The “International Research Journal of Applied...

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Aim and Scope The “International Research Journal of Applied Sciences” is a bi-Annual,

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INTERNATIONAL RESEARCH JOURNAL OF APPLIED SCIENCES President

G. Madegowda, Ex. MP and Founder of Bharathi Education Trust EDITORIAL BOARD

Patron: Prof. S. Nagaraju,

Principal, Bharathi College, Bharathinagara.

Editor-In-Chief: Dr. P. Nagendra M Sc. Ph.D.

Associate professor, Dept. of Chemistry, Bharathi College, Bharathinagara

Associate Editors: Dr. T. Tamizh Mani Principal, Bharathi College of Pharmacy

Dr. Suyoga Vardhan D. M Assistant professor, Dept. of Chemistry, Bharathi College, Bharathinagara

Dr.Gurukar Mathew Department of Botany

Dr.G. S. Sandesh., MRCS University Hospital, UK.

Dr. Rajesha Department of Chemistry

Dr. R. L. Jagadish Department of Polymer Chemistry

Dr.Babu Antharavally University of Wisconsin (USA),

Dr. Puttaswamy. S Professor, Central College Campus Bangalore University

Dr. H. L Ramesh Associate Professor, Bangalore University

Dr.Shivaswamy. S Dept. of Sericulture

Dr. M. Raju Department of Botany

Dr. Lakshminarayana Department of Biotechnology

Dr. K.H.Vekatesh Department of Life science ,Bangalore University

Dr.H.B. Mahesha Department of sericulture , Yuvaraja’s College,Mysore

Advisory Committee: Madhu G. Madegowda, BE, MBA Hon. Executive Trustee, BET.

B. M. Nanjegowda, MA., Hon. Secretary,BET

Prof. M. Venkatareddy Head Dept. of Physics

Dr.Sathish Reddy Pecking University, Beijing, China

Chandrashekar Pecking University, Beijing, China

Dr. B. P. Siddaraju Dept. of Chemistry

SENIOR ADVISORY COMMITTEE Dr. D. Channe Gowda, Professor,DOS in Chemistry, University of Mysore, Mysore.

Dr. H. S. Yathirajan Professor and Chairperson, DOS in Chemistry,University of Mysore, Mysore

Prof. K. N. Thimmaiah Northwest Mississippi Community College Southaven, MS. United States

Prof. S. Shashikanth DOS in Chemistry. Manasagangothri, Mysore

Dr. S. Anand Professor,DOS in Chemistry, University of Mysore, Mysore.

Dr. K. M. LokanathRai Professor, DOS in Chemistry. University of Mysore, Mysore

Editorial Office: BETAHE, Bharathi College, Bharathinagara, Maddur, Mandya – 571422 INDIA.

iii

INTERNATIONAL RESEARCH JOURNAL OF APPLIED SCIENCES

CONTENTS

Vol. 1, Issue 2. July – Dec. 2014

Sl. No. Title of the Paper Page No.

1 GENETIC RELATIONSHIP BETWEEN INVERSION KARYOTYPES, MORPHOMETRIC TRAITS OF DROSOPHILA ANANASSAE

M. Prathibha and S.C. Jayaramu

1-8

2 ROLE OF INVERSION SYSTEM ON MATING ACTIVITIES AND FITNESS TRAITS IN DROSOPHILA ANANASSAE

S.C. Jayaramu and M. Prathibha

9-15

3 COMPARATIVE STUDIES ON THE MORPHOLOGICAL AND REPRODUCTIVE TRAITS OF FOUR MULBERRY VARIETIES (MORUS SPP.)

K. H. Venkatesh, S. Shivaswamy and Munirajappa

16-21

4 CHROMOSOME NUMBERS, STOMATAL FREQUENCY AND KARYOTYPE STUDIES OF THREE MULBERRY GENOTYPES (MORACEAE)

K. H. Venkatesh, S. Shivaswamy and Munirajappa

22-27

5 ENVIRONMENTALLY FRIENDLY SYNTHESIS OF BIS (INDOLYL) METHANES CATALYSED BY NITROPHTHALIC ACID

Sudhakara A, Nataraja G, Rajesha, Ramesha S

28-34

6 ANTIMICROBIAL STUDY ON THIAZOLIDINONES OF SUBSTITUTED N'-BENZYLIDENE-2-(PHENYLAMINO) ACETOHYDRAZIDES

K. C. Chaluvaraju, B. Shalini, P. Nagendra, G. Pavithra and R. D. Rakesh

35-39

7

STUDIES ON PHYTOCHEMICAL INVESTIGATION OF LEAF EXTRACT OF ACALYPHA INDICA

Rajesha, P. Nagendra and B. P.Siddaraju

40-47

8 CRYSTAL AND MOLECULAR STRUCTURAL STUDIES OF 3-(5H-DIBENZO[B,F]AZEPINE-5-YL)-N,N-DIMETHYL PROPAN-1-AMINE CHLORIDE

P. Nagendra, Rajesha, S. Madan Kumar, B.P. Siddaraju and N. K. Lokanath

48-51

Int. Res. J. App. Sci., Vol. 1, Issue 2. July – Dec. 2014 1

GENETIC RELATIONSHIP BETWEEN INVERSION KARYOTYPES, MORPHOMETRIC TRAITS OF DROSOPHILA ANANASSAE

M. Prathibha1 and S.C. Jayaramu1*

1&2Department of Zoology, Yuvaraja’s College, University of Mysore, Mysore, India

*Corresponding Author Email id: [email protected]

Manuscript received 7th August 2014, revised 17th October 2014, Accepted 20th November 2014

Abstract Drosophila ananassae flies collected from four geographical areas namely, Mysore, Bellur, Manglore and Dharwadwere used in the present study. Inversion frequencies and morphometric traits were analyzed here. For study of inversion frequencies, 2LA, 3LA, 2LA+3LA and those without inversion were used. Variation in the morphometric traits analyzed include, head width, wing length, number of sternopleural and scutellar bristles. These studies indicate that D.ananassae carries significant geographic variation in all these traits. The study thus confirms the hypothesis that intra specific variation is inherent in different geographical strains of D. ananassae. Key words: Drosophila ananassae, Courtship, Geographical populations, inversion frequencies,

Morphometric traits.

Introduction: Inversions in Drosophila with reference to seasonal, geographic, altitudinal and latitudinal variations have been well documented [49]. In certain species, north south clines in inversion frequencies (increase towards equator) have been reported [19,13,5]have found a good correspondence between the mean number of heterozygous inversions and an index expressing environmental heterogeneity in natural populations of D. willistoni. Superiority of inversion heterokaryotypes over homokaryotypes has been demonstrated by Dobzhansky [12]. This led Dobzhansky and coworkers [13] to suggest that chromosomal polymorphism is a device to cope with the diversities of environments. Dobzhansky [12] has opined that heterotic balancing selection and perhaps other forms of selections are responsible for the maintenance of most of the inversion polymorphisms. Further, Dobzhansky [11] classified the polymorphic system of a species as either “rigid or flexible” based on its apparent responsiveness or otherwise to environmental change.

Genetic variations due to point mutations could also occur at morphological traits. Although a considerable amount of genotypic variation exists at

these loci in natural populations, very few attempts have been made to analyze it. It has been suggested that the genetic basis of phenotypic changes is fundamental for understanding the mechanism of evolution in natural and experimental populations [20] and the study of quantitative characters in wild populations could prove to be interesting for evolutionary studies [8]. Drosophila populations have been surveyed in order to study the mechanisms of maintaining genetic variability of quantitative characters particularly morphological traits [7, 15, 35, 40].

The genetics of quantitative traits has been extensively studied in D.melanogaster by using different bristle phenotypes, particularly sternopleural and abdominal bristle number [21, 37, 38]. Sternopleural bristle phenotypes in D. melanogaster have been frequently employed to study the effect of artificial and natural selection and to throw light on the genetic constitution of natural population [1, 2, 9]. Influence of different chromosomes on sternopleural bristle number have been detected and different genetic factors controlling sternopleural bristle number have been located in different chromosomes by using marker strains [37, 38, 48]. Genetic

mailto:[email protected]


heterogeneity for sternopleural bristle number has been found in Indian populations of D. melanogaster[41]. Individuals showing intermediate phenotypes for sternopleural bristle number were significantly more heterozygous at certain allozyme loci than more extreme ones, on observation which supports additive and homeostatic models for gene action and this could explain the higher heterozygosity of central phenotypes [36]. In certain cases, association between chromosomal inversion polymorphism and morphometric characters has been reported [9, 16, 40, 52]. How this association between the inversions and morphometric traits is spread among the species of Drosophila is not known. Therefore the present study has been carried out in order to analyze1)inversion polymorphism (frequency) of four different geographic populations of D. ananassaeand 2)compare morphometric traits of inversion free strain and strains carrying 2LA, 3LA and 2LA+3LA inversions of D. ananassae.

Materials and Methods

Analysis of inversion frequencies in natural populations:

D. ananassae flies collected from Dharwad, Bellur, Manglore and Mysore following the procedure described by Hegde et al[17] was used for the present study. After the flies were brought to the laboratory, the females were individually placed in glass vials (2.5cm x 8.5cm) containing wheat cream agar medium and males were used for identification. These flies were then maintained at constant temperature of 22 ± 1°C and relative humidity of 70%. When larvae appeared, eight third instar larvae from each isofemale line were used for analysis of inversion frequency and others were allowed to continue their development. The polytene chromosomes were prepared using the procedure described by Reddy and Krishnamurthy [34].

Analysis of morphometric characters among four inversion phenotypes

To analyze the role of inversions on morphometric traits three different strains carrying 2LA, 3LA and 2LA+3LA inversions and one

inversion free strain were built up in the laboratory using the female flies collected from natural habitat at Dharwad. These females were individually placed in vials containing wheat cream agar media (isofemale line) and when larvae appeared, eight larvae from each vial were sacrificed to check for presence or absence of inversions in their salivary gland chromosomes. D. ananassae populations collected fromDharwad carries two common inversions namely, 2LA and 3LA. The wild caught individuals therefore would be either without inversion, or carry 2LA alone, or 3LA alone or both 2LA+3LA. When all the eight larvae carried a given inversion, then that individual (their mother) was designated as the strain carrying that particular inversion. The adult progenies which appeared from such mothers were classified as inversions free, 2LA, 3LA, and 2LA+3LA strains.

For the sake of convenience, these strains were designated as IA, IB, IC and ID respectively. IA is monomorphic (inversion free), IB is with 2LA, IC is with 3LA, and ID is with 2LA+3LA strains. These strains were separately maintained for six generations and at each generation, three to five larvae were used to check for the presence or absence of respective inversions. Although in each generation, the polytene chromosomes showed the presence of either inversion loop or absence of loop, because they originate from same isofemale line all progeny contained only that particular inversion homokaryotype or heterokaryotype. The adults emerged from these strains were used to build up populations for the study of variation in morphometric traits.

Four morphometric traits viz., sternopleural bristles, scutellar bristles, head width and wing length were studied in IA, IB, IC and ID strains as per procedure described by [27]. Four morphometric characters namely, sternopleural bristles, scutellar bristles of the left side of the body, head width and wing length were selected for this study (Fig. 1-4). Both large and small bristles present on the sternopleural plate and scutellum were considered for counting the bristle number. Measurements of head and wing were made using an ocular micrometer (1 unit =100μm) under 100X magnification. A total of 30 males and females


were used from each strain for the

morphometric study.

Results and Discussion:

Fig. 2. Counting sternopleural bristles

(a=Dorsal view, b=Front view) Fig.1. Points of measurement of head width of D. ananassae

Fig. 3. Counting scutellar bristles Fig. 4. Points of measurement of winglength

Table 1: Inversion frequency (%) in different geographic Populations of D. ananassae

Inversion frequency (%)

Strains N 2LA 3LA 2LA+3LA Inversion free (Monomorphic)

Dharwad 38 20.0 50.0 20.0 10.0

Mangalore 42 36.7 53.3 6.7 3.3

Mysore 40 33.3 43.4 10.0 13.3

Bellur 32 16.7 40.0 26.7 16.6

Table 2: Morphometric traits in different inversion strains in D. ananassae (Values are Mean ± SE)


Strain → ↓Parameters

IA (Inversion

free)

IB (2LA

inversion)

IC (3LA

inversion)

ID (2LA+3LA inversions)

F

value

P value

Sternopleural bristles

6.26±0.102a 6.70±0.086b 6.84±0.082b 7.74±0.14c 24.243 0.000**

Scutellar bristles

4.30±0.06a 4.44±0.07a 4.56±0.07b 4.64±0.06b 4.614 0.004*

Head width 0.72±0.007a 0.75±0.009b 0.77±0.011b 0.82±0.003c 21.082 0.000**

Wing length 1.82±0.02a 1.83±0.02a 1.84±0.03a 1.89±0.02a 1.544 0.204NS

Same superscript in each row indicates that the value is non significant by DMRT. NS- non significant *P<0.05; **P< 0.001.

Variation in inversion frequencies

Table 1 show that the frequencies of inversions differ in different geographical populations of D.ananassae. In all populations studied, highest number of individuals carried 3LA inversion while least number of individuals was inversions free (monomorphic). In Dharwad and Mangalore populations the inversion frequency was found to be in the following increasing order, 3LA >2LA >2LA+3LA > monomorphic. The percentage of different inversions in Mysore population decreased in the following order, 2LA+3LA < monomorphic < 2LA < 3LA, while in Bellur populations; the situation was as follows, 3LA > 2LA+3LA >2LA > monomorphic. This shows that the populations of D.ananassae are highly polymorphic. Moreover, the same inversion was found to be present in different frequencies in different populations. For example, the frequency of 2LA inversion in Dharwad population was 20 percent while in Mangalore population it was 36.7 percent. It was noticed that in all the population studied, 3LA inversion was most frequent while 2LA+3LA was least. The difference in the percentage of inversions in different populations suggests that the frequency of each of the three paracentric inversions was not the same in all the populations studied. This agrees with earlier studies of inversion frequency in different species of Drosophila[18, 32, 39, 46, 47]. It has been demonstrated that certain inversions heterozygotes have selective advantage over homozygotes [10]. The persistence of inversion polymorphism

observed here in these populations could be explained by an advantage of inversion heterozygotes over corresponding homozygotes. The authors in the present study have noticed more heterokaryotypes than homokaryotypes. This confirms the fact that the inversion polymorphism is adaptive and balanced due to higher Darwinian fitness on inversion heterozygotes [10].

