + 2.0 mg/l BAP + 0.5 mg/l NAA and 0.4

Sucrose (3.0 %) G)

culture tubes and

further promoted for easy translocation and

assimilation of these energy sources available in

the media by the explants resulting in cell division

igorous growth. Similar type of

Decalepis hamiltonii

Vanilla planifolia (Giridhar et

Acclimatization and hardening: The well rooted

shoots were removed from the culture tubes and

ed thoroughly to remove the traces of agar.

grown Solanum nigrum

with well developed roots and shoots were

transplanted to plastic cups containing autoclaved

vermiculite and soil (1:1). About 90% of the

transplanted plantlets survived after

59


acclimatization and showed healthy growth

without any morphological variations. Finally after

one month the hardened plants were transferred to

pots containing garden soil and sand (2:1) and

were allowed to grow under nursery shade

conditions. These plants were watered at 3 days

intervals and were finally planted in field

condition. All the plantlets were phenotypically

indistinguishable from the parent plants.

Table-2: Effect of AgNO3 on different concentrations of IBA, NAA and IAA on in vitro rooting

using half strength MS medium. Observation: After 8 weeks, values are mean ± S.E. of 20

independent determinants.

Plant growth regulators (mg/l) Concentration

of

AgNO3 (mg/l)

Regeneration

frequency

(%)

Mean no. of

roots/shoot

Mean root

length (cm) IBA NAA IAA

1.0 - - - 80 15.4 ± 0.39 3.69 ± 0.26

1.0 - - 0.4 90 28.6 ± 0.25 4.85 ± 0.10

2.0 - - - 85 17.7 ± 0.32 4.26 ± 0.17

2.0 - - 0.4 98 39.4 ± 0.33 5.4 ± 0.39

3.0 - - - 75 13.9 ± 0.26 2.73 ± 0.15

3.0 - - 0.4 85 25.7 ± 0.89 3.4 ± 0.29

- 1.0 - - 75 11.6 ± 0.3 2.2 ± 0.51

- 1.0 - 0.4 85 22.4 ± 0.39 4.61 ± 0.33

- 2.0 - - 82 13.8 ± 0.47 3.1 ± 0.66

- 2.0 - 0.4 95 27.5 ± 0.2 3.65 ± 0.28

- 3.0 - - 72 10.7 ± 0.26 1.4 ± 0.15

- 3.0 - 0.4 80 20.3 ± 1.30 2.9 ± 0.65

- - 1.0 - 65 9.5 ± 0.37 1.70 ± 0.27

- - 1.0 0.4 75 16.7 ± 0.16 3.6 ± 0.22

- - 2.0 - 70 12.3 ± 0.30 2.79 ± 0.36

- - 2.0 0.4 80 20.1 ± 0.52 4.26 ± 0.81

- - 3.0 - 62 9.4 ± 1.04 2.3 ± 0.78

- - 3.0 0.4 78 14.4 ± 0.14 2.39 ± 0.44

60


Fig.4. Rooting and acclimatization of Solanum nigrum: A, B) Root formation from shootlets inoculated

on MS media with IBA (1.0 mg/l) and AgNO3 (0.4 mg/l) C) Plantlet showing elongated root system D)

Hardened plantlet in polybags containing soil and vermiculate in 1:1 ratio E) Plantlet in field condition

Conclusion

It can be concluded that among the different

carbon sources tested, fructose along with silver

nitrate (0.4mg/l) showed better response followed

by sucrose, glucose and maltose in terms of

multiple shoot induction. Since fructose and

sucrose are the better carbohydrate sources for in

vitro shoot multiplication of Solanum nigrum.

However, further research is required to explore

the effect of different carbon sources on in vitro

plant regeneration.

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62


Synthesis and Spectral characterization and DNA Binding properties of Copper(II) complexes with Nicotinoyl and Isonicotinoylhydrazones

S. Chandrasekhar & K. Hussain Reddy*

Department of Chemistry, Sri Krishnadevaraya University, Anantapur- 515 003, India *For Correspondence: [email protected]

Abstract

Copper (II) complexes with series of ligands viz.

