Research Article
ejbps, 2016, Volume 3, Issue 9, 564-568.
Shashank et al.
SJIF Impact Factor 3.881
ISSN 2349-8870
European Journal
Biomedical
Europeanof
Journal
of Biomedical and Pharmaceutical Sciences
Volume: 3
Issue: 9
AND Pharmaceutical sciences
564-568
Year: 2016
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THE GENETIC FIDELITY ASSESSMENT OF AILANTHUS EXCELSA’S LEAVES
BETWEEN MANDSAUR (M.P.) AND RATLAM (M.P.) DISTRICTS
Bhatt Shashank*1, Dhyani Suresh2 and Rajesh Kumar Kesharwani 3
1,3
Department of Biotechnology, NIET, NIMS University, Jaipur, Rajasthan -303121, India.
2
Himgiri Zee University, Dehradun, Uttarakhand-248197, India.
Corresponding Author: Rajesh Kumar Kesharwani and Shashank Bhatt
Department of Biotechnology, NIET, NIMS University, Jaipur, Rajasthan -303121, India.
Article Received on 09/07/2016
Article Revised on 10/08/2016
Article Accepted on 01/09/2016
ABSTRACT
The Ailanthus excelsa Roxb. is found in different states in India and their leaves are used as fodder for cattle. 95%
ethanolic extract was shown as good solvent having a much extraction capacity of secondary metabolites from
dried leaves. The glycosides, phenol, lignin, saponins and tannins were present in the 95% ethanolic extract of
dried leaves. The concentrations of DNA were found 0.0218, 0.0210, 0.0207, 0.0215 and 0.0212 mg/100gm
respectively and their mean value was found 0.0212 mg/100 gm in Mandsaur area of Madhya Pradesh where as in
the area of Ratlam, the DNA concentrations were found 0.0218, 0.0212, 0.0218, 0.0218 and 0.0223mg/100gm
respectively and the mean value was 0.0218 mg/100 gm. The comparative study between Mandsaur (M.P.) area
and Ratlam (M.P.) area were calculated by ANOVA-CRD in which the S.EM. value was 0.006289 and CD 5%
0.018553. These datas showed a significant value.
KEYWORDS: Secondary Metabolites, Genomic DNA, Ailanthus excelsa Roxb.
INTRODUCTION
Plants have an important place in the medicinal world
since ancient times. Traditional herbal plants have been a
great source of therapetic agents. The plants are the main
source of metabolites that have various pharmacological
activities. These contents have the effectivity against
various insects and bacteria etc. The different types of
natural hormones, vitamins, and proteins contents can be
extracted and used in the pharmaceutical company for
the preparation of antibiotics. The allopathic medicines
have side effect but Ayurvedic medicines have not any
side effect (okigbo et al.2008).
The world Health Organization (WHO) has estimated
that near about 80% population of developing countries
depend on traditional medicines for primary healthcare.
These traditional medicines are prepared by plants’ bark,
roots, fruits and leaves which play a major role in the
preparation of medicines. Herbal drugs play a major role
in all the traditional systems of medicine. Plant products
can be used for the semi synthetic preparation of other
drugs. For e.g., plant steroids are used in the manufacture
of oral contraceptives and other sterocidal hormones.
Introduction of selected plant
The Ailanthus excelsa Roxb. was selected for the study
of genetic fidelity. The common name of this plant is
Mahanimba and their leaf shape is similar to Azadirachta
indica (neem). The leaves of Ailanthus excelsa is a good
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source of metabolic contents. The glycosides, phenols,
lignins, tannins etc. have been detected in leaves of
Ailanthus excelsa (Bhatt S. et.al.2012).
The environmental conditions in which soil and climatic
conditions are involved and these conditions differ in
each states and countries by which the plant growth and
shape of leaves may be different.
MATERIAL AND METHODS
Collection of identification Ailanthus excelsa Roxb.
The plant parts were collected from Mandsaur. The plant
material was taxonomically identified, confirmed and
authenticated by Dr. Rakesh Mohan Painuli, In charge,
Herbarium, Department of Botany, HNB Garhwal
University, Srinagar (Garhwal), Uttarakhand (HNB
20705). The leaves of Ailanthus excelsa Roxb. were
collected for isolation of genomic DNA from Mandsaur
(M.P.) and Ratlam (M.P.).
Phytochemical Study of Secondary Metabolites
The shaded dried leaves were used for the experiments
that were collected from Mandsaur (M.P.). The different
compounds were extracted from leaves by 18 hours cycle
of soxhlet apparatus. Petroleum ether, chloroform, 95%
ethanol and distilled water solvents were used for the
extraction of compounds. Various tests were used for the
identification of secondary metabolites. Each test has
specific reaction with plant extract and produces colour
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European Journal of Biomedical and Pharmaceutical Sciences
complex by which primary and secondary metabolites
can be identified.
Extraction and Estimation of Nucleic Acid from
Leaves of Ailanthus excelsa Roxb.