Variation in morphometric traits

Mean number of sternopleural bristles of ID strain was highest while, it was lowest in IA (Table 2). The data on mean sternopleural bristles when subjected to f value and p value showed that mean sternopleural bristle number varied significantly between different inversion strains. The mean number of sternopleural bristles of IA (Inversion free strain) strain was significantly lesser when compared to IB, IC and ID strains (Table 2). This means the number of sternopleural bristle is characteristic of a given karyotype. The scutellar bristle distribution was also varied in different karyotypes. For example, Mean number of scutellar bristles in ID strain was highest while it was lowest in IA strain (Table 2). Mean number of scutellar bristles of IA strain was significantly lesser when compared to the remaining inversion bearing strains. Mean number of scutellar bristles in IB strain was not significant with IA and significantly lesser when compared with IC and ID strains. The IC and ID strains had significantly greater number of scutellar bristles when compared


to IA and IB strains but non significant with each other.

A review of table 2 also revealed that head width was highest in ID strain and least in IA. The mean head width of IA (Inversion free strain) strain was significantly lesser when compared to IB, IC and ID strains (Table 2). Mean head width of IB and IC strains was significantly greater when compared with IA strain and non significant with each other and significantly lesser when compared with ID strain. Similarly, ID inversion strain had largest head width compared to all others. Wing length and wing width also showed similar variation among different karyotypes. Thus we observed significant variation of all the morphometric traits which suggests differential genotypic influences on these traits. As early as Mather[22], had noticed significant genetic variability affecting chaetae number present in all major chromosomes of D. melanogaster. No proper methodology to assess the variability of quantitative or morphometric traits was available then, yet polygenic activity had been suggested when Mather [22] made this observation.

In the present study the authors have analyzed the differences in four morphometric traits in four different genetic strains of D. ananassae viz., inversion free strain (IA), the second with a sub-terminal inversion on the left arm of second chromosome (2LA) - IB strain, third with a terminal inversion on the third chromosome (3LA)-IC strain and the fourth with two inversions, one on second chromosome and another on the third chromosome (2LA+3LA) - ID strain. Among the four morphometric traits analyzed, sternopleural and scutellar bristles are the polygenic traits whose expression is under the influence of the environmental conditions [22, 23, 24, 31, 50]while the head width and wing lengths are polygenic traits that determine the body size of the flies [4, 15, 44, 45, 51, 52]. This study thus permits the analysis of relationship between these morphometric traits and inversions strains.

The number of sternopleural bristles was highest in the strain with 2LA+ 3LA inversion (strain ID) and lowest in the strain with inversion

free (IA). The number of sternopleural bristles was intermediate in the strain which carried 2LA and 3LA the inversions. Although the inversion free strain had lower number of sternopleural bristles than inversion strains. This shows that the presence of inversion 2LA+ 3LA produces extra bristles on the sternopleural plate. The presence of extra bristles in the inversion karyotypes of D. melanogaster has also been noticed by Das and Singh [6]. Thus the present study confirms the observation of these authors. In contrast to sternopleurals, the mean number of scutellar bristles was lowest in the inversion free strain. The bristle number increased in strain with 3LA and highest number of bristles was found in flies with both 2LA and 3LA. The increase was found to be statistically significant. This observation provides an evidence for the association of extra scutellar bristles with inversions confirming such an association between scutellar bristle number and inversion frequencies as also been demonstrated by Das et al[7]in D. ananassae and Singh and Das [40] in D. melanogaster.

A careful scrutiny of the table 2 also shows that the inversion free strain carries lesser number of scutellar bristles than those with inversions. Even among the strains which carried inversions, the strain with double inversion had the highest number. While discussing the adaptive significance of inversion polymorphism, Muller [26] has argued that heterotic makeshifts that arose in the stress of comparatively rapid evolutionary flux and that are due to be rectified ultimately, when longer term natural selection repairs its short term imperfections and miscarriages. On the other hand Dobzhansky and his associates [14] demonstrated heterozygote superiority and argued that the inversion heterokaryotypes might have been favoured by natural selection due to this superiority. The reason for the presence of extra bristles might be due to overdominance which is one of the features of heterozygote superiority. The maintenance of inversion polymorphism in natural populations of D. ananassae seems to be associated with many other adaptive functions in terms of sexual behaviour, fitness and morphometric traits.


Most importantly, in the present studies, inversion frequency was also found to be variable in four different populations of D. ananassae, with a correlation to bristle number. Detailed studies on association of inversion frequencies with extra bristles and other parameters have been carried out in D. melanogaster[6, 15, 40]. Mather [22] reported significant genetic activity affecting chaeta number present on all chromosomes of D. melanogaster and polygenes determining the extra bristles were detected in all three major chromosomes [35]. Whittle [50] found an influence of chromosome 3 on the increase in number of extra scutellar bristles which was later supported by the localization of polygenes for extra bristles [33]. Based on the results in the present study the authors suggest that polygenes concerned with extra bristles could be located on both chromosome 2 and 3 because of the association of these bristles with both inversion 2LA and 3LA.

Even the head width differs in flies carrying different inversions. It was noticed that head width was more in strains carrying both 2LA and 3LA inversion and less in strains carrying single inversion (either 2LA or 3LA). Furthermore, the head width of strain without inversion was significantly different from that of the strain with double inversion. The authorsare of the opinion that with reference to the bristle number; the inversion strains show superiority over those without inversions. Even the mean wing length of the strains with double inversion was found to be greater than others. The mean wing length of inversion free strain was lowest and it

was not significantly different from inversion strains. The wing length of double inversion strain (2LA+3LA) was highest and it was not significantly different from other strains. Thus the heterotic effect of inversion could be seen with regard to this trait also. As wing length is an index of body size [25, 42, 43], the present study indicates that the flies of the strain carrying double inversion are larger than the others. The present study thus supports the observation of Ombo et al., [30] in Leptysma argentina with regard to body size and chromosomal polymorphism. According to them Leptysma argentina constitutes an interesting case because it has a polymorphism for a centric fusion whose presence is strongly and systematically correlated with increased body size [28, 29]. They have concentrated their study on sexual selection on chromosome and phenotypic traits and detected significant differences between successful and unsuccessful males with three morphometric traits. Moreover a significant difference in fusion dosage was noticed, before and after directional sexual selection the fusion homozygotes being the most favoured karyotypes.

Acknowledgments

The authors are grateful to the Principal, Yuvaraja’s College, Department of Zoology, University of Mysore, Mysore-570005 for providing facilities, Dr. S. N. Hegdeand Dr. M.S. Krishna for help during the investigation. Authors are also grateful to UGC financial support for Minor research project to Dr. S.C. Jayaramu.

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ROLE OF INVERSION SYSTEM ON MATING ACTIVITIES AND FITNESS TRAITS IN DROSOPHILA ANANASSAE

S.C. Jayaramu1* and M. Prathibha1

1 Department of Zoology, Yuvaraja’s College, University of Mysore, Mysore, India.

*Corresponding Author Email id: [email protected]

Manuscript received 10th August 2014, revised 15th November 2014, Accepted 6th December 2014

Abstract Natural populations are endowed with large amount of chromosomal and genetic variation. The role of inversions on mating behavior and fitness has been studied. Three different strains of D. ananassae carrying 2LA, 3LA and 2LA+3LA inversions and one inversion free strain were built up in the laboratory using the female flies collected from natural habitat at Dharwad, India. Mating behaviors such as courtship latency, mating latency and copulation duration, fitness characters such as Fecundity and Fertility were studied in these inversion and inversion free stocks using no choice experiment. The mating behaviors pattern in three different inversions and inversion free strains of D. ananassaewere quantified and compared between different strains. The carrier of two inversions (2LA+3LA) took more time to copulate but had higher fitness than inversion free stock. The concept of inversion heterokaryotype superiority is confirmed with reference to both inversions. Key words: Drosophila ananassae, inversions, heterokaryotype, mating behavior, fitness.

Introduction: Studies have documented that

inversions in Drosophila influence fitness and

heterokaryotypes are superior over homokaryotes [3].

The relationship between sexual activity and

chromosomal polymorphism has also been found in

D. persimilis [25, 26, 27], in D. pseudoobscura [14];

D. pavani [5], D. subobscura [24] and D. robusta

[15]. In addition to inversions, isozyme variants also

have influence on sexual behaviour, body size and

fitness. The body size, an observable and measurable

phenotypic trait also affects the fitness [9, 16, 17].

Both laboratory and field studies have shown

influence of male size on male mating success and

other fitness characters [10, 12]. In Drosophila,

Anderson and Brown [1] and Ehrman et al [8] have

shown rare male mating advantage for inversion

karyotypes. The question is whether inversion

polymorphism, enzyme polymorphism and

morphometric variation are inter related to have effect

on the fitness or each one is independent of one

another? Whether, this superiority is limited only to

inversion polymorphs or seen in enzyme and other

polymorphs? Whether, inversion polymorphism has

any influence on the expression of allozyme alleles?

The role of inversion polymorphism on fitness has

been well established. How about the allozyme

polymorphism? The sexual behavioural traits such as

mating speed, copulation duration, fecundity, fertility

etc which are considered as fitness characters are of

quantitative nature. What is the role of inversion or

allozyme polymorphism on these traits? In the

present studies, the authors have tried to address the

above questions. For this purpose D. ananassae has

been selected as the experimental model because of its

following characteristics. It is a cosmopolitan

domestic species belonging to melanogaster group of



ananassae sub group and ananassae species complex

[4]. This species occupies a unique status in the

whole of genus Drosophila due to certain peculiarities

in its genetical behavior [18, 19]. Absence of male

crossing over, high level of inversion polymorphism

and high mutability are the features which make it

useful for certain genetic studies. The species harbors

large number of inversions and carries three well-knit

co-extensive inversions namely 2LA on the left arm of

the 2nd chromosome, 3LA on the left arm of the 3rd

chromosome and 3RA on the right arm of the 3rd

chromosome. The frequency of these inversions

varies in different geographical populations and hence

they can be subjected to different types of genetic

analysis on inversions.

The author in the present studies has made

mating behaviour and fitness are studied in an

inversion free strain and strains carrying 2LA, 3LA

and 2LA+3LA inversions of Dharwad population of

D. ananassae.

Material and methods

To analyze the role of inversions on mating

behaviour of three different strains carrying 2LA,

3LA and 2LA+3LA inversions and one inversion

free strain were built up in the laboratory using the

female flies collected from natural habitat at

Dharwad. These females were individually placed

in vials containing wheat cream agar media

(isofemale line) and when larvae appeared, eight

larvae from each vial were sacrificed to check for

presence or absence of inversions in their salivary

gland chromosomes. D. ananassae populations

collected from Dharwad carries two common

inversions namely, 2LA and 3LA. The wild caught

individuals therefore were either without inversion

(designated as IA), or carry 2LA alone (IB), or

3LA alone (IC) or both 2LA+3LA (ID) (Fig. 1-3).

When all the eight larvae carried a given inversion,

then that individual (their mother) was designated

as the strain carrying that particular inversion. The

adult progenies which appeared from such mothers

were classified as inversions free, 2LA, 3LA, and

2LA+3LA strains. These strains were separately

maintained for six generations and at each

generation, three to five larvae were used to check

for the presence or absence of respective

inversions. Although in each generation, the

polytene chromosomes showed the presence of

either inversion loop or absence of loop, because

they originate from same isofemale line all progeny

contained only that particular inversion

homokaryotype or heterokaryotype. The adults

emerged from these strains were used to build up

populations for the study of variation in mating

behaviour, morphometric traits and isozymes.

Analysis of mating behaviour and fitness

among four inversion phenotypes

Different traits of mating behaviour viz.,

courtship latency, mating latency and copulation

duration, and courtship acts like tapping,

scissoring, vibration, circling, licking, ignoring,

extruding and decamping were recorded for the

mated pairs of IA, IB, IC and ID strains. The

terminologies used here are similar to those used

by Hegde and Krishna [11]. Virgin females and

bachelor males of D. ananassae were isolated from

the adults which developed from the larvae left out

after inversion analysis. The virgin and bachelor

isolation was made within 3 hours of exclusion and

these flies were kept separately for the study of

mating behavior. After 5 days a virgin female


along with a bachelor male was placed in an Elens-

Wattiaux mating chamber (a circular chamber with

a diameter of 9 cms) to study mating behaviour.

Each pair was observed for 1hr and if there was no

mating, then the pair was discarded. The mating

behavior and the individual courtship traits were

recorded following the procedure of Hegde and

Krishna [11]. These observations were made

simultaneously for each pair by two observers and

then the data were pooled. A total of 30 pairs were

observed in this way.

These copulated pairs were separately

transferred to vial (3”x1”) containing wheat cream

agar medium; and transferred to fresh food vials

every day (once in 24hrs) to study fertility of these

strains. Number of adults emerged were counted

for fifteen days. The means and standard errors of

fertility were calculated. One way ANOVA

followed by Duncan multiple range test (DMRT)

was applied to the data on fertility.

To analyze fitness each mated pair was

transferred into a vial containing wheat cream agar

medium. After 24 hours, the pairs were transferred

to fresh food vial, and the eggs laid in the previous

vial were counted. This procedure was continued

for 15 days and the total number of eggs laid and

the adults emerged from each pair was recorded to

determine fecundity and fertility of these strains.

The data on the mean courtship traits, fecundity

and fertility was statistically analyzed by One way

ANOVA followed by DMRT.