2-formylpyridine nicotinoylhydrazone (FPNH), 2-

acetylpyridine nicotinoylhydrazone (APNH) and

2-benzoylpyridine nicotinoylhydrazone (BPNH)

2-formylpyridine isonicotinoylhydrazone

(FPINH),2-acetylpyridine isonicotinoylhydrazone

(APINH) and 2-benzoylpyridine isonicotinoyl-

hydrazone (BPINH) have been synthesized and

characterized based on physico-chemical and

spectral data. The complexes are characterized

based on electronic, IR and ESR spectral studies.

Molar conductivity data suggest that the

complexes are 1:2 electrolytes IR data suggest that

the ligands act as neutral tridentate ligands in all

copper complexes. Copper complexes are

investigated both in solid state and in solution state

at room temperature and at liquid nitrogen

temperature. The spin Hamiltonian, orbital

reduction and bonding parameters of complexes

are calculated. The redox behavior of the

complexes has been investigated by cyclic

voltammetry. Cu (II) complexes undergo one

electron reduction to their respective Cu(I)

complexes. The non-equivalent current in cathodic

and anodic peaks (ic/ia = 0.465–0.728 at 100 mV s-

1) indicate quasi-reversible behavior. Structures of

the complexes are proposed based on physico-

chemical and spectral data.

Keywords: Copper (II) Complexes, Isonicotinoyl

hydrazones, Spectral Characterization and DNA

Binding.

Introduction

Interaction of transition metal complexes with

nucleic acids and investigation of the cleavage

mechanisms have gained much more attention due

to their great importance in the design of new

chemotherapeutic agents, manipulation of genes,

development of tools or probes for the study of

nucleic acid structures (Sigman and Chen 1990;

Cowan, 2001). Chemical nucleases have some

advantages when compared with enzymatic

nucleases. They are smaller in size and thus they

can reach more sterically hindered regions of a

nucleic acid molecule. In addition, the efficiencies

of chemical nucleases in binding followed by a

scission reaction (Sigman et al., 1979; Travers et

al., 1993) making them popular in pharmacology

and biotechnology. Investigations of isonicotinoyl

hydrazones is of interest, especially due to their

pharmacological properties (Shechter et al., 2003;

Rehder, 2001; Thompson and Orvig, 2001).

Isonicotinic acid hydrazide (INH) is the first line

medication in the prevention and treatment of

tuberculosis. It is one of the first anti-depressive

drugs discovered. It is also used in the treatment of

a wide range of bacterial diseases (Rollas and

Güniz, 2007, Mazza et al., 1992 Ianelli et al.,

1995; Cesur et al., 1990; Bottari et al., 2000) .

Hydrazones derived from condensation of

isonicotinylhydrazine with pyridine carbonyls

have been found to show better anti-tubercular

activity (Kümmerle et al., 2009) rather than INH.

Metal complexes of isonicotinoyl hydrazones

exhibit increased antitumour (Verquin et al., 2004)

and antibacterial activity (N. Raman et al 2004).

63


Copper complexes are known to have a broad

spectrum of biological actions (Crouch et al.,

1986). Many copper complexes are used as anti-

inflammatory, anti-arthritic, anti-ulcer, anti-

convulsant and anti-tumour agents (Sorenson et

al., 1982; Apelgot et al., 1986). It has been shown

that copper accumulates in tumours due to the

selective permeability of cancer cell membranes to

copper compounds (Renade and Panday, 1984) .

Because of this, a number of copper complexes

have been screened for anti-cancer activity and

some of them were found active both in vivo and

in vitro (Hall et al., 1997)

In the light of the above and in continuation

of our ongoing research work (Murali Krishna et

al., 2008; Pragathi and Hussain Reddy, 2014;

Moksharagni et al., 2015; Chandrasekhar and

Hussain Reddy 2016), a series of heterocyclic

isonicotinoyl hydrazones (Figure 1) and their

copper(II) complexes have been synthesized and

characterized. DNA binding properties of Copper

(II) complexes are also uncovered using

absorption spectrophotometry.