Reagents
Diphenylamine Reagent
1.5 g diphenylamine (Merck) was dissolved in 100 ml
acetic acid and 1.5 ml of conc. H2SO4 was added. It was
stored at 4ºC in dark coloured bottle. In 20 ml of the
reagent, 0.1 ml of 1.6% aqueous acetaldehyde solution
was added just before use to induce the colour
development.
Acetaldehyde solution
Redistilled acetaldehyde solution at a concentration of
1.26% was prepared as an aqueous solution and then
stored at 4ºC.
Perchloric acid
PCA: 5% PCA was prepared by dissolving 5 ml of 73%
PCA in 68 ml of distilled water.
DNA Standard
The calf thymus (HiMedia, MB103) was used for
standard curve. 2 mg calf thymus DNA was dissolved in
10 ml of 5% PCA and prepared stock solution and stored
at 4ºC when not in use.
Extraction Method of DNA
Nucleic acid (DNA) was extracted by the method of
Schneider (1945). Firstly, 500 mg shade dried leaves
material of A. excelsa was weighed and added into test
tube then 5 ml of 10% cold trichloroacetic acid was
added into it. The tube was kept under cool condition.
The tube material was homogenized with a polytron
homogenizer and then allowed to stand for 30 minutes at
room temperature and supernatant was discarded. The
pellet was collected and again added 3 ml of 10% cold
TCA and mixed it thoroughly with the help of
cyclomixer. This mixture was centrifuged at 2500 rpm
for 10 min. and supernatant was discarded.
After the removal of supernatant, 3 ml of isopropanol
was added into pellet and mixed thoroughly in
cyclomixture. The mixture was recentrifuged at 2500
rpm for 10 min. and then supernatant was again
discarded. The pellets were saved and washed thrice with
1ml isopropanol. After washing the pellets, 5 ml of 5%
PCA was added and heated in boiling water bath for 15
min. After it, the tubes were centrifuged at 3000 rpm for
25 min. The supernatant was collected and used for the
estimation of DNA.
Estimation of DNA
The DNA estimation was measured by diphenylamine
method Burton (1956).
Took the 1.5 ml of PCA (Merck) extract into test tube
and added the 3 ml of diphenylamine reagent. The tubes
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were kept in a water bath and maintained temperature at
70ºC for 20 min and then cooled at room temperature.
The colour development of sample extract was measured
at 595 nm wavelength. The calf thymus of DNA was
used as standard. The DNA content was expressed in
mg/100 gm of the leaf tissue.
RESULT AND DISCUSSION
DNA is the most important genetic material that carries
genetic information from one generation to the next
generation. The five replicates were collected from each
place. The concentrations of genomic DNA in
mg/100gm were calculated from the standard curve of
calf thymus DNA. In table 1, the samples were collected
from Mandsaur (M.P.). The concentrations of DNA were
found 0.0218, 0.0210, 0.0207, 0.0215 and 0.0212
mg/100gm respectively and their mean value was found
0.0212 mg/100 gm in Mandsaur area (M.P.) The table 2
showed the concentration of DNA in mg/100gm that was
collected from Ratlam (M.P.). The concentration values
were 0.0218, 0.0212, 0.0218, 0.0218 and 0.0223
respectively. The mean value was 0.0218 mg/100 gm.
Figure 1 and 2 indicated the concentration of genomic
DNAs (mg/100gm) in leaves of Ailanthus excelsa
collected from Mandsaur (M.P.) and Ratlam (M.P.)
areas. Figure 3 indicated the comparative study of
genomic DNA concentration (mg/100gm) between
Mandsaur (M.P.) and Ratlam (M.P.). The comparative
study between Mandsaur (M.P.) area and Ratlam (M.P.)
area were calculated by ANOVA-CRD in which the
S.EM. value was 0.006289 and CD 5% 0.018553. These
datas showed a significant value.
Temperature is an important factor for changes in outer
morphological conditions and inner metabolic contents.
The samples collection places have also different in
temperatures. Mandsaur (district Mandsaur) maximum
temperature 39.8ºC and minimum temperature 25.4ºC
while Ratlam (district Ratlam) maximum temperature
44ºC and minimum temperature 28.0ºC. In the present
findings, there were significant variations observed in the
temperature, climatic conditions and types of soil which
alter the size, length protein contents and quantity of
DNA in the leaves of A. excelsa. The findings clearly
indicate that there were alterations in morphological and
environmental factors of A. excelsa leaves at different
areas. I have also observed the geographical areas
(latitude and longitute) of Mandsaur and Ratlam that are
different. The geographical findings of Mandsaur
(District Mandsaur) 23º45’50” North and 25º2’55” North
latitude and 74º42’30” East and 75º50’20” East
longitude, Ratlam (District Ratlam) 23º52’ North and
23º05 North latitude and 74º31’ East to 75º41’ East
longitude.