Results and Discussion

Fig.1. 2LA inversion of D. ananassae Fig. 2. 3LA inversion of D. ananassae

Fig. 3. 2LA+ 3LA inversion of D.ananassae


Table 1: Qualitative and quantitative courtship traits in different inversion

strain of Drosophila ananassae (Values are Mean ± SE)


IA (Inversion free)

IB (2LA inversion)

IC (3LA inversion)

ID (2LA+3LA inversions)

F value

P value

Mating latency 27.48±1.21d 24.36±.79c 18.82±.34b 12.60±.41 a 71.54 0.000**

Copulation duration 3.37±0.02a 3.68±0.05b 4.07±0.04c 4.43±0.03d 127.09 0.000**

Tapping 7.18±0.17a 8.86±0.32b 11.86±0.35c 13.30±0.30d 87.07 0.000**

Scissoring 7.62±0.22a 9.56±0.28b 12.94±0.41c 14.06±0.44d 71.28 0.000**

Vibration 8.72±0.31a 9.96±0.24b 12.56±0.31c 13.52±0.43d 44.43 0.000**

Circling 3.12±0.13a 3.76±0.17b 5.06±0.16c 6.48±0.21d 70.97 0.000**

Licking 2.82±0.12a 3.58±0.12b 4.88±0.17c 6.06±0.20d 71.18 0.000**

Ignoring 6.32±0.27d 5.40±0.21c 4.48±0.17b 3.24±0.12a 37.98 0.000**

Extruding 6.42±0.24d 4.92±0.23c 3.72±0.15b 2.46±0.11a 72.88 0.000**

Decamping 5.68±0.24d 4.30±0.16c 3.58±0.20b 2.72±0.18a 39.46 0.000**

Note: 1) Same superscript in each row indicates that the value is non significant by DMRT. 2) Mating latency and copulation duration are measured in minutes. 3) **P< 0.001.

Table 2: Fecundity and fertility of different inversion strains of D. ananassae (Values are Mean ± SE)


IA (Inversion

free)

IB (2LA

inversion)

IC (3LA

inversion)

ID (2LA+3LA inversion)

F

value P

value

Fecundity 172.06±3.21a 187.84±4.57b 189.58±2.87b 210.82±6.02c 13.35 0.000**

Fertility 134.56±2.81a 147.48±4.28b 150.94±3.21b 165.52±3.29c 13.59 0.000**

Same superscript in each row indicates that the value is nonsignificant by DMRT. **P< 0.001.

In the present study we noticed that mating

latency was highest in strain IA of D.ananassae

while it was lowest in ID (Table 1). The study also

shows mean copulation duration of different

inversion strains revealed that mean copulation

duration was highest in strain ID while lowest in IA


strain. All these values observed are statistically

significant by ANOVA followed by DMRT (Table

1). Copulation duration of IA strain was

significantly lesser than IB, IC and ID strains.

Mean copulation duration of IB was significantly

greater than IA strains but lesser than IC and ID

strains. Mean copulation duration of IC strain was

significantly lesser than ID strains but greater than

IA and IB strain. On the other hand, mean

copulation duration was significantly greater in ID

strain compared to all others.

Male courtship activities of ID strain was

significantly highest while of IA strain was lowest

(Table 1). Male courtship activities such as

tapping, scissoring, vibration, circling and licking

of IA strain was significantly less than those strains

that carried inversions. The courtship activities of

IB strain were greater in IA and less in IC and ID

strains. It was noticed that the male courtship

activities of tapping, scissoring, vibration, circling,

and licking activities were performed in the

following decreasing order was, IA < IB <IC < ID.

This indicates that the inversion free strain showed

the lowest activity and the carriers of inversion

exhibited the same parameter more number times.

The nonreceptive females also behaved in the same

manner as those of males. The females of

inversion free strain showed greater repulsion than

the carrier of inversions. The strains carrying

double inversion was least repulsive than those of

carriers of single inversion and accepted males

more quickly.

Similar observations were made with

reference to fecundity also (Table 2). Fecundity

was highest in ID strain and lowest in IA strain and

the values were significant (Table 2). The

fecundity of IB strain was significantly greater than

IA strain and lesser with ID strain but not

significant with IC strain. The fecundity of IC

strain was significantly greater than IA strain and

lesser with ID strain but not significant with IB

strain. The fecundity was significantly greater in

ID strain compared to all others. Thus the mean

fecundity of the carrier of double inversion was the

highest while the strain with no inversion was

lowest. Table 2 also shows mean fertility of

different strains. Highest fertility was noticed in ID

strain and least in IA strain. The fertility also

varied in the same pattern. Thus the fertility of the

carrier of double inversion was the highest while it

was lowest in strain with no inversion.

In Drosophila many adaptive functions has

been found to be associated with inversion

polymorphism. Morphometric traits, mating

activities, fitness and certain genetic loci are

associated with inversion polymorphism. A review of

Table 1 revealed that mating latency was highest in

strain carrying inversion free while it was lowest in

strain with 2LA+ 3LA inversion, while strain with

double inversion has longest copulation duration than

others. The mean copulation duration was lowest in

the inversion free strain. Although the double

inversion strain had long courtship and mating

latencies, with reference to copulation duration it is

performing well. Similarly we also noticed highest

fecundity and fertility in double inversion strain than

the others. Thus the double inversion strain exhibits

heterotic effect with regard to copulation duration.

Evan the courtship display the males of double

inversion strain showed greater courtship acts

(tapping, scissoring, vibration, circling and licking)

than the other strains (Table 1). On the other hand

female showed less rejection responses to males of


double inversion strain than the others. This agrees

with work of Hegde and Krishnamurthy, [10];

Singh and Chatterjee, [20]; Hegde and Krishna, [11];

Sisidia and Singh, [21, 22, 23].

Fecundity is one of the fitness characters

which has relevance to the reproductive success and

survival of a given species [13]. In Drosophila it is

one of the less known quantitative traits. Fecundity is

extremely sensitive to a great variety of direct

environmental factors [2]. It also has a direct bearing

on the number of offspring produced by the female.

Scrutiny of Table 2 that shows differential mean

fecundity of females of different inversion strains of

D. ananassae reveals that fecundity was highest in

strain carrying double inversions than others. The

strain without inversion had the lowest fecundity.

With reference to fecundity, the 2LA and 3LA

inversion strains non significant and have higher

fitness than the inversion free strains thereby

exhibiting heterotic effect which confirms the

observations of Dobzhansky and Levene [7].

Fertility is another fitness character which

determines the reproductive success of a species. In

the present study, the author has noticed highest

fertility in the double inversion strain of D. ananassae.

The same kind of result was also noticed with

reference to fecundity. The fertility of without

inversion strain was lowest when compared to the

carry single or double inversion strains. The four

strains of D. ananassae used in the present study are

derived in the laboratory. The present findings of the

author thus agrees with that of Dobzhansky [6] who

has noticed the heterotic effect both in the natural

populations and in the population cages of D.

pseudoobscura that are not directly brought from

natural environments.

Acknowledgments

The authors are grateful to the Principal,

Yuvaraja’s College, Department of Zoology,

University of Mysore, Mysore - 570005 for providing

facilities, Dr. S. N. Hegde and Dr. M. S. Krishna for

help during the investigation. Authors are also grateful

to UGC financial support for Minor research project

to Dr. S.C. Jayaramu.

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COMPARATIVE STUDIES ON THE MORPHOLOGICAL AND REPRODUCTIVE TRAITS OF FOUR MULBERRY VARIETIES (MORUS SPP.)

K. H. Venkatesh*1, S. Shivaswamy2 and Munirajappa1

1 Mulberry Breeding & Genetics Laboratory, Department of Sericulture/Life Science, Bangalore

University, Bangalore-56006, India. 2 Associate professor, Department of Sericulture, Bharati College, Bharati Nagara, Mandya-571422

Karnataka, India.

*Corresponding author: Email id: cytogenetics1 @ gmail.com

Manuscript received 12th July 2014, revised 23th October 2014, accepted 26th December 2014

Abstract Four popular mulberry verities, viz., S799, S1635, Morus macroura and S36 were selected for morphological traits like height, internodal distance, leaf area and colouration, stomatal frequency and reproductive parameters included length and number of flowers per inflorescence and pollen fertility were evaluated. Mulberry varieties exhibited considerable variations with regard to morphological traits and observed stomatal frequency and staining ability higher in diploid and uneuploid compared to their counterparts. Reduced height, number of branches internodal distance and staining ability of pollen noticed in tetraploid and triploid. Key words: Mulberry (Morus spp.), Micro morphology, Diploid, Triploid, Tetraploid, Uneuploid. Introduction: Genus Morus to which all mulberry

plants belong to forms an economically important

group of family Moraceae with more than 60 species

found distributed in both the hemispheres [13]. The

foliage of the plant is used mainly as a unique source

of silkworm (Bombyx mori L.). Most of the cultivated

varieties of mulberry are diploid with 2n=28

chromosomes, a few are polyploids [8, 19]. The

Meiotic studies of some varieties of Morus were

studied [23] and confirmed the extreme difference

between the 13 small pairs and one large pair of

chromosomes. Micro morphology and reproductive

traits of different ploidy level of the mulberry varieties

were studied and are considered diploid parents are

superior to triploids and tetraploids [22]. Stomatal

frequency and karyotypic studies have been studied

[23, 24] and karyotypes of these taxa are symmetric,

only metacentric and submetacentric chromosomes

are found in the somatic complement. Triploid

varieties have higher leaf yield as well as better

nutritive qualities from the point of silkworm rearing

when compared to diploid varieties [1, 14, 15].

Natural tetraploid varieties are occurring in the wild

and in the cultivated forms in the eastern Himalayas

[4, 5] its leaves are unsuitable for silkworm feeding.

In the present study is focused on the comparative

account of morphological and reproductive traits of

four popular mulberry varieties.


Table 1. Comparison of morphological characters in diploid, triploid, tetraploid and uneuploid mulberry varieties.

Characters Variety S799 Variety

S1635 M.

cathyana Variety S36

Growth habit Height (cm) 229 219 197 231 Number of branches 09 07 05 08

Intermodal distance (cm) 4.0 4.3 5.0 3.9

Leaf Leaf size 197.35 182.76 170.45 197.00 Length of petiole (cm) 4.0 4.2 4.6 4.3

Width of petiole 0.40 0.37 0.31 0.39

No. of stomata per unit area (mm2)

180.81 118.22 110.78 190.00

Width of stomata (µm) 13.9 15.8 17.6 13.2

Leaf texture Thick, Green,

Chartaceous

Thin, Green,

Chartaceous

Thin, Green,

Coriaceous Thick, Green, Chartaceous

Flower Length of inflorescence (cm)

3.5 3.3 3.0 3.6

Diameter of inflorescence (cm)

1.2 1.1 1.0 1.2

No. of flowers per inflorescence (cm)

38 33 29 39

Length of flower (cm) 0.64 0.61 0.60 0.61

Pollen stain ability (%) 98.26 92.62 90.44 98.00


Materials and methods

Four mulberry varieties, namely S799, S1635, Morus

macroura and S36 which are maintained in the

mulberry germplasm bank of Department of

Sericulture, Bangalore University, Bangalore, India,

were taken for present study. Cuttings of these

varieties were planted in pots for experimental use.

Morphological characters and reproductive traits are

critically examined at different stages of growth and

development. Following the procedure laid down in

the mulberry descriptor [3].

Stomatal frequency

Stomatal frequency and size were calculated by using

the formula and expressed as number of stomata/mm2

[2, 15] and pollen fertility was also assessed by

staining of pollen grains with 2% aceto- carmine.

Stomatal frequency = Number of Stomata x mm2

Area of microscopic field

Results and discussion

Comparative accounts of morphological and

reproductive traits in diploid, triploid, tetraploid and

uneuploid mulberry varieties are summarised in

Table 1.

VarietyS799. It is an evolved male variety from

Berhampore Institute. This variety best suited for

irrigated condition. It has better rooting and sprouting

abilities and it is capable of thriving well both in

temperate and tropical conditions. It revealed diploid

chromosome number of 2n=28. Stem is light green to

brown in colour. Leaves are larger, thick, dark green,

coriaceous, chordate, unlobed serrate and acuminate.

This variety exhibited maximum height, short and

thick petiole, short internodal distance and increase in

number of stomata per unit area. Stomatal frequency

and pollen staining ability was found to be

210.62/mm2 and 97.22% respectively (Fig. 1-3).

Variety S1635.It is also evolved male variety through

ployploidization, mutation breeding and selection.

Stem is green to greyish brown in colour. Leaves are

medium, deep green, unlobed, chordate, dentate and

acuminate. This variety revealed triploid chromosome

number of 2n=42 [19]. This variety exhibited medium

in height and internodal distance and number of

stomata per unit area are decreased when compared to

diploid variety. Stomatal frequency and pollen

stainability was found to be 180.44/mm2 and 94.66%

respectively (Fig. 4-6).

Morus macroura. It isan owing to the deficiency of

observation on the pubescence of the plant; it is being

one of the important characteristic for identifying this

variety. It revealed tetraploid chromosome number of

2n=56 [21]. Stem is purple green to grey brown in

colour. The leaves are smaller, thin, upper surface is

dark green and lustrous with a pale green under

surface, lobed, margin is crenate-dentate, acuminate

and having thin long internodes. Many minute

pubescences were found on young stem and leaves.

This variety exhibited reduction in height and number

of branches when compared to diploids and triploids.

Stomatal frequency and pollen stainability was found

to be 179.44/mm2 and 92.48% respectively (Fig.7-9).

Variety S36. This variety is best suited for irrigated

condition. Under ideal agro climatic conditions this

variety yields 45 tonnes of leaf yield per hectare in

one year. It is an uneuploid mutant with chromosome

number of 2n=30 [19].Stem is light green to brown in

colour. Leaves are larger, thick, deep green, chordate,

unlobed serrate and acuminate. This variety exhibited

maximum height, short and thick petiole, reduction in


internodal distance and increase in number of stomata

per unit area. Stomatal frequency and pollen stain

ability was found to be 230.22/mm2 and 98.00%

respectively (Fig.10-12).