Fig.1. A general structure for six

hydrazone ligands

Where,

R1 R2

H nicotine 2-form ylpyridine nicotinoylhydrazone (FPNH)

CH3 nicotine 2-acetylpyridine nicotinoylhydrazone (APNH)

C6H5 nicotine2-benzoylpyridine nicotinoylhydrazone (BPNH)

H isonicotine2-formylpyridine isonicotinoylhydrazone (FPINH)

CH3 isonicotine2-acetylpyridine isonicotinoylhydrazone (APINH)

C6H5 isonicotine2-benzoylpyridine isonicotinoylhydrazone (BPINH)

Experimental

Materials and Methods: Analytical grade

Cu(NO3)2. 3H2O was obtained from Merck. The

solvents used for synthesis of copper(II)

complexes were distilled before use. Calf thymus

DNA (CT-DNA) was purchased from Genie Bio

labs, Bangalore, India. Solutions of CT-DNA in

50 µM Tris-HCl (pH, 7.0) gave the ratio of UV

absorbance at 260 and 280 nm of 1.8 indicating

that the DNA was sufficiently free of protein. The

DNA concentration was determined by UV

absorbance at 260 nm using molar absorption

coefficient 6600 M-1. Stock solutions were kept at

4oC and used after not more than four days. DNA

binding studies were performed in 50 mM

NaCl/5mM Tris- base, pH, 7.0 buffer.

Physical measurements: The molar conductance

of the complexes in DMF (10 −3 M) solution was

measured at 28 °C with a Systronic Model 303

direct reading conductivity bridge. The electronic

spectra were recorded in DMF with a Perkin

Elmer UV Lamda−50 spectrophotometer. FT–IR

spectra in KBr disc were recorded in the range

4000–400 cm−1 with a Perkin Elmer spectrum 100

Spectrometer. The cyclic voltammetry was

performed with a CH instruments 660C

electrochemical analyzer and a conventional three

64


electrodes, Ag/AgCl reference electrode, glassy

carbon working electrode and platinum counter

electrode. Nitrogen gas was purged and

measurements were made on the degassed (N2

bubbling for 5 min) complex solution in DMF

(10−3 M) containing 0.1 M tetrabutylammonium

hexaflourophosphate (TBAHEP) as the supporting

electrolyte. The ligands(FPNH, APNH, BPNH

FPINH, APINH and BPINH) are synthesized as

described before (Moksharagni et al., 2015).

Synthesis of copper complexes: An ethanolic

solution (20 ml) of hydrazone ligand (2

mmol) was added slowly to a methanolic solution

(10 ml) of the Copper(II) chloride dihydrate (1

mmol) in a clean 100-ml round bottom flask and

the contents were heated under reflux on water

bath for 2-4 hrs. The reaction solution was allowed

to stand at 25oC for 10-12 hrs.. Dark green

coloured complex was formed. It was filtered off,

washed with small quantity of methanol.

Analytical data of copper(II) complexes are given

in Table 1.

DNA binding study: The electronic spectra of

metal complexes in aqueous solutions were

monitored in the absence and in the presence of

CT-DNA. Absorption titrations were performed by

maintaining the metal complex concentration at 20

× 10−6 M and varying the nucleic acid

concentration (0–7.36 × 10−6 M). The titrations

were carried out by gradually increasing the

concentration of CT-DNA with each addition of

10 µL DNA. The ratio(r) of [complex]/[DNA]

value vary from 23.41 to 2.60. Absorption spectra

were recorded after each successive addition of

DNA solution. The intrinsic binding constant (Kb)

was calculated by using the equation,

[DNA]/(εa-εf) = [DNA]/(εb-εf) + 1/ Kb(εb-εf) - (1)

where [DNA] is the molar concentration of DNA

in base pairs, εa, εb and εf are apparent extinction

coefficient (Aabs/[M]), the extinction coefficient for

the metal (M) complex in the fully bound form

and the extinction coefficient for free metal (M)

respectively.


Synthesis and characterization of new ligands

(FPINH, APINH, BPINH, FPNH, APNH and

BPNH) based on IR, NMR and mass spectral data

are reported by us (Moksharagni et al., 2015).

All the complexes are stable at room temperature,

non-hygroscopic, sparingly soluble in water,

soluble in methanol, ethanol and readily soluble in

CH3CN, DMF and DMSO. The analytical data are

consistent with the proposed molecular formulae

of complexes. Physical properties viz., colours,

melting points, percentage of yield and molar

conductivity of the complexes are given in Table

1. Molar conductivity data suggest that the

complexes are 1:2 electrolytes (Geary, 1971).

