TISSUE CULTURE AND THERAPEUTIC CAPABILITIES OF Syzygium alternifolium (Wight) Walp. - AN EASTERN GHAT ENDANGERED ENDEMIC AT ALAGAR HILLS OF MADURAI DISTRICT Synopsis submitted to MADURAI KAMARAJ UNIVERSITY in partial fulfilment of the requirements for the award of the degree of DOCTOR OF PHILOSOPHY IN BOTANY By S. MOHAN (Reg. No. P4098) Under the Guidance of Dr. G.C.ABRAHAM, M.Sc., M.Phil., Ph.D., Associate Professor & Head Department of Botany The American College Madurai. MADURAI KAMARAJ UNIVERSITY (University with Potential for Excellence) Madurai-625 002, Tamil Nadu, India June - 2020
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Syzygium alternifolium (Wight) Walp. - AN EASTERN GHAT
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TISSUE CULTURE AND THERAPEUTIC CAPABILITIES OF
Syzygium alternifolium (Wight) Walp. - AN EASTERN GHAT
ENDANGERED ENDEMIC AT ALAGAR HILLS OF
MADURAI DISTRICT
Synopsis submitted to
MADURAI KAMARAJ UNIVERSITY in partial fulfilment of the requirements for the
award of the degree of
DOCTOR OF PHILOSOPHY IN BOTANY
By
S. MOHAN
(Reg. No. P4098)
Under the Guidance of
Dr. G.C.ABRAHAM, M.Sc., M.Phil., Ph.D., Associate Professor & Head
Department of Botany The American College
Madurai.
MADURAI KAMARAJ UNIVERSITY
(University with Potential for Excellence)
Madurai-625 002,
Tamil Nadu, India
June - 2020
TISSUE CULTURE AND THERAPEUTIC CAPABILITIES OF
Syzygium alternifolium (Wight) Walp. - AN EASTERN GHAT
ENDANGERED ENDEMIC AT ALAGAR HILLS OF
MADURAI DISTRICT
SYNOPSIS
Submitted by
S.MOHAN
(Reg.No.P4098)
S.MOHAN Dr.G.C.ABRAHAM
(Research Scholar) (Research Supervisor)
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A. Thesis Title