Fig. 1-12. 1, 2 & 3, Twig, stomatal frequency and pollen stain ability of variety S799, 7, 8 &9, Twig,stomatal frequency and pollen stain ability of Morus macroura

4, 5&6, Twig,stomatal frequency and pollen stain ability of variety S1635, 10, 11 & 12, Twig , stomatal frequency and pollen stain ability of variety S36

Comparative morphological and reproductive traits

on four mulberry varieties, some variations are

recorded with respect ploidy level, stem color, leaf

color and texture, stomata frequency and pollen

staining ability, etc. These variations are largely due

to genetic flux operating on the evolution of

different mulberry variants [16].


Varieties, S799, S1635, Morus macroura and S36

belonging to Morusalba are morphologically

distinct and some similarities in their adaptation,

good rooting and leaves with identical leaf margin

and dissimilarities in there, leaves texture, height,

internodal distance, stem colour, inflorescence,

pollen stain ability and stomatal frequency were

recorded. Cytologically S799, S1635, Morus macroura

and S36 showed 2n=28, 2n=42, 2n=56 and 2n=30

chromosomes respectively. Leaves of Morus

cathyana are light green and coarse in texture.

Diploid forms are grow more quickly and possesses

larger dark green leaves when compared to triploids

and tetraploids.

Morphological characters of uneuploid variety S36

resembled diploid in their adaptation i e. leaf yield,

leaf size, leaf margin ,unlobed and light green

leaves, good rooting, etc. Observation of uneuploid

number along with the normal ones may be due to

the vegetative propagation. The partial adaptation of

a vegetative propagation has resulted in the

maintenance of such altered nuclei in the somatic

tissues as stated [9]. However, leaves of Morus

macroura are hirsute, pubescence on the under

surface, lustrous, coarse in texture. Hence these

leaves are unsuitable for silk worm feeding. The

frequency of stomata per unit area is significantly

less in triploid and tetraploid compared to diploid

and uneuploid. The present findings are in

agreement with the reports of Tikadar etal., [17].

Stomatal frequency is an important parameter in

selecting drought resistant genotype [7]. Stomatal

frequency correlated with drought and disease

resistant [10, 12]. Further lesser frequency per unit

area is more suitable for rain fed conditions.

However, reduction in the internodal and number of

stomata per unit area indicates that the increased

dosage of genes does not always increase in size but

may also reduce it [8]. In the present study diploid

mulberry variety showed marginally higher pollen

fertility when compared to triploid, tetraploid and

uneuploid varieties. The reduced pollen fertility in

triploid and tetraploid can be attributed to various

meiotic anomalies which invariably result in the

loss of chromatin materials [9].This information

will be of much use in establishing a phylogenetic

relationship and evolution of mulberry and will also

help in selecting mother plants for hybridization

based on ploidy level, morphology and reproductive

traits. These variations may be even attributed to

genetic drift. Finally it can be resolved that

morphological variations and evolution of Morus

spp. is mainly due to structural changes in the

ployploidization and mutation.

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polyploidy mulberry tree (111). The evaluation

of breeded polyploidy mulberry leaves and the

results of feeding silkworms on them Research

Reports of Faculty of Textile and Sericulture

Shinshu University, 1959, 9,6. Ueda, Japan.

15) A. K. Sikdar, M. S. Jolly, B. N. Susheelamma

and K. Giridhar Indian J .Seric., 1986,25,2-88

16) T. Sugiyama, On the breeding of triploid

mulberry by diploidizing gamete cells (A

Preliminary Note) Jpn. J. Breed1959, 9,41.

17) A. Tikadar and S. B. Dandin, Current Science,

2007, 92 (12), 1729.

18) A. Tikadar, A. Ananda Rao and P. Mukherjee,

Indian J. Seri.,1999, 38(2), 160.

19) K. H. Venkatesh, Cytogenetical investigations

in the Genus Morus L. Ph.D thesis, 2007,

Bangalore University, Bangalore.

20) K.H. Venkatesh and Munirajappa, J. Cytol. &

Gen. 2012, 13 (NS), 29.

21) K. H. Venkatesh, S.Shivaswamy and

Munirajappa, Comparative micromorphology

andreproductive studies in three mulberry

varieties (Moraceae). International Journal of

Science and Nature, 2013, 4 (4), 608.

22) K. H. Venkatesh, R. Nijagunaiah and

Munirajappa, Cytogenetical studies in some

diploid mulberry varieties (Moraceae).

Cytologia, 2013, 78,1- 69.

23) K. H. Venkatesh, N. Venu,B. Dinesh and

Munirajappa, J. Cytol Gen., 2013, 14 (NS), 101.

24) K. H. Venkatesh, Munirajappa and S.

Shivaswamy, Ind. App. Res., 2014, 4,6-35.


CHROMOSOME NUMBERS, STOMATAL FREQUENCY AND KARYOTYPE STUDIES OF THREE MULBERRY GENOTYPES (MORACEAE)

K. H. Venkatesh1*, S. Shivaswamy2 and Munirajappa1

1Department of Sericulture/Life Science, Bangalore University, Bangalore -560056, India

2 Department of Sericulture, Bharathi College, Bharathinagara, Mandya -571422,

Karnataka, India.

*Corresponding author: Email id: [email protected]

Manuscript received 17th October, revised 12th November, accepted11th December 2014

Abstract Three mulberry varieties, viz., RFS135, S36 and S1708 were selected. Stomatal frequency, somatic chromosome numbers, ploidy level, range of chromosome length, arm ratio and haploid chromatin length were studied for these varieties. RFS135 is diploid with 2n=28, S36 is uneuploid with 2n=30 and S1708 is triploid with 2n=42 somatic chromosomes numbers respectively.Somatic chromosome length ranges from 1.66 휇m to 3.16 휇m where as an arm ratio ranges from 0.39 to 1 .00 휇m.Stomatal frequency was lesser in triploid variety when compared to diploid and uneuploid varieties. Their karyotypes were commonly bi-modal, decreasing in length from the longest to the shortest chromosomes.

Key words: Mulberry (Morus spp.) stomatal frequency, mitosis, karyotype analysis

Introduction: In mulberry cultivation, attention

must be given to both quality and quantity of

mulberry leaves. They must be high yielding with low

inputs. Among the related fields, information on

cytology viz., chromosome number, chromosome

morphology, ploidy level, meiotic behaviour etc.

provide more dependable information to classify the

available material into taxa of different magnitude.

Most of the cultivated varieties of mulberry are

diploids with 2n=28 chromosomes, but a few are

polyploids [9 14]. Meiotic studies of diploid (2n=28)

varieties of Morus were studied and confirmed the

extreme difference between 13 small pairs and one

large pair of chromosomes[18].Many triploid varieties

are considered to superior than diploids in leaf yield

and nutritive qualities of leaf. Micro morphology and

reproductive characteristics of different ploidy level of

the mulberry varieties were studied [16] and are

considered diploid parents are superior to triploid and

tetraploid. Stomatal frequency and karyotype analysis

of few mulberry genotypes were studied [15, 17].

Morphological, anatomical and reproductive

parameters in different ploidy levels of mulberry

varieties were studied [19]. These different

chromosomes numbers has reflected on their

micromophology and reproductive characters of

diploid, triploid and tetraploid varieties. In this report

chromosome numbers, stomatal frequency and

karyotype of three mulberry varieties have been

discussed.



Materials and methods

Root tips were collected between 10.00 to 10.30

a.m. and pre-treated with saturated solution of 0.002

M 8 - hydroxyquinoline at 100C for 3 hours and then

fixed in 1:3 glacial acetic acid: alcohol. They were

transferred to 2% aceto-orcein: 1N HCl (9:1) for

seven minutes and squash preparations were made in

45% of acetic acid. Photomicrographs and drawings

were made on the same day of preparation. For each

variety number of preparations was made to ascertain

the chromosome number and their morphology.

Ideograms were drawn using suitable scale.

Karyotype classicifications were made according to

Levan et al. [11].

Stomatal frequency

Stomatal frequency was determined by nail polish

impression method. Stomatal frequency was

calculated by using the formula and expressed as

number of stomata/mm2 [1].

Stomatal frequency = Number of Stomata

Area of microscopic field

Results and discussion

Details of the stomatal frequency, somatic

chromosome number, ploidy level, range of

chromosome length, karyotype formula, arm ratio and

haploid chromatin length are presented in Table 1.

Table.1. Karyotype analysis in RFS135, S36 and S1708 mulberry varieties.

M. varieties

Stomatal frequency/ mm2

2n Chromosme number

Ploidy level

Karyotype

Chromosome size range (흁m)

Arm ratio (흁m)

Haploid chromatin Length(흁m)

RFS135

260.40

28

Diploid

2n=28=10Bm+6Bsm+4Cm+8Csm

1.79 - 2.90

0.39-1.00

29.20

S36

290.00

30

Uneuploid

2n=30=10Bm+10Bsm

+2Cm+8Csm

1.66 - 2.50

0.41-0.97

31.40

S1708

150.60

42

Triploi

d

2n=42=22Bm+20Bsm

2.00-3.16

0.63-1.00

52.33


Variety RFS135

This variety has been recommended for cultivation

under rainfed condition. Under ideal agro-climatic

conditions this genotype yields 28-30 tonnes of leaf

yield/ha/year. The stomatal frequency was found to be

260.40/mm2 (Fig. 1). Chromosomes are very small

(1.79 to 2.90휇m) in size. This taxon revealed diploid

chromosome number of 2n=28 (Fig. 2) with ten

medium chromosomes with median primary

constriction, six medium chromosomes with sub

median primary constriction, four short

chromosomes with median region primary

constriction and eight short chromosomes with sub-

median primary constriction. The karyotype formula

of this taxon is 2n=28=10Bm+6Bsm+4Cm+8Csm (Fig.

7). The karyotype is symmetrical with an arm ratio

ranging from 0.39 to 1.00. The total chromatin length

of haploid complement was 29.20휇m.

Variety S36

This variety is best suited for both rainfed and

irrigated conditions. Under ideal agro-climatic

condition this genotype yields 38-40 tonnes of leaf

yield/ha/year. It is a fast growing taxon exhibits good

rooting and sprouting ability. The stomatal frequency

was found to be 290.00/mm2 (Fig. 3). Chromosomes

are small (1.66 to 2.50 휇m) in size. This taxon

revealed uneuploid chromosome number of 2n=30

(Fig. 4) ten medium chromosomes with median

primary constriction, ten medium chromosomes with

sub median primary constriction, two short

chromosomes with median region primary

constriction and eight short chromosomes with sub-

median primary constriction. Only metacentric and

sub metacentric chromosomes are found in the

somatic complement. The karyotype formula of this

taxon is 2n=30=10Bm+10Bsm+2Cm+8Csm (Fig. 8). The

karyotype is symmetrical with an arm ratio ranging

from 0.41 to 0.97. The total chromatin length of

haploid complement was 31.40휇m.

Variety S1708

It is triploid mulberry variety. It is being cultivated as

perennial bush especially in hilly tract. The stomatal

frequency was found to be 150.60/mm2 (Fig. 5).

Chromosomes are very small (2.00 to 3.16 휇m) in

size. This taxon revealed triploid chromosomes

number of 2n=42 (Fig. 6) with twenty two medium

chromosomes with median primary constriction and

twenty medium chromosomes with sub-median

primary constriction. Only metacentric and sub

metacentric chromosomes are found in the somatic

complement. The karyotype formula of this taxon is

2n=42=22Bm+20Bsm (Fig. 9). The karyotype is

symmetrical with an arm ratio ranging from 0.63 to

1.00. The total chromatin length of haploid

complement was 52.33휇m.


Fig :1 & 2, Stomatal frequency and somatic chromosomes (2n=28) of variety RFS135 3 & 4, Stomatal frequency and somatic chromosomes (2n=30) of variety S36

5 & 6, Stomatal frequency and somatic chromosomes (2n=42) of variety S1708


Fig : 7, 8 & 9, Ideogram of varieties RFS135, S36 and S1708 .

To evolve dependable system of classification, a

study of all the three types of relationship viz.,

Phylogenetic, Phenotypic and Geotropic is

imperative as stated [4]. Stated that determination

of biological species in cultivated plants the

observational basis are full description of

chromosome number, karyotype analysis and

evidence of natural hybridization [2].

Stomatal frequency and size are considered as two

important parameters in characterization of

mulberry genotypes. These two characters are

having positive correlation with drought resistance.

The observed small size and lesser frequency of

stomata in triploid than diploid and uneuploid

varieties. Established that stomatal frequency and

size decreases with increase in ploidy level [13].

The observed genotypic level differences in

stomatal frequency are in agreement with various

other reports [8], [7] and [20]. The present findings

also clearly showed frequency of stomata per unit

area is significantly less in triploid compared to

diploid and uneuploid. Moisture retention capacity

will be higher in those mulberry varieties

possessing smaller and lower stomatal frequency

[3].

Basic chromosome number of the genus Morus

L., as x=14 for majority of the species have been

reported [5] and [10]. In the present study the three

mulberry varieties belong to Morus alba. Three

varieties showed diploid with 2n=28, uneuploid

with 2n=30 and triploid with 2n=42 chromosomes.

Mulberry variety S36 has displayed the uneuploid

chromosome number of 2n=30. The observation of

uneuploid number like 2n=30 for S36 mulberry

variety confirm the observations made by earlier

workers suggesting the inconsistency of

chromosome numbers and the probable reason cited

for the same is high degree of vegetative


propagation which invariably results in polysomaty

[6]. The partial adaptation of a vegetative

propagation has resulted in the maintenance of such

altered nuclei in the somatic tissues [10]. In general

chromosomes are smaller with a close range of

length variation. Confirming the earlier reports,

cytologically they showed similar karyotype with

only two types of chromosomes, equal chromatin

length and also length range. Occurrence of good

number of natural triploids has been attributed to

the process of fertilization between an unreduced

female gamete and reduced pollen [12]. In all above

cases the cytological identity of each cultivar has

been represented in phenotypic variation.