Table 1. Analytical and physico – chemical properties of Copper(II) complexes

S.No. Complex Colour Decomp. Temp. ºC Molar

conductivity*

1 [Cu(FPNH)2]Cl2 Light Green 238- 240 0 C 89.1

2 [Cu(APNH)2]Cl2 Green 252-256 0 C 88.1

3 [Cu(BPNH)2]Cl2 Brownish Green 188-190 0 C 88.6

4 [Cu(FPINH)2]Cl2 Light Green 258-260 0 C 90.1

5 [Cu(APINH)2]Cl2 Green 236-238 0 C 88.8

6 [Cu(BPINH)2]Cl2 Brownish Green 184-186 0 C 89.1

* Units for molar conductivity, Ohm-1 cm2 mol-1

65


Electronic spectral studies: The electronic

spectral data of copper (II) complexes are recorded

in dimethylformamide (DMF). Typical electronic

spectrum of [Cu (APNH)2]Cl2 complex is shown

in Fig. 2. The complexes show strong intense

bands in the range 34589-38150 cm-1 are assigned

to intra ligand transitions. One medium intensity

band observed in the range 24235-26320 cm-1 is

due to metal to ligand charge transfer transition

(MLCT). Whereas one broad band observed in the

region 14500-16718 cm-1 is assigned to d-d

transition (Lever, 1984)26 in favour of octahedral

structure. Important electronic spectral bands of

copper (II) complexes are given in Table 2.

Fig. 2. Electronic spectrum of [Cu(APNH)2]Cl2 complex

Table 2. Electronic spectral data (cm-1) of copper (II) complexes.

S.No. Complex π-π*

Transition

CT

Transition

d-d

Transition

1 [Cu(FPNH)2]Cl2 37615 25324 15275

2 [Cu(APNH)2]Cl2 37250 26320 14500

3 [Cu(BPNH)2]Cl2 36500 26145 16325

4 [Cu(FPINH)2]Cl2 34569 24235 16378

5 [Cu(APINH)2]Cl2 34589 24582 16718

6 [Cu(BPINH)2]Cl2 38150 25575 16587

66


Infrared spectral studies: IR spectra of

hydrazone ligands are compared with those of

copper complexes to determine donor atoms of

ligand. Important IR spectral bands and their

assignment are given in Table 3. The IR spectra of

the ligands have several prominent bands due to

νN-H and νC=O and νC=N stretching modes. The νN-H

bands are appeared in spectra of all complexes

indicating that the ligands do not undergo

enolization due to complexation. The bands due to

νC=O and νC=N are shifted to lower frequency

suggesting the involvement of azomethine

nitrogen and amide oxygen in chelation. IR data

suggest that the ligands act as neutral tridentate

ligands in all the copper complexes. Based on

molar conductance, electronic and IR spectral

data, a tentative and general structure for the

copper (II) complexes are assigned. A general

structure for complexes is given in Figure 3.

Table 3. IR spectral data of ligands and their copper (II) complexes

Ligand/Complex ν (N-H) ν (C=O) ν (C=N)

FPNH 3496 1670 1610

[Cu(FPNH)2]Cl2 3370 1665 1595

APNH 3211 1662 1605

[Cu(APNH)2]Cl2 3280 1625 1585

BPNH 3352 1691 1602

[Cu(BPNH)2]Cl2 3350 1675 1590

FPINH 3294 1668 1615

[Cu(FPINH)2]Cl2 3362 1662 1601

APINH 3189 1668 1607

[Cu(APINH)2]Cl2 3385 1650 1590

BPINH 3434 1690 1628

[Cu(BPINH)2]Cl2 3385 1675 1605

N

C=N

R1

HN

C

O

Cu

C

O

NH

R1

N

2 Cl-

R2

R2

2+

N=C

Fig.3. A general structure for Copper(II) complexes

67


ESR Spectral studies: ESR spectra of copper

complexes were recorded in solid state and in

DMF solution at room temperature and at liquid

nitrogen temperature. X-band powder and DMF

solution ESR spectra of [Cu(APNH)2]Cl2

complex are given in Figure 4 .