The soil also affects the plant growth and quantity of
primary and secondary metabolic contents. Jat et al.,
2010 have been studied on the plant growth activity and
genotypic activity and found ‘Sel 9’ and ‘Sel 7’genes
were responsible for plant height. 40.7, 100.7, 150.0 and
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Shashank et al.
European Journal of Biomedical and Pharmaceutical Sciences
251.3 cm plant’s height had been recorded and ‘Sel 9’
was responsible for this height. ‘Sel’ 7 is also responsible
for higher growth characteristic and have 3.92, 5.73, 9.34
cm crown diameter.
Table-1: Quantitative Estimation of Genomic DNA in Leaves of Ailanthus excelsa’s Roxb. Sample Collection
Place- Mandsaur (Madhya Pradesh)
O.D. at Concentration
Sample Quantity
Distilled Water
DPA without
Replication No.
595
Genomic DNA
(ml.)
(ml)
Aldehyde (ml.)
nm
(mg/100 gm)
M-1
0.2
1.8
3
0.082
0.0218
M-2
0.2
1.8
3
0.079
0.0210
M-3
0.2
1.8
3
0.078
0.0207
M-4
0.2
1.8
3
0.081
0.0215
M-5
0.2
1.8
3
0.080
0.0212
S.EM.
0.00011
CD5%
0.000326
M-It indicates Mandsaur.
Figure-1 Concentration of genomic DNA in leaves of Ailanthus excelsa collected from Mandsaur (M.P.) areas.
Table –2: Quantitative Estimation of Genomic DNA in Leaves of Ailanthus excelsa’s Roxb.
Sample Collection Place- Ratlam (Madhya Pradesh)
Replication No.
Sample
Quantity
(ml.)
R-1
R-2
R-3
R-4
R-5
S.EM.
CD5%
R-It indicates Ratlam.
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0.2
0.2
0.2
0.2
0.2
Distilled
Water
(ml.)
DPA without
Aldehyde
(ml.)
1.8
1.8
1.8
1.8
1.8
3
3
3
3
3
O.D. at
595 nm
0.082
0.080
0.082
0.082
0.084
0.002501
0.007377
Concentration
Genomic DNA
(mg/100 gm)
Mean of Genomic
DNA Concentration
in A. excelsa Leaves
(mg/100 gm)
0.0218
0.0212
0.0218
0.0218
0.0223
0.0218
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Shashank et al.
European Journal of Biomedical and Pharmaceutical Sciences
Figure-2 Concentration of genomic DNA in leaves of Ailanthus excelsa collected from Ratlam (M.P.) areas.
Figure-3 Comparative study of genomic DNA concentration (mg/100gm) between Mandsaur (M.P.) and Ratlam
(M.P.).
CONCLUSION
On the basis findings it is clearly Indicated that the
climatic and environmental conditions affect the genetic
material, primary and secondary metabolites of plant.
Thus, it was concluded that Ailanthus excelsa’s leaves
are a good source of primary (proteins and DNAs) and
secondary metabolites (glycosides, saponins, phenol,
tannins, lignin etc.) by which various types of various
Ayurvedic and allopathic medicines can be prepared and
diseases be treated.
ACKNOWLEDGEMENT
Praying and dedicating my research article to Maa
Saraswati, the goddess of knowledge and wisdom, I am
unable to find words to express my deepest gratitude to
my parents, Mr. Krishna Kumar Bhatt and Mrs.
Subhadra Bhatt whose encouragement helped me to go
ahead on this bright path. I share the credit of my work to
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my respectable elder brother, Mr. Mayank Bhatt who
very often guided me in the work.
The research work would have only been a dream, had
my way not been enlightened, by my well wishers and
the above respectables. Last but not least, the Almightly
God is unforgettable without whose kindness and grace
nothing could have happened.
REFERENCES
1. Bhatt S., Dhyani S., Preliminary Phytochemical
screening of Ailanthus excelsa Roxb. Int J Curr
Pharm Res., 2012; 4(1): 87-89.
2. Burton K.A Study of the conditions and mechanism
of the diphenylamine reaction for the colorimetric
estimation of deoxyribonucleic acid, Biochem. J.,
1956; 62(2): 315-323.
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3.
4.
5.
European Journal of Biomedical and Pharmaceutical Sciences
Jat HS, Mann JS, Sharma SC, Chand R.
Identification of best performing ardu (Ailanthus
excelsa) genotypes in semi-arid ecosystems of
Rajasthan. The Indian Journal of Agricultural
Sciences, 2010; 80(3) 227-30.
Okigbo R.N., Eme U.E., Ogbogu S., Biodiversity
and conservation of medicinal and aromatic plants in
Africa. Biotechnol. Mol. Biol. Rev., 2008; 3(6): 127134.
Schneider W.C. Phosphorus compound in animal
tissue. I. Extraction and estimation of desoxypentose
nucleic acid and of pentose nucleic acid. J. Biol.
Chem., 1945; 161: 293-303.
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