References

1) K. R., Aneja, Experiments in microbiology, plant

pathology, tissue culture and mushroom

production technology (3rd edition) new age

international publishers, 2001, New Delhi.

2) H. C. Parker, Taxonomy and biological species

concept in cultivated plants. In Genetic Resources

in Plants, their Exploration Conservation. Eds. O.

H. Frankel and E. Bennet. Blackwell Scientific

Publications, Oxford and Edinburgh, 1970, 49, 68.

3) Basavaiah and TCS. Murthy, Natl. Semi. Mulb.

Seri. Res. 26-28, KSSR&DI, Bangalore, 2001,

India, 98.

4) M. S. Chennaveeraiah. Biosystematics-A

Presidential Address. 70th Indian Science Congress,

Section of botany, 1983.

5) B. C. Das, Cytological studies on Morus indica

Land M laevigata Wall Caryologia, 1961, 14,

159.

6) B.C. Das, Sci, and Cult., 1963, 29, 250.

7) M. K. Dwivedi, A. K. Sikdar, M. S. Jolly, B. N.

Susheelamma and N. Suryanarayana, Indian J.

Genet., 1988, 48(3), 305.

8) M. S. Eswar Rao, Improvement of mulberry through

ployploid breeding. Ph.D. Thesis, Bangalore

University, Bangalore, 1996.

9) B. S. Gill and R. C. Gupta, Cur. Sci. 1979, 48, 1-

35.

10) D. Kundu and A. Sharma, Chromosome studies in

some Indian Moraceae In P. Kachroo (ed) Recent

advances in Botany Bishen Singh Mahedra Pal

Singh Dehradum, 1976, 348.

11) A. K. Levan, Fredga, and A. A. Sandberg,

Hereditas, 1964, 52, 201.

12) H. Seki, Cytological studies on Morus Part-1

Polyploidy of mulberry tree with special reference

to spontaneous occurrence of triploid plant. J. Fact.

Sci. Sinshu Univ, 1961, 20, 1.

13) A. Tikader, Indian J. Forestry, 2001, 24,3-, 344.

14) K. H. Venkatesh, “Cytogenetical investigation in

the Genus Morus L.” Ph. D., Thesis Bangalore

University, Bangalore, 2007.

15) K. H. Venkatesh, N. Venu, B. Dinesh and

Munirajappa, J Cytol Gen., 2013c,14 (NS), 101.

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Int. .Sci. and Nat.2013b, 4, 4-608.

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Ind. J. App. Res., 2014a, 4,4-35.

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20) K. Vijayan, P. K. Sahu, S. P. Chakraborti and B. N.

Roy, Indian J. Genet., 1999, 59(14), 512.


ENVIRONMENTALLY FRIENDLY SYNTHESIS OF BIS (INDOLYL)

METHANES CATALYSED BY NITROPHTHALIC ACID

Sudhakara A1, Nataraja G1, Rajesha2, Ramesha S3

1Department of Chemistry, Jain Institute of technology, Bada Cross, Davanagere

Karnataka India-577005 2BET Academy of Higher Education, Bharathi college, Bharathi Nagara , Madur Taluk, Mandya

Karnataka, India- 571422 3 R&D Center, Department of Chemistry, Rajarajeswari College of Engineering, Mysore Road,

Bangalore

Karnataka, India – 560074

Corresponding Author: E-mail id: [email protected]

Manuscript received 17th June, revised 12th August, accepted13th November 2014

Abstract A general, mild and efficient synthesis of bis (indolyl) methanes via electrophilic substitution reaction of indole with various aldehydes and ketones under catalysis of nitro phthalic acids as potential green catalyst have been described in good yield. Key words: Aldehydes; Phthalic Acids; Ketones; bis (indolyl) methanes; Isatin.

Introduction: Bis(indolyl)alkanes and their derivatives are more attractive compounds as the bioactive metabolites of terrestrial and marine origin1. This unit has found to exhibit important biological activity. Vibrindole A 1 was demonstrated for the first time to exhibit antibacterial activity against Staphylococcus aureus, S. albus, and B. subtilis.2 Recently 2,2-Di(3-indolyl)-3-indolone 2 was isolated from the toxic mucus of the boxfish ostracion cubicus and reported to active against Staphyloccous aureus3.

Consequently, numerous methods have been reported

for the preparation of bis (indolyl) methanes.4 Of these

methods, the acid-catalyzed condensation of indoles

with carbonyl compounds is one of most simple and

straightforward approaches for the synthesis of

bis(indolyl) methanes. The acids utilized in this type

of reaction are protic acids such as CH3COOH5, HCl6,

sulphamic acid7, H3PMo12O40 . H2O8 and lewis acids

sush as InCl39, ZrCl4,10 InF3,11 FeCl3,12 In(OTf)3,13

CuBr214. Generally, these lewis acid catalysts are

moisture sensitive and are easily decomposed or

deactivated in the presence of a small amount of water

and are thus difficult to handle, further disposal of

these acids leads to environmental pollution.

At present, with the rapid development in the field of

catalytic and synthetic chemistry, researchers have

started to pay more attention to develop some eco-

friendly catalyst to avoid or minimize these harmful

NH

NH

NH O

2NH

NH

H

1



effects. Particularly, the condensation of indoles and

carbonyl compounds has been carried out successfully

using KHSO4,15 I2,16 NBS,17 LiClO4,18 CAN,19

etc.Similarly few environmentally friendly catalyst

such as ion exchange resin,20 montmorillonite K-10

clay21 and rare earth catalysts,22 zeolites,23

NaHSO4.SiO2,24 ionic liquid25,were also appeared in

literature.

Encouraged by the above survey, in the present study

we investigated the catalytic activity of various

phthalic acids in the synthesis of bis(indolyl|)

methanes formed by the condensation reaction of

indole with various aldehydes and ketones.

Nevertheless we have all ready explored the

possibility of use of nitro phthalic acid as catalyst in

the Imino Diels alder reaction25.Thus Phthalic acid,

isophthalic acid, terephthalic acid and their nitro

derivatives have found to catalyze the reaction in the

synthesis of various bis (indolyl) methanes. This is the

first report for the use of these acids as catalysts in bis

(indolyl) methane synthesis.

All the melting points were recorded in open capillary

and were compared with the literature.5,15 the purity of

the compounds was checked by TLC on silica gel and

were purified by column chromatography. 1H NMR

spectra were recorded on a Bruker-400 Hz

spectrometer using TMS as an internal standard. IR

spectra were obtained using a FTS-135 spectrometer

instrument. Mass spectra were recorded on a JEOL

SX 102/DA-6000 (10 kV) FABmass spectrometer.

Solvents, Chemicals and reagents were purchased

from Merck chemical company in high-grade quality.

Experimental

Synthesis of Bis(indolyl)methanes

4-nitro phthalic acid (1.0 mmol) was added to a

mixture of indole (2.0 mmol) and aldehydes or

ketones (1.0 mmol) in ethanol (10 mL). The reaction

mixture was stirred at room temperature for the

appropriate time (Table 3). After the completion of the

reaction, it was quenched with water (10mL) and

extracted with ethyl acetate (2 X 15 mL). And

combined organic layer were dried over anhydrous

sodium sulphate, concentrated and the crude product

was purified by silica gel column chromatography and

eluted with an ethyl acetate and petroleum ether

mixture to afford bis (indolyl) methane.

Spectral data

3,3’-bis-indolyl (phenyl) methane (3a)

Pink solid; mp 124–125 oC;IR (KBr) 3415, 3025,

1631, 1380, 1265, 1008, 734 cm-1:1H NMR (CDC13)

ppm: 7.89 (brs, 2H, NH), 7.38 (d, J=7.8 Hz, 2H),

7.33–7.35 (m, 4H), 7.19–7.30 (m, 5H), 7.00 (m, 2H),

6.64 (d, J=1.1 Hz, 2H),5.88 (s, 1H); MS(EI, 70eV):

m/z (%):322 (M+).

4-Chlorophenyl-3, 3’-bis(indolyl)methane (3c)

Pink solid; mp 78-80 oC;IR (KBr): 3411, 3055, 2923, 2848, 1617, 1417, 1327, 1013, 743 cm-1;1H NMR (CDCl3) ppm: 7.93 (brs, 2H, NH), 7.26–7.38 (m, 8H), 7.18 (t, J=7.8 Hz, 2H), 7.02 (t, 2H, J=7.6 Hz, 2H), 6.65 (s, 2H), 5.86 (s, 1H),MS(EI,70eV): m/z (%): 356 (M+).

4-Methoxyphenyl-3,3’-bis(indolyl)methane (3h)

Pinkish solid; mp 192-193 oC;IR (KBr): 3392, 3055,

2933, 2838, 1610, 1507, 1320, 1023, 743 cm-1; 1H

NH

NH

NH

Ph

1 2

EtOH / rt

3

Scheme 1

4 - NPAPhCHO+


NMR (CDCl3): 1H NMR (CDCl3): ppm: 7.93 (brs,

2H, NH), 7.26–7.38 (m, 8H), 7.18 (t, J=7.8 Hz, 2H),

7.02 (t, 2H, J=7.6 Hz, 2H), 6.65 (s, 2H), 5.86 (s, 1H);

3.77 (s, 3H); MS (EI, 70eV): m/z (%): 352 (M+).

1-(di-1H-indol-3-ylmethyl)-2-naphthol (3i)

Yellow solid; mp 203-205 0C;IR (KBr): 3415,

3020,1605, 1460, 1290, 1068, 1004, 750 cm-1; 1H

NMR (CDCl3) ppm: 12.2 (s, 1H), 8.15 (d, J=8.6 Hz,

1H), 8.06 (brs, 2H, NH), 7.83 (d, J=8.0 Hz, 1H), 7.73

(d, J=8.0 Hz, 1H), 7.3-7.45 (m, 7 H), 7.2 (t, J=7.2 Hz,

2H), 7.02 (t, J=7.6 Hz, 2H), 6.82 (s, 1H), 6.76 (s, 1H),

6.5 (s, 1H, CH),MS (EI, 70eV): m/z (%): 388 (M+).

3,3'-(2,3-dihydro-1,4-benzodioxin-6-ylmethanediyl) bis (1H-indole) (q)

Pink red: mp 238-240 0C.

1H NMR (DMSO) ppm: 10.76 (br, NH,2H), 7.3 (m,

2H), 7.03 (t, J=7.08 Hz, 2H), 6.8 (d,J= 7.07 Hz, 2H),

6.7 (m, 6H), 5.6 (s, 1H), 4.1 (s, 4H).13C NMR:

143.4,141.1,

136.4,132.6,139.9,121.7,120.5,119.6,115.5,113.7,112.

1, 111.0, 75.2, 44.2,MS (EI,70eV): m/z(%): 379(M -).

1H,1''H-3,3':3',3''-terindol-2'(1'H)-one (3r)

Brown Solid: mp 248-250 oC;1H NMR (DMSO)

ppm: 10.92 (brs, s 2H), 10.56 (s,1H),7.34 (d, J=8.01

Hz, 2H), 7.23 (t, J=6.3 Hz, 4H), 6.99 ( m, 4H), 6.8( m,

4H).

13C (75, MHz, CDCl3) 196.5, 143.2, 135.5, 133.7,

132.1, 130.5, 122.2, 121.7, 121.2, 120.5, 119.6, 116.8,

112.2, 112.1, 111.0, 86.5,MS (EI, 70eV): m/z (%):

363.1 (M -).

5'-fluoro-1H,1''H-3,3':3',3''-terindol-2'(1'H)-one (3s)

Brown Solid: mp 245-247 oC;1H NMR (DMSO)

ppm: 10.97 (brs, s, 2H), 10.60 (s, 1H), 7.35 (d, J=8.1

Hz, 2H), 7.21 (d, J= 8.34 Hz, 2H), 7.05 (m, 4H), 6.87(

d, J=2.55 Hz, 2H), 6.82 (t, J=7.23 Hz, 2H).13C (75,

MHz, CDCl3) 196.5, 142.6, 134.2, 133.5, 132.7,

130.6, 123.4, 122.1, 121.7, 120.5, 119.6, 112.2, 111.4,

111.0, 86.1,MS (EI, 70eV): m/z (%): 381 (M+).

5'-bromo-1H,1''H-3,3':3',3''-terindol-2'(1'H)-one(t)

Yellow Solid: mp 248-250 oC,1H NMR (DMSO)

ppm: 10.99 (brs, 2 NH), 10.72 (s, NH), 7.36 (d,

J=8.07 Hz, 2H), 7.26 (d, J=19.53 Hz, 3H), 7.16 (d,

J=2.37 Hz, 3H), 7.03 (m, 5H) 6.87 (d, J=3.6 Hz, 2H ),

6.82 (d, J=7.89 Hz, 2H).13C (75, MHz, CDCl3)

194.5,141.5,136.8, 136.2, 132.8, 131.6, 122.8, 122.1,

121.5, 120.5, 119.6, 112.1, 111.2, 111.0, 86.2,MS (EI,

70eV): m/z (%): 441 (M+).