The spin Hamiltonian, orbital reduction and

bonding parameters of complexes are given in

Table 4. The g|| and g┴ are computed from the

spectra using TCNE free radicals as g marker. The

observed g|| values for complexes {BPNH Cu}, is

less than 2.3 suggesting significant covalent

character of metal ligand bond in agreement with

observation of Kivelson. The g|| and g┴ were more

than 2, corresponding to an axial symmetry. The

trend The g|| > g┴ >ge (2.0023) observed for these

complexes suggests that the unpaired is localized

in the dx2-y

2 orbital (Kivelson et al., 1961) of the

copper ion. The axial symmetry parameter G is

defined as (Narang and Singh, 1996)

G = [�║��.��]

[�┴��.��]

The calculated G values for these complexes

indicate that there are strong interactions between

the copper centers in DMF medium. g||, g┴ , A||, A┴

of complexes and the energies of the d–d

transitions are used to calculate the orbital

reduction parameters (K║, K┴), the bonding

parameter(α2).The factor α2 which is usually taken

as a measure of covalency is evaluated by the

expression,

α2 = A║/ p + ( g|| -2.0023)3/7(g┴ -2.0023) + 0.004

The observed K║ > K┴ relation indicates the

significant in-plane π-bonding.

Fig. 4. X-band powder and DMF solution ESR spectra of [Cu(APNH)2]Cl2 complex at (A) 300K and

(B) at LNT (C) at 300K and (D) at LNT.

Cyclic voltammetric studies: The redox

behavior of the complexes has been investigated

by cyclic voltammetry in DMF using 0.1 M

tetrabutylammonium hexafluorophosphate as

supporting electrolyte. The cyclicvoltammogram

of [Cu (FPNH)2]Cl2 complex is given in Figure

5 and the electrochemical data of copper(II)

complexes are summarized in Table 5.

68


Table 4. Spin Hamiltonian and orbital reduction parameters for Copper(II) complexes at 300K and 77K

in solid state and in DMF solution

Complex

In solid state

In DMF solution

g║ g┴ gavg G g║ g┴ gavg G A║X10-5

A┴X10-5

K║ K┴ α2 λ *

[Cu(APNH)2]Cl2 2.48 2.06 2.08 1.61 2.12 2.01 2.04 0.829 0.0169 0.0184 0.740 0.893 0.056 152

[Cu(BPNH)2]Cl2 2.14 2.05 2.07 2.88 2.12 2.04 2.08 3.122 0.0128 0.0115 1.530 0.711 0.284 461

[Cu(FPNH)2]Cl2 2.15 2.04 2.12 3.91 2.20 2.09 2.14 2.254 0.0138 0.0158 1.661 0.6105 0.138 172

[Cu(APINH)2]Cl2 2.14 2.06 2.09 2.38 2.12 2.03 2.07 4.291 0.0187 0.0248 2.575 1.032 0.116 176

[Cu(BPINH)2]Cl2 2.13 2.05 2.10 2.67 2.16 2.04 2.10 4.183 0.0287 0.0785 1.307 0.826 0.310 585

[Cu(FPINH)2]Cl2 2.14 2.07 2.11 2.03 2.21 2.08 2.14 2.671 0.0145 0.0458 1.860 0.769 0.285 374

Fig.5. Cyclic voltammetric profile of [Cu(FPNH)2]Cl2 complex.

The cathodic peak current function values were

found to be independent of the scan rate. Repeated

scans at various scan rates suggest the presence of

stable redox species in solution. It has been

observed that cathodic (Ipc) and anodic (Ipa) peak

currents were not equal. The E1/2 values are

observed in 0.349–0.628 V potential range for all

copper complexes. It may be concluded that all the

Cu(II) complexes undergo reduction to their

respective Cu(I) complexes. The non-equivalent

current in cathodic and anodic peaks (ic/ia =

0.465–0.728 at 100 mV s-1) indicate quasi-

reversible behavior (Khumhar et al., 1991). The

difference, ΔEp in all the complexes is found to be

greater than the Nerstian requirement 59/n mV

(n = number of electrons involved in oxidation

reduction). This observation suggests quasi-

reversible character of reduction. The complexes

show large separation (∆Ep) between anodic and

cathodic peaks indicating quasi-reversible

character.