5'-chloro-1H,1''H-3,3':3',3''-terindol-2'(1'H)-one (3u)

White Solid: mp 260-262 oC,1H NMR (DMSO)

ppm:10.99 (s, 2H, NH), 10.72 (s, 1H, NH), 7.36 (d, J

= 8.07 Hz, 2H), 7.26 (d, 1H), 7.19 (d, J = 8.55 Hz,

3H). 7.03 (m, 3H), 6.87 (d, J=2.4 Hz, 2H), 6.80 (d,

J=7.89 Hz, 2H).13C(75,MHz,CDCl3)196.2, 141.2,

136.5, 134.1, 131.2, 129.7, 122.2, 121.9, 120.9, 120.5,

119.6, 113.6, 112.1, 111.0, 86.1,MS (EI, 70eV): m/z

(%): 397 (M+)

Initially, we examined the 4-Nitro Phthalic acid (4-

NPA) in the model reaction of indole with

benzaldehyde (Scheme 1) in different reaction media

to investigate the solvent effect. The results are

summarized in Table 1 and shows that polar solvents

are much better than nonpolar solvents. Remarkably,

the condensation proceeded smoothly in water and to

afford desired product in good yield (80%). However,


the ethanol was found to be best for the catalytic

reaction at room temperature in terms of yield,

reaction time and product isolation.

The catalytic activities of different nitro derivatives of

phthalic acids were also tested and the results are

shown in Table 2. Interestingly, the reaction time as

well as the yield differs for each derivative. 4-nitro

phthalic acid (4-NPA) is found to be good catalyst

compared to other phthalic acids. The minimum

activity concentration of 4-nitrophthalic acid was also

tested. It was observed that, the yield depended on the

amount of catalyst loading, and in the presence of 25

mol% (based on the amount of indole) of 4-nitro

phthalic acid, the reaction afforded 95% yield of the

corresponding bis(indolyl)methanes in 3h in case of

benzaldehyde. Furthe studies shows that increasing

amounts of catalyst did not give better yield but

reduced the reaction time (Table 2).

Table 1 .Effect of Solvents in the Reaction of Indole with Benzaldehyde Catalyzed 4-nitro phthalic acid at room temperature. Solvents Yield (%)a

EtOH 95 MeOH 94 CH3CN 85 C2H5OC2H5 80 Toluene 30 Benzene 20 H2O 80 a Isolated yields

Using these optimized conditions, we examined the

reaction of various aldehydes and ketones with indole

in the presence of 4-NPA to afford bis (indolyl)

methanes (Scheme 2).

The results are summarized in Table 3. In all cases,

the electrophilic substitution reaction of indoles with

aldehydes could proceed smoothly at room

temperature to produce the corresponding bis (indolyl)

methanes in good yield in shorter times. Whereas the

reaction of the ketones and indole took longer time

when compared with aldehydes, and unreacted

ketones and indole remained the same.

Further, we also tried to explore the catalytic activities

of 4-NPA, to seek the general, mild and efficient of

this method of accessing the bisindolylalkane

framework. We then sought to apply the methodology

to preparing a variety of naturally-occurring

compounds. We first targeted vibrindole A 1,which

was obtained in 80% yield from the reaction of

acetaldehyde and indole. Then trisindolylalkane 3

(Scheme-3) isolated from the bacterium Vibrio

parahaemolyticus,26was prepared by the reaction of

indole-3- carboxaldehyde and indole in 75% yield.

We were also able to obtain the compound 4

(Scheme-4), from the coupling of isatine and two

equivalents of indoles, which is structurally very close

to 2,2-Di(3-indolyl)-3-indolone 2 was isolated from

the toxic mucus of the boxfish ostracion cubicus.

Thus 5-Fluro isatin (0.1gm 0.60 mmol) underwent

smooth condensation with indole (0.141gm 1.20

mmol) to produce 6-fluoro-1,1-di-1H-indol-3-yl-1,3-

dihydro-2H -inden-2-one with good yields without the

need for column chromatography. This clearly shows

that 4-NPA is not only suitable to activate indoles and

simple aldehydes, but it is also suitable to activate

stearically congested keto group of isatin.

NH

CHO

2 eq of indole

4-NPA / EtOH rtNH

NH

NH

3

Scheme-3

NH N

H

NH

R2R2

O 50 mol% 4 - NPA

R1

1 23

R1 EtOH / rt

Scheme 2

+

R1 = aliphatic or aromatic substitutes

R2= H, CH3


Table 2. Effect of Catalysts in the Reaction of Indolewith Benzaldehyde in EtOH at room temperature

Entry Catalyst Time (h) Yield (%)a

1. Phthalic acid 8 78

2 4-nitro phthalic acid 3 95

3 3-nitro phthalic acid 8 85

4 3,5-dinitro P. acid 6 86

5 Isophthalic acid 10 55

6 Terephthalic acid 48 38

7 2-nitrotere. P.acid 38 52

8 4-Nitrophthalic acidb 2 95

aIsolated yields; b50 mol% of catalyst.

Table 3. Scope of Bisindolylalkane formation Catalyzed by 4-NPAa

Time Yield Product Indole Carbonyl (h/min) (%)b

3a 3.00

95

3b 1.30 96

3c 1.45 95

3d 2.30 90

3e 2.30 92

3f 3.30 88

3g 1.15 94

3h 4.00 90

3i 3.30 86

3j 4.00 85

3k 3.45 88

3l 24.00 48

3m 2.00 96

NH

1

EtOH / rt

Scheme 4

4 - NPA

NH

O

OF

+

NH

NH

NH

O

F

2 4

CHO

NH

NH

CHO

H3CO

CHO

CH3

NH

CHO

ClNH

CHO

BrNH

CHO

ClNH

CHO

OHNH

CHO

O2NNH

OH

CHO

NH

NH

.

CHO

O CHONH

COCH3

NH

NH

CHO

O


3n 16.00 78

3o 2.30 90

3p 2.30 84

3q 2.00 90

3r 1.30 94

3s 0.45 95

3t 4.00 90

3u 4.30 92

a The reaction was carried out in EtOH at r. t., b

Isolated yields.

Conclusion

The catalytic properties of novel phthalic acid,

isophthalic acid, terephthalic acid and its nitro

derivatives in the condensation of indoles with

aldehydes and ketones were investigated. 4-nitro

phthalic acid served as mild and effective catalyst for

the condensation of the indole with aldehydes and

ketones in EtOH at room temperature in the presence

of both moisture and air, to afford bis (indolyl)

methanes in high yields compared to other acid

derivatives. This method offers several significant

advantages such as high conversions; easy handling,

cheaper catalyst, cleaner reaction profiles, short

reaction time and the reaction conditions are amenable

to scaling since the catalysts used are environmentally

friendly.

Acknowledgments

We thank to Principal and Management of Jain

Institution of technology for their valuable support

and encouragement of this research work and Indian

Institute of Science Bangalore,for Spectral studies.

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NH

CHO

H3CONH

MeO

CHO

ClNH

MeO

OO

CHO

NH

NH

O

ONH

NH

O

OF

NH

NH

O

OBr

NH

NH

NH

O

OCl


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ANTIMICROBIAL STUDY ON THIAZOLIDINONES OF

SUBSTITUTED N'-BENZYLIDENE-2-(PHENYLAMINO)

ACETOHYDRAZIDES K C Chaluvaraju*1, B Shalini1, P Nagendra2, G Pavithra1and R D Rakesh1

1Department of Pharmaceutical Chemistry, Govt. College of Pharmacy, Bangalore-560 027.

2Department of Post Graduate Studies and Research in Chemistry, BET Academy of Higher

Education, Bharathinagara, Mandya – 571 422.

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

Manuscript received 21th July, revised 15th September, accepted12th December 2014

Abstract In the present study about four thiazolidinones (2a-2d) were synthesized from substituted N’- benzylidene-2-(Phenylamino)acetohydrazides. These were characterized using physical data (m.p, Rf ), spectral studies and are in good agreement with the proposed ones. Antimicrobial studies of these compounds were carried out against bacteria Staphylococcus aureus, Bacillus substilis, Pseudomonas aerogenosa, Escherischia coli and pathogenic fungi Aspergillus niger and Candida albicans. Ciprofloxacin and Griseofulvin were used as standard antibacterial and antifungal drugs for comparison. Keywords: Antimicrobial studies, Ciprofloxacin, Griseofulvin, Thiazolidinones etc.,

Introduction: Microbial infections are

ubiquity and now a day’s their treatment with

the existing antimicrobial agents is becoming

challenging due to the emergence of resistance

[1]. Hence there is a need for the search of an

effective antimicrobial drug to counteract

these infections as the quest for a more reliable

and suitable drug is always fascinating and

challenging. A number of drugs containing

simple heterocycle or a combination of

different heterocyclic moieties have been in

use these days. Thiazolidinones are one such

important class of heterocycles and they drawn

the attention of chemist over the years because

of their biological importance [2]. They found

to possess antibacterial, antifungal,

anticonvulsant, antidiabetic, antiulcer,

antimycobacterial, antiviral, anti-

inflammatory, analgesic, radical scavenging,

anticoagulant and antithrombic activities[3-

13]. Famotidine, Nizatidine, Pyridothiazole,

Rosiglitazone etc., are some of the drugs

currently used in medicine and they found to

contain thiazolidinone nucleus in them[14].

However there is a paucity of literature on

their antimicrobial activities. In view of this

and in continuation of our previous work on

biologically important thiazolidinones, it was

thought worthwhile to synthesise some more

thiazolidinones from substituted N’-

benzylidene-2-(Phenylamino) acetohydrazides

for their possible antimicrobial activities as the

results obtained from our laboratory proved



the biological potentiality of thiazolidinones in

terms of their antiulcer activity[18].

Materials and Methods

The melting point of the synthesized

compounds were determined in open capillary

using LABHOSP melting point apparatus and

recorded without correction. Progress of the

reaction and the purity of the compounds were

checked using precoated silica gel TLC plates

(60 GF, 254 MERCK) and a mixture of n-

hexane and ethyl acetate (4:1) as a mobile

phase[15]. The IR spectra of the synthesized

compounds were recorded on SHIMADZU

FTIR 8400 spectrometer by KBr pellet

technique[16]. The 1HNMR spectra of the

synthesized compounds were taken using

BRUKER SPECTROSPIN-400MHz

spectrometer using CD3OD/CD3COCD3 as

solvent and TMS as internal standard [17].

The chemical shift data’s were expressed as δ

ppm.

Synthesis of substituted N'-benzylidene-2- (phenylamino) acetohydrazides (1a-1d):

The compounds 1a-1d required for the

synthesis of Thiazolidinones (2a-2d) were

synthesized from substituted 2- (phenylamino)

acetohydrazides according to the procedure

described in our previous study[18].

1a:N’-[(2-hydroxybenzylidene)-2-

(phenylamino)]acetohydrazide:Yield:55%;

m.p:1980C; Rf:0.25; IR (KBr cm-1): 1460(C=C

Ar), 1672(C=N), 3307(O-H), 1444(C-H bend); 1HNMR(CD3OD, δ ppm): 3.9(s, 2H, -CH2),

4.0(s, 1H, N-H), 5.0(s, 1H, Ar-OH), 6.4-7.1(m,

9H, Ar-H), 8.0(s, 2H, N-H & -CH2).

1b:N’-(3-hydroxy-4-methoxybenzylidene)-2-

(phenylamino)acetohydrazide:Yield:53%;

m.p:2150C; Rf:0.34; IR (KBr cm-1): 1211(C-

O), 1647(C=N), 3385(Ar-OH), 748(C-H),

1450(C-H bend); 1HNMR(CD3COCD3, δ

ppm): 3.7(s, 3H, Ar-OCH3), 3.9(s, 2H, -CH2),

4.0(s, 1H, N-H), 5.0(s, 1H, Ar-OH), 6.4-7.0(m,

8H, Ar-H), 8.0(s, 2H, N-H & -CH2).

1c:N’-[4-(dimethylamino)benzylidene)]-2-

(phenylamino] acetohydrazide: Yield:60%;

m.p:1780C; Rf:0.30; IR (KBr cm-1):

1664(C=N), 1602(C=C Ar), 1394(C-H bend);

1HNMR(CD3COCD3, δ ppm): 2.8(s, 6H, -

N(CH3)2, 3.9(s, 2H, -CH2), 4.0(s, 1H, N-H),

6.4-7.4(m, 9H, Ar-H), 8.0(s, 2H, N-H & -

CH2).

1d: N’-(3-fluorobenzylidene)-2-(phenylamino)

acetohydrazide: Yield:52%; m.p:1880C;

Rf:0.62; IR (KBr cm-1): 1668(C=N),

1473(C=C Ar), 1458(C-H bend), 1226(C-F); 1HNMR(CD3OD, δ ppm): 3.9(s, 2H, -CH2),

4.0(s, 1H, N-H), 6.4-7.4(m, 9H, Ar-H), 8.0(s,

2H, N-H & -CH2).

Synthesis of Thiazolidinones (2a-2d).

To an equimolar mixture of (0.01 mol)

acetohydrazides(1a-1d) and thioglycholic acid

(0.01mol , 0.7 ml) in 20 ml of DMF, a

catalytic amount of anhydrous ZnCl2 was

added and the reaction mixture were refluxed

for 8 hours. The reaction progress was

monitored by TLC using a mixture of n-

hexane and ethyl acetate (4:1) as a mobile


phase. The solvent was recovered under

reduced pressure and the respective residues

thus resulted were dissolved in

dichloromethane and washed with 10%

sodium bicarbonate solution, dried over

anhydrous sodium sulphate and the solvent

was recovered under reduced pressure. The

obtained residues were then recystallized from

ethanol (Scheme 1).

2a:N-[2-(2-hydroxyphenyl)-4-oxo-1,3-

thiazolidin-3-yl]-2-(phenylamino)acetamide:

Yield:64%; m.p:1400C; Rf:0.36; IR (KBr cm-

1): 1672(C=O), 1311(C-N), 1672(C=C Ar),

3015(Ar-OH); 1HNMR(CD3OD, δ ppm):

3.2(s, 2H, S-CH2), 3.9(s, 2H, -CH2), 4.0(s, 1H,

-NH), 5.0(s, 1H, Ar-OH), 5.9(s, 1H, C-H), 6.4-

7.0(m, 9H, Ar-H), 8.0(s, 1H, CONH).

2b:N-[2-(4-hydroxy-3-methoxyphenyl)-4-oxo-

1,3-thiazolidin-3-yl]-2-

(phenylamino)acetamide: Yield:57%; m.p:

1280C; Rf:0.6; IR (KBr cm-1): 1772(C=O),

1444(C=C Ar), 3057(Ar-OH); 1HNMR(CD3COCD3, δ ppm): 3.2(s, 2H, S-

CH2), 3.7(s, 3H, -OCH3). 3.9(s, 2H, -CH2),

4.0(s, 1H, -NH), 5.0(s, 1H, Ar-OH), 5.9(s, 1H,

C-H), 6.4-7.0(m, 7H, Ar-H), 8.0(s, 1H,

CONH).

2c: N-{2-[4-(dimethylamino)phenyl]-4-oxo-

1,3-thiazolidin-3-yl}-2-(phenylamino)

acetamide: Yield:61%; m.p: 1620C; Rf:0.7; IR

(KBr cm-1): 1685(C=O), 1315(C-N),

1458(C=C Ar), 1363(C-H bend); 1HNMR(CD3COCD3, δ ppm): 2.8(s, 6H,

N(CH3)2, 3.2(s, 2H, S-CH2), 3.9(s, 2H, -CH2),

4.0(s, 1H, -NH), 5.0(s, 1H, Ar-OH), 5.9(s, 1H,

C-H), 6.4-7.0(m, 9H, Ar-H), 8.0(s, 1H,

CONH).

2d:N-{2-[4-(fluoro)phenyl]-4-oxo-1,3-

thiazolidin-3-yl}-2-(phenylamino)acetamide:

Yield:56%; m.p: 1720C; Rf:0.4; IR (KBr cm-

1): 1678(C=O), 1226(Ar-F), 1506(C=C Ar); 1HNMR(CD3OD, δ ppm): 3.2(s, 2H, S-CH2),

3.9(s, 2H, -CH2), 4.0(s, 1H, -NH), 5.9(s, 1H,

C-H), 6.4-7.0(m, 9H, Ar-H). 8.0(s, 1H,

CONH).

Antimicrobial activity

Antimicrobial activity of the synthesized

compounds 2a-2d were carried out using

bacterial strains Staphylococcus aureus,

Bacillus substilis, Pseudomonas aeurgenosa,

Escherischia coli and pathogenic fungal

strains Aspergillus niger and Candida albicans

by agar well diffusion method[19]. Nutrient

agar and dextrose sabouraud’s agar medium

were used for antibacterial and antifungal

studies respectively. The plates were incubated

at 370C(±10C) for 24 h. Ciprofloxacin and

Griseofulvin were used as standard

antibacterial and antifungal drugs respectively

for comparison. The inhibition zones caused

by the compounds (2a-2d) on the

microorganisms were examined, measured in

mm and tabulated (Table 1).


Scheme 1: Synthetic Protocol of thiazolidinones (2a-2d)

NH

O NH

NH2

R1

Ar - CHOR2 NH

O NH

N

Ar-

R1

R2

(1a - 1d)

SHCH2COOH

Anhydrous ZnCl2

NH

O NH

N

S

O Ar- R2

R1

(2a - 2d)

Where

Table1: Antimicrobial activity of compounds 2a-2d

Compound R1 R2 1a O-OH H 1b 3-OCH3,

P-OH H

1c P-N(CH3)2

H

1d P-F H

Compounds Microorganisms

Zone of inhibition in mm

S.aureus B.subtilis P.aerogenosa E. coli A.niger C.albicans 2a 12 14 09 08 09 06

2b 11 12 07 12 08 07

2c 12 13 10 08 08 06

2d 13 10 12 11 09 07

Ciprofloxacin 20 23 22 21 - -

Griseofulvin - - - - 19 17


Results and Discussions

The percentage yield of the synthesized

compounds 2a-2d was found to be between

50-65%. The IR and 1HNMR Spectra of these

compounds clearly indicate that the assigned

structures are in good agreement with them.

Of the four compounds tested the compound

1a showed maximum antibacterial activity (14

mm) against Bacillus substilis, compound 1d

(13 mm) against Staphylococcus aureus

among the bacterial strains used. Also the

compound 1a and 1d exhibited maximum

antifungal activity (9 mm) among the fungal

strains used. From the literature, it is revealed

that the antibacterial activity of thiazolidinones

may be due to their inhibitory activity of

enzymes Mur B which is a precursor acting

during the biosynthesis of Peptidoglycan[20].

Therefore it may be concluded that alterations

in the electron density of Aniline nucleus with

electron withdrawing groups is influencing

antimicrobial property rather with electron

donating groups. However these compounds

are less active compare to standard drugs used.

Acknowledgement

The authors are thankful to the Principal,

Govt. College of Pharmacy, Bangalore for

providing laboratory facilities to carry out this

work and are also thankful to the Director,

IISc, Bangalore for providing spectral details

required for the study.

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STUDIES ON PHYTOCHEMICAL INVESTIGATION OF LEAF

EXTRACT OF ACALYPHA INDICA

*Rajesha, P. Nagendra,1 and B. P.Siddaraju2 1Department of Post Graduate Studies and Research in Chemistry, BET Academy of Higher

Education, Bharathinagara,Mandya Dist.-571422 KARNATAKA . 2Department of Chemistry,G.Madegowda Institute of Technology,Bharathi nagara-571 422.

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

Manuscript received 25th June, revised 10th August, accepted 20th August 2014

Abstract The goal of this study was to determine the preliminary antibacterial activity of crude extract of Acalypha Indica. Shade dried leaves powder was used to prepare extracts by using different solvents like petroleum ether, chloroform, ethyl acetate and methanol. The antibacterial activity of the ethyl acetate extract was done on some standard and wild pathogenic bacterial strains such as Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Bacillus subtilis Escherichia coli and Salmonella typhi. The testing was done by the agar cup plate method using sterile top agar. Zone of inhibition of extract (50, 100 and 150 mg/ml) was compared with that of standard Amoxicillin (0.5 and 1 mg/ml) prepared in DMSO. The extract shows potential antibacterial properties comparable with that of standard amoxicillin against the organisms tested. Extract obtained by ethyl acetate extracted has been studied the antibacterial activity. Comparative results obtained from the above methods indicate that ethyl acetate extract shows better activity.

Keywords: Antibacterial Activity, cup plate method, Acalypha indica, Soxhlet.

Introduction: Respiratory tract infections are an important cause of morbidity and mortality for all age groups. Each year approximately seven million peoples are died as direct consequences of acute and chronic respiratory infection. Bronchitis and pneumonia are the most common infection. Respiratory pathogens like lebsiella pneumoniae, Pseudomonas aeroginosa and Staphylococcus aureus are some of the causative agents responsible for bronchitis and pneumonia [1]. In recent years multiple drug resistance in human pathogenic microorganisms has developed due to indiscriminate use and commercial antibacterial drugs commonly used in treatment and injections diseases. This situation forced scientists for searching new antimicrobial substances from various sources

like medicinal plants which are the good sources and novel antimicrobial chemotherapeutic agents [2]. The toxicity produced by the antimicrobial

agents can be cured or prevented or antagonize

with herbs [3]. Herbal molecules are safe, will

overcome the resistance produced by the

pathogens. Some herbs have antibacterial

properties, which will be useful to clinical use

[4]. World Health Organization [5] describes

a medicinal plant as any plant in which one or

more of its organs contains substances that can

be used for therapeutic purposes or which are

precursors for the synthesis of useful drugs.

Today, nearly 88% of the global populations

mailto:[email protected].


turn to plant derived medicines as their first

line of defense for maintaining health and

combating diseases. Currently, people of Asia

and India are utilizing plants as part of their

routine health management [6].

Herbal medicines have been the basis of

treatment and cure for various diseases and

physiological conditions in traditional methods

practiced in India such as Ayurveda, Unani

and Siddha. Medicinal components from

plants play an important role in conventional

as well as western medicine. Plant derived

drugs have been a part of the evolution of

human healthcare for thousands of years. Plant

based drugs were commonly used in India and

China [7]. Plants produce a diverse range of

bioactive molecules, making them a rich

source of different types of medicines. The

most important of these bioactive constituents

of plants are alkaloids, tannins, and flavonoids

and phenolic compounds [8]. These substances

are usually found in several parts of plants like

root, leaf, shoot and bark. The effects of plants

extracts on microbes have been studied by a

very large number of researchers in different

parts of the world [9]. Plants produce a diverse

range of bioactive molecules; require the most

common source of antimicrobial agents. Their

usage as traditional health remedies is the most

popular for 80% of world population [10]. In

recent years, multiple drug resistance has

developed in many microbes, which has

resulted in search for new antibiotic sources.

Acalypha indica Linn belongs to the family

Euphorbiaceae. It is a common weed in many

parts of Asia .It is an annual herb, about 80 cm

high and commonly found in waste places or

fields. It is locally known as“kucing galak” or

“rumput lis-lis”, “kuppaimeni” in India and

“t’ie han tsai” in China [11].This plant is used

as diuretic, antihelmintic and for respiratory

problems such as bronchitis, asthma and

pneumonia [12]. According to the Siddha text,

‘Pathartha Guna Chinthamani’ (page no: 179),

Acalypha cures diseases of the teeth and gums,

burns, toxins of Plant and mixed origin,

stomach pain, diseases due to Pitha, bleeding

piles, irritations, stabbing pain, wheezing,

sinusitis and neutralizes predominance of the

Kabha factor. According to Siddha Materia

Medica the leaf powder when given in the

dose of 950 mg to 1300 mgs, cures respiratory

diseases.In the present study, an attempt has

been made to enrich the knowledge of anti

bacterial activity of Acalypha indica leaves

extracts against pathogenic bacteria like,

Escherichia coli, Klebsiella pneumoniae,

Pseudomonas aeruginosa, Staphylococcus

aureus, Streptococcus pyogenes which cause

respiratory diseases. A. indica L. commonly

known as “Kuppai meni” in Tamil, and

Kuppesoppu in Karnataka belongs to the

family Euphorbiaceae. It has been used

traditionally for the treatment of throat

infections, wound healing, anti-venom and

migraine pain relief. There are various clinical

constituents namely kaempferol glycoside,

mauritianin, clitorin, nicotiflorin and biorodin

that have been isolated from the flower and

leaves of A.indica [13]. The presence of these

phytochemicals could be responsible for the

wide range of antimicrobial activities. The

leaves of A.grandishave also been reported to

possess many medicinal properties including


contraceptive activity [14]. Hence, a study was

carried out to analyse qualitatively the

presence of various medicinally important

phytochemical constituents occurring in

different types of extracts prepared from the

leaves of A. indica. Themedical significance of

various phytochemicals for microbial control

and therapeutics is discussed.

Acalypha indica Linn: A. indica is an

important medicinal plant in the Indian Ocean

islands as well as in India for its expectorant

properties. It also has significant antibacterial

and antifungal activities, both against human

and plant pathogens. The leaves, root, stalks

and flower are used in medicine. It has

cathartic, anthelmintic, expectorant, anodyne

and hypnotic properties. The leaves possess

laxative properties and are used in chronic

bronchitis, asthma, consumption, syphilitic

ulcers and Candidal vaginal infections. The

leaves, root, stalks and flower are used in

medicine. The plant contains the alkaloid

acalyphine which is an active principle; Indian

acalypha is a well known remedy in

rheumatism [15]

Our present study was undertaken to

antibacterial studies in leaf ethyl acetate

extracti of Acalypha indica because the

particular leaf of the plant has been widely

used for cough and other respiratory problems.

Shade dried leaves powder was used to

prepare extracts by using soxhlet method using

different solvents like petroleum ether,

chloroform, ethyl acetate and methanol. For all

the solvents obtained by extraction procedure

.Comparative study of the results obtained

from the above methods indicates that the

ethyl acetate extract shows better. The medical

significance of various phytochemical

constituents identified in the leaf extracts of

A.indica and their potential antimicrobial and

therapeutic application was discussed.

Antimicrobial activity of ethyl acetate, extract

of Acalypha indica was revealed the presence

of very active component.

Experimental Materials and Methods: Collection and authentification Plant Material:

Bharathinagara, Mandya district Karnataka is

one among the rich biodiversities of the world.

It attracts attention from people mainly due to

its pleasing climate and wealth of herbal

medicines present in the blossom of its rural

forest. Bharathi College is situated in the

heart of the Mandya district in the state of

Karnataka.

The flora around the college has spurred

young researchers into action for finding out

new medicinal plants for various ailments.

Survey of literature revealed that not much

work has been done on medicinal plants of

Mandya district.

Thus, there is a plenty of scope for research

work to be carried out on medicinal and

aromatic plants available in this area. Hence,

minor research program has been undertaken

in our laboratory in this regard. In

continuation of this research program, first it

was thought to collect different medicinal

plants useful in curing various disorders and

then subject them to various methods of


extraction, phytochemical investigation and

antimicrobial activity evaluation.

Discussion with Ayurvedic pandiths and local

herbal healers of bharathinagara region gave a

enormous information regarding the medicinal

plants available in this area, which are being

used to treat respiratory disorders. A detailed

literature survey of the medicinal plants was

done. The plant selected for the study is

Acalypha indica because this particular plant

has been enormously used for the cough

treatment. The plant material was collected

from in and around Bharathinagara, Karnataka

India. The plant was authenticated by Prof.

Nagendra, head of the department Botany,

Bharathi College Bharathi nagara. Mandya

District, Karnataka, India.

EXTRACTION OF PLANT MATERIALS Hot (Soxhlet) method of Extraction

The collected plant materials were

washed with running water. The washed

material was tapped dry and chopped into

pieces. The plant material was then sprayed

with ethanol in order to arrest any enzymatic

degradation, shade dried and coarsely

powdered. Weighed amount of this material

was successively extracted using Soxhlet

extractor with solvents of varying polarity

starting from pet-ether (60-80 0C), chloroform,

ethyl acetate and methanol. Each extraction

was carried out for 18 hrs (approximately 45

cycles). Concentrated extracts were obtained

as above and preserved.