DNA binding studies: The binding interactions of

the complexes with CT-DNA were monitored by

comparing their absorption spectra with and

without CT-DNA. In the presence of increasing

amounts of DNA, the spectra of all complexes

showed a strong decrease (hypochromicity) in

intensity with shift in absorption maxima towards

higher (bathochromic shift) wavelengths. Fig 6

shows absorption spectra of [Cu(APNH)2]Cl2

complex in the presence of increasing amounts of

DNA.

69


Table 5. Cyclic voltammetric profile of copper (II) complexes

Fig. 6. Absorption spectra of [Cu(APNH)2]Cl2 complex in the absence and in the presence of increasing

concentration of CT-DNA; [top most spectrum is recorded in the absence of DNA and below spectra on

addition of 10µl DNA each time. A plot of [DNA] / (Ea - Ef) vs [DNA] is shown in the insert]

The binding constant values are given in Table 6.

On addition of DNA, the absorbance of the

complexes decreases (hypochromism) and

absorption maximum of all complexes is shifted to

higher wavelength (bathochromism).The binding

of an intercalative molecule to DNA is generally

characterized by large hypochromism and

significant red shift due to strong stacking

interactions of aromatic chromophore between

base pairs of DNA. The extent of hypochromism

and red shift is commonly consistent with the

strength of intercalative interaction (Usha and

Palaniander, 1994). However, in the present case,

the magnitude of hypochromism (up to 25.62 %)

is as expected for typical classical intercalators.

The binding constants (Table 6) suggest that the

complexes bind DNA very strongly via

intercalation.

S.

No.

Complex Redox

couple Peak Potentials (V) ΔEP

(mv)

E1/2 -ic/ ia log kca ΔG◦b

Epc Epa

1 [Cu(APNH)2]Cl2 II/I 0.368 0.549 181 0.458 0.692 0.186 1067

2 [Cu(BPNH)2]Cl2 II/I 0.382 0.492 110 0.628 0.719 0.301 1728

3 [Cu(FPNH)2]Cl2 II/I 0.292 0.516 224 0.404 0.679 0.152 872

4 [Cu(APINH)2]Cl2 II/I 0.212 0.487 275 0.349 0.728 0.124 712

5 [Cu(BPINH)2]Cl2 II/I 0.301 0.556 255 0.428 0.465 0.138 1136

6 [Cu(FPINH)2]Cl2 II/I 0.312 0.462 150 0.387 0.558 0.221 1286

70


Table 6. Electronic absorption data upon addition of CT-DNA to Cu(II) complexes.

Conclusions

Copper(II) complexes of a series of nicotinoyl and

isonicotinoyl hydrazones have been synthesized.

These complexes are characterized based on

conductivity measurements, UV-Vis, IR and ESR

spectral studies. Electrochemical behavior of these

complexes has been studied by using cyclic

voltammetry. DNA binding properties are

uncovered using UV-Visible spectrophotometry.

Absorption spectral data and binding constants

(Kb) suggest that the complexes bind DNA

strongly via intercalation.

Acknowledgements:

One of the authors (Chandrasekhar) is thankful to

University Grants Commission, New Delhi, India

for the award of BSR Junior Research Fellowship.

The authors are thankful to UGC, New Delhi

(Sanction No. Lr.No.F40-80/2011 (SR)) for

financial support. The authors also thank UGC and

DST for providing equipment facility under SAP

and FIST programs respectively. KHR is thankful

to UGC for the sanction of one-time grant

(Sanction Lr. No.F.19-106/2013 (BSR)) for

financial support.

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72


Total Coloring and Chromatic Number of Strong Intuitionistic Fuzzy Graph

S. Narayanamoorthy∗ and P. Karthick Department of Mathematics, Bharathiar University, Coimbatore - 641 046, India.


Abstract

In this paper, we introduce the concept of k-fuzzy

total coloring on strong intutionistic fuzzy graph.

We determine the chromatic number for strong

intuitionistic fuzzy graph G∗ k, with intuitionistic

fuzzy set of vertices and intuitionistic fuzzy set of

edges in terms of family of intuitionistic fuzzy

sets.