ANTIBACTERIAL ACTIVITY

Microorganisms Standard cultures of following

microorganisms were obtained from Kiran

diagnosis centre Shimogga. The

microorganisms were identified by staining

techniques. The organisms were maintained by

sub culturing at regular intervals in nutrient

agar medium. Gram +ve bacteria:

Staphylococcus aureus Staphylococcus

epidermidis Bacillus cereus, Bacillus subtilis

Gram -ve bacteria: Escherichia coli

Salmonella typhi

Preparation of inoculums The suspension of all organisms were prepared

by inoculating one colony of the strain in 20

ml of nutrient broth in conical flask and

incubated at 37oC for 24 hours to activate the

strain. The suspension is adjusted such that it

contained approximately 1 x 106 cells/ml. It

was obtained by calculating the cells by

Neubers chamber. Nutrient agar (HiMedia)

was prepared for the study.

Culture medium: The medium was

prepared by dissolving 13 gm of nutrient broth

in 1000ml of distilled water pH to (7.3 ± 0.2),

and subjecting it to sterilization in an

autoclave at 121oC for 15 min.

Antimicrobial Agent: The reference standard amoxicillin was purchased from Hindustan Antibiotics Ltd., Determination of minimum inhibitory

concentration: The molten nutrient agar

media was prepared and distributed in Mc

cartney bottles, 20 ml each, prior to

sterilization. A measured amount of the


methanol extract was added to each bottle in

such a manner that the final concentration per

ml of the agar medium was 0 (control), 5, 15,

25, 50 and 100 mg. the final mixture was

poured individually into 100 mm sterile

petriplates. For uniform diffusion of the drug

throughout the medium, the nutrient agar

plates containing different concentrations of

the drug were refrigerated overnight at 4˚ C

and then dried for 24h at 37˚ C before

inoculation. One loopful (loop diameter –

2mm) of an overnight grown bacteriological

culture of the test organism at concentration ~

106 colony forming units (cfu/ml) was placed

in all the petriplates marked by checkerboard

technique8. The spot inoculated plates were

incubated at 37˚ C for 24h and then observed

for any growth of microorganisms. The

minimum concentration of extract which

prevent bacterial growth was taken as MIC

(Table 1). The antibacterial growth was

observed by formation of bacterial colony or

turbidity around the inoculum’s spot.

Determination of zone of inhibition by cup plate method The antibacterial activity of methanolic extract

was performed using Agar cup-plate method.

20ml of sterile nutrient agar medium was

pored into sterile petri-dishes and allowed to

solidify. The petri dishes were incubated at

37oC for 24 hours to check for sterility. The

medium was seeded with the organisms by

pour plate method using sterile top agar (4 ml)

contained 1 ml culture. Bores were made on

the medium using sterile borer. Dried ethyl

acetate extract of leaf of Acalypha indica was

dissolved in Dimethyl sulfoxide (DMSO) to

obtained different concentration (50, 100 and

150 mg/ml) and sterilized by filtration through

a Whatman filter paper no. 1, and 0.1 ml of the

different concentrations of extract were added

to the respective bores. 0.1ml of Amoxicillin

at a concentration of (0.5 mg/ml, 1mg/ml) was

taken as standard reference. The plates were

incubated overnight at 37oC with appropriate

positive and negative controls. The petri-

dishes were kept in refrigerator at 4oC for ½

hour for diffusion. After diffusion the petri-

dishes were incubated at 37oC for 24 hours

and zone of inhibition were observed and

measured. Dimethyl sulfoxide was used as the

control


Table 1. Determination of MIC of methanolic leaf extract of Acalypha indica against different bacteria.

Table 2. Determination of MIC of methanolic leaf extract of Acalypha indica against different bacteria.

‘0’ – Control (without extract); ‘+’ – Growth; ‘-‘ – No growth

Name of bacteria

Growth in nutrient agar containing different concentration of extract in mg/ml 0

5

15

25

50

100

S. aureus + + + - - - S. epidermidis + + + - - - B. subtilis + + + + - - B. cereus + + + + - - E. coli + + + - - - S. typhi + + + - - -

Microorganism

Zone of inhibition in mm Extract Conc. mg/ml

Amoxicillin Conc. mg/ml

50

100

150

0.5

1

S. aureus 13 ± 0.45

17 ± 0.58

19 ± 0.10

22 ± 0.33

27 ± 0.55

S. epidermidis 15 ± 0.52

16 ± 0.17

17 ± 0.55

21 ± 0.52

25 ± 0.72

B. subtilis 8.5 ± 0.22

10 ± 0.92

11.5 ± 0.21

13 ± 0.61

16 ± 0.11

B. cereus 7 ± 0.31

8.0 ± 0.16

9 ± 0.43

13.5 ± 0.48

17± 0.62

E. coli 22 ± 0.73

21.5 ± 0.41

23 ± 0.61

21 ± 0.15

24 ± 0.69

S. typhi 16.0 ± 0.48

18 ± 0.63

22.5 ± 0.12

19 ± 0.23

28 ± 0.71


Table 2. Antibacterial activity of Amoxicillin and leaf extract of Acalypha indica

Results and Discussion

The observations of the MIC study has been

tabulated in table 1 and it was found that the

minimum inhibitory concentration for ethyl

acetate leafextract against E. coli is 15 mg/ml,

where as for Salmonella typhi, Staphylococcus

aureus and Staphylococcus epidermidis it was

25 mg/ml and for, Bacillus cereus and Bacillus

subtilis were inhibited at 50 mg/ml. From the

data it is evident that the ethyl acetate extracts

is active against both Gram positive and

bacteria but more active against Gram negative

at low concentration. The results of zone of

inhibition of the ethyl acetate leaf extract and

comparison with standard antibiotic

amoxicillin were recorded in Table 2. The

result shows that the ethyl acetate extract of

Acalypha indica displayed concentration

dependent antibacterial activities. It indicates

that Acalypha indica shows antibacterial

activity towards all six investigated

phytopathogenic bacteria. The highest

antibacterial activity was found towards E.

coli, while it was less active against S. aureus.

The compounds responsible for this

antimicrobial property were not investigated.

The ethyl acetate extract of Acalypha indica

had impressive antibacterial and could lead to the

discovery of new antibiotics. This becomes

more relevant as the current antibiotics in use

are fast loosing effectiveness due to emergence

of resistant microorganisms. The isolation of

components of leafs of Acalypha indica ethyl

acetate extract is in progress as very potent

antimicrobial agents.

Conclusion The phytochemical investigation of ethyl

acetate extracts of Acalypha indica reveals

that, the The ethyl acetate extract of Acalypha

indica had impressive antibacterial and could

lead to the discovery of new antibiotics. This

becomes more relevant as the current

antibiotics in use are fast loosing effectiveness

due to emergence of resistant microorganisms.

The isolation of components of leafs of

Acalypha indica ethyl acetate extract is in

progress as very potent antimicrobial agents

Acknowledgement

The authors are grateful to Vision group of

Science and technology, Karnataka, for

providing fund through Spice project. The

authors also thankful to BET Academy of

Higher Education for providing necessary

facilities.

References 1) V. Ponni., S. Thenmozhi. S. Rajan. J.

basic. App. Bio. 2000; 3(3&4), 134-136. 2) F. Karaman, M. Sahin. H. Gulluse, M.

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3) W.S. Lin, and X.Z. Song, Zhong Xi Yi Jie He Za Zhi.(Chinease article. 1989; 9. 338, 402-404.


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6) Bibitha, et.al, Indian J.Microbiol, 2002, 42, 361-363.

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14) V. Duraipandiyan, S. Ignacimuthu 2007. J. Ethnopharmacol. 112, 590-594.


CRYSTAL AND MOLECULAR STRUCTURAL STUDIES OF 3-(5H-DIBENZO [B,F]AZEPINE-5-YL)-N,N-DIMETHYLPROPAN-1-AMINE

CHLORIDE

P. Nagendraa, Rajesha, S. Madan Kumar c, B.P. Siddarajub, and N. K. Lokanathc

aDepartment of Chemistry, BET Academy of Higher Education, Bharathi College, Bharthi Nagara, Mandya - 571422, India.

bDepartment of Engineering Chemistry, Cauvery Institute of Technology, Mandya-571402 cDepartment of Studies in Physics, University of Mysore, Manasagangotri, Mysore-570 006,

Correspondence email: [email protected]

Manuscript received 13th August, revised 15th November, accepted 23rd December 2014

Abstract

Imipramine derivatives were taken up for crystal and molecular structure studies by single crystal x-ray diffraction studies. In the title salt, C19H22N2+·Cl−, crystallize in the monoclinic space group P21/c and the azepine ring adopts boat conformation and the dihedral angle between benzene ring fused to azepine ring is 51.11°. The molecules are connected with C17-H17A···Cl1 and C18-H18B···Cl1 hydrogen bonds. In addition short contacts of the type N2···Cl1 is observed. Overall packing of the molecules shows three - dimensional architecture. Key words: single-crystal X-ray study; T = 296 K; Imipramine derivatives

Introduction: The title compound, Imipramine is used in the treatment of depression and enuresis, such as depression associated with agitation or anxiety and has similar efficacy to the antidepressant drug moclobemide. Imipramine (Tofranil), also known as melipramine, is a tricyclic antidepressant (TCA) of the dibenzazepine group. It has also been used to treat nocturnal enuresis because of its ability to decrease the delta-wave stage of sleep where this occurs (Delini-Stula,et. al.,). The other dibenzoazepine derivatives crystal structure are reported from our group

(Abdoh et al., 2013; Manjunath et al., 2013)

N

NCl_

+

Chemical structure of the compound Data collection: CrysAlis PRO (Oxford Diffraction, 2010); cell refinement: CrysAlis PRO; data reduction: CrysAlis RED (Oxford Diffraction, 2010);



program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXTL. Experimental The title compound is a gift sample from R.L. Fine Chem. Bangalore. X- Ray quality Single crystals were obtained from slow evaporation of a solution of ethanol (m.p.:198-210°C)

The molecular structure of the title molecule is shown in Fig-1. The seven-membered azepine ring adopts a boat conformation with puckering parameters Q2 = 0.735 (5) Å, Q3 = 0.315 (5) Å, φ2 = 331.3 (4)°, φ3 = 57.7 (9)°, and total puckering amplitude QT = 0.800 (5) Å (Cremer & Pople, 1975). The dihedral angle between the two benzene rings, (C1—C6) and (C9—C14), fused to the azepine ring is 51.11 (1)°.

Fig-1: Molecular structure of the title compound showing the atom labelling scheme and 50% probability displacement ellipsoids


Special details Geometry: All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement: Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Table 1. Experimental details Crystal data Chemical formula C19H22N2+·Cl− Molecular weight 313.84 Crystal system, Monoclinic Space group P21/c Temperature 296 (K) a 14.3698 (4)Å b 9.1625 (3)Å c 13.8335(4) Å β β 96.868 (2)° V 1808.30 (9) Å3 Z 4 Radiation type Cu Kα µ 1.84 mm−1

Crystal size 0.23 × 0.22 × 0.21mm Data collection Bruker X8 Proteum Diffractometer diffractometer Absorption correction SADABS (Bruker, 2013) Tmin, 0.677 Tmax 0.699 No. of measured, independent 11102 Observed [I > 2σ(I)] 2960 Reflections 1850 Rint 0.044 (sin θ/λ)max (Å −1 ) 0.587 Refinement R[F 2 > 2σ(F 2 )], wR(F 2 ),S 0.074, 0.241, 1.05 No. of reflections 2960 No. of parameters 202 H-atom treatment H-atom parameters constrained Δρmax 0.37 Δρmin (e Å−3 ) - 0.37 Table 2. Hydrogen-bond geometry (Å,o) D—H···A D-H H···A D···A D-H···A C17—H17A···Cl1i 0.97 2.65 3.574 (4) 159

C18—H18B···Cl ii 0.96 2.76 3.698 (5) 166

Symmetry codes: (i) x, −y+1/2, z+1/2; (ii) −x+1, y−1/2, −z+3/2

Fig-2: Packing diagram of the title compound viewed along the a-axis.


In the crystal, C17—H17A···Cl1

and C18—H18B···Cl1 hydrogen bonds connects the molecules (Table-1 and Fig- 2). The short contact N2···Cl1 observed have a distance of 2.97 Å. With all these interactions the packing of the molecules shows three-dimensional architecture. References 1) M.M.M. Abdoh, S. Madan Kumar, K.S.

Vinay kumar, B.C. Manjunath, M.P.

Sadashiva, N. K. Lokanath, Acta Cryst

2013. 69, 17.

2) Bruker (2013). APEX2, SAINT and

SADABS. Bruker AXS Inc., Madison,

Wisconsin, USA. Cremer, D. & Pople,

J. A. (1975).

3) A. Delini-Stula, H. Mikkelsen, J. Angst,

J. Am. Chem. Soc. 1995, 97, 1354–

1358.

4) B.C. Manjunath, K.S. Vinay kumar, S.

Madan Kumar, M.P. Sadashiva, N.K.

Lokanath, Acta Cryst., 2013, 69, 1233.

5) C.F. Macrae, I. J. Bruno, J. A.

Chisholm, P.R. Edgington, P. McCabe,

E. Pidcock, L. Rodriguez-Monge, R.

Taylor, J. van de Streek, Wood, P. A. J.

Appl. Cryst., 2008, 41, 466–470.

6) B.C. Manjunath, K.S. Vinay kumar, S.

Madan Kumar, M.P. Sadashiva, N.K.

Lokanath, Acta Cryst., 2013, 69, 1233.

7) C.F. Macrae, , I.J. Bruno, J.A.

Chisholm, P.R. Edgington, P. McCabe,

E. Pidcock, L. Rodriguez-Monge, R.

Taylor, J. van de Streek, P.A. Wood, J.

Appl. Cryst. 2008. 41, 466–470.

8) Sheldrick, G. M. Acta Cryst. 2008, A,

64, 112–122.


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