Keywords: intuitionistic fuzzy graph, fuzzy total

coloring, chromatic number.

2010 Mathematics Subject Classification:

05C65, 68R10, 05C72.

Introduction

Graph coloring serves as a model for conflict

resolution in problems of the combinatorial

optimization. A k- coloring φk is a coloring

function with no more than k different colors φk :

X → {1, 2, ..., k}. A graph is k-colored if it admits

a k - coloring. The chromatic number χ (G) of a

graph G is the minimum k for which G is k-

colorable. As an advancement fuzzy coloring of a

fuzzy graph was defined by authors Eslahchi and

Onagh in 2004, and later developed by them as

fuzzy vertex coloring in 2006. This fuzzy vertex

coloring was extended to fuzzy total coloring in

terms of family of fuzzy sets by S. Lavanya and R.

Sattanathan in 2009. The k-fuzzy total coloring to

complete fuzzy graph introduced by V. Nivethana

and A. Parvathi (2013).

In 1986, K.T. Atanassov (Atanassov, 1999)

introduced the concept of intuitionistic fuzzy set as

a generalization of fuzzy sets. Intuitionistic fuzzy

set has been applied to a wide variety of fields

including computer science, engineering,

mathematics, medicine, chemistry and economics.

Muhammad Akram, Bijan Davvaz introduced the

notion of strong intuitionistic fuzzy graph in

(Akram and Bijan, 2012). In this paper, we define

fuzzy total coloring to strong intuitionistic fuzzy

graph satisfying certain conditions. The fuzzy total

chromatic number is the minimum value of k such

that k-fuzzy total coloring exists. Here we consider

strong intuitionistic fuzzy graph by taking

intuitionistic fuzzy set of vertices and intuitionistic

fuzzy set of edges.

Preliminary definitions

In this section, we discuss some basic notations

and definitions used throughout this paper, G

denotes graph, Gˆ denotes fuzzy graph, Gˆk

denotes complete fuzzy graph, G∗ denotes

intuitionistic fuzzy graph, G∗k denotes strong

intuitionistic fuzzy graph.

Definition.1

Let X be a nonempty set. A fuzzy set A in X is

characterized by its membership function

µA : X → [0, 1] and µA(x) is interpreted as the

degree of membership of element x in fuzzy set A

for each x ∈ X.

Definition.2

Let X be a finite nonempty set. The triple G^ = (X,

σ, µ) is called a fuzzy graph on X, where σ

and µ are fuzzy sets on X and E (X × X)

respectively, such that µ({x, y}) ≤ min{σ(x),

σ(y)}for all x, y ∈ X. We use µ(xy) for µ({x, y}) .

73


Definition.3

An intuitionistic fuzzy graph is of the form G∗ =

(X, E), where

1. X = {x1, x2, ..., xn} such that µ1 : X → [0, 1] and

ν1 : X → [0, 1] denote the degree of

membership and non membership of the element

xi ∈ X, respectively such that 0 ≤ µ1(x) +

ν1(x) ≤ 1 for all xi ∈ X (i=1, 2, ..., n),

2. E ∈ X × X where µ2 : X × X → [0, 1] and ν2 : X

× X → [0, 1] are defined by µ2(xi, xj) ≤ min(µ1(xi),

µ1(xj)), ν2(xi, xj) ≥ max(ν1(xi), ν1(xj)) such that 0 ≤

µ2(xi, xj) + ν2(xi, xj) ≤ 1 for all (xi, xj) ∈ E (i, j=1,

2,..., n).

Definition.4

A family Γ = {γ1, γ2, ..., γk} of fuzzy sets on X S E

is called a k-fuzzy total coloring of Gˆk =

(X, σ, µ) if

a) max{γi(x)} = σ(x) and max{γi(xy)} = σ(x) ∧

σ(y) for all x, y ∈ X, xy ∈ E and 1 ≤ i ≤ k.

b) γi ∧ γj = 0 for 1 ≤ i, j ≤ k.

c) For every strong edge xy of Gˆk, min{γi(x),

γi(y)} = 0 and for any set of incident edges xy on

vertex x ∈ X of Gˆk , min{ γi(xy)} = 0, 1 ≤ i ≤ k.

k-fuzzy total coloring on strong intuitionistic

fuzzy graph

We extend the definition of k- fuzzy total coloring

on the strong intuitionistic fuzzy graph in the

definition given below. Since we deal with strong

intuitionistic fuzzy graph for which µ2(xi, xj)

strictly equals to min (µ1(xi), µ1(xj)) and ν2(xi, xj)

strictly equals to max(ν1(xi), ν1(xj)), the definition

can be stated as follows:

Definition.1

A family Γ = {γ1, γ2, ..., γk} of fuzzy sets on X S E

is called a k-fuzzy total coloring of G∗k = (X, E) if

a) max{γi(xi)} = (µ1(xi), ν1(xi)) for all xi ∈ X, 1 ≤ i

≤ k and max{γi(xixj)} = (µ2(xixj), ν2(xixj)

= {min(µ1(xi), µ1(xj)), max(ν1(xi), ν1(xj))} for all

edge xixj ∈ E and 1 ≤ i, j ≤ k.

b) γi ∧ γj = 0 for 1 ≤ i, j ≤ k.

c) For every strong edge xixj of G∗k, min{γi(xi),

γi(xj)} = 0 and for any set of incident edges on

xixj on vertex xi ∈ X of G∗k, min{ γ(xixj)} = 0, 1 ≤

i, j ≤ k.

Fuzzy total chromatic number of strong intuitionistic fuzzy graph

Fig.1. Strong intuitionistic fuzzy graph G∗

k

Consider the fig-1, a strong intuitionistic fuzzy graph G∗k = (X, E) with vertex set X = {x1, x2, x3, x4, x5,

x6} and edge set E = {xixj| ij = {12, 13, 14, 15, 16, 23, 24, 25, 26, 34, 35, 36, 45, 46, 56}

the membership function defined as follows :

74


75


Conclusion

In this research paper, we introduced the concept

of k- fuzzy total colring on strong intutionistic

fuzzy graph. We determined the chromatic number

for strong intuitionistic fuzzy graph G*k, with

intuitioistic fuzzy set of vertices and intuitionistic

fuzzy set of edges in terms of family of

intuitionistic fuzzy sets. Graph coloring is one of

the most useful models in graph theory. It has been

used to solve problems in school timetabling,

computer register allocation, electronic bandwidth

allocation, and many other applications.

76


References

1. A. Shannon, K.T. Atanassov, A first step to a

theory of the intuitionistic fuzzy graphs,

Proceeding of FUBEST (Lakov, D., Ed.),

Sofia, 1994, 59-61.

2. Eslahchi, B. N. Onagh, Vertex Strength of

Fuzzy Graphs, International Journal of

Mathematics and Mathematical Sciences,

2006.

3. H. P. Yap, Total Colorings of Graphs, Lecture

Notes in Mathematics, Springer-Verlag,

Berlin, 1996.

4. K. T. Atanassov, Intuitionistic Fuzzy Sets,

Theory and Applications, Springer, 1999.

5. M. Akram, D. Bijan, Strong intuitionistic

fuzzy graphs, Filomat 26:1(2012), 177-196.

6. S. Lavanya, R. Sattanathan, Fuzzy Total

Coloring Of Fuzzy Graphs, International

Journal of Information Technology and

Knowledge Management, Vol. 2, 2009, 37-39.

7. Siamak Firouzian, Mostafa Nouri Jouybari,

Coloring fuzzy graphs and traffic light

problem, The Journal of Mathematics and

Computer Science, Vol. 2, 2011, 431-435.

8. Susana Munoza, M. Teresa Ortunoa, Javier

Ramrezb, Javier Yaneza, Coloring fuzzy

graphs, 33, 2005, 211-221.

9. V. Nivethana, A. Parvathi, Fuzzy total

coloring and chromatic number of a complete

fuzzy graph, International Journal of

Emerging Trends in Engineering and

Development, Vol. 6, 2013, 377-384.

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vide an opportunity for Scientists and young researchers, an official scientific journal entitled “Science Spectrum” has been started. The researchers are requested to contribute Research papers/Reviews/Short Communications in various fields of Science. The details

Research Articles are invited from researchers in Academic Institutions, Scientific laboratories and other industry pertaining to any of the following broad scientific fields including interdisciplinary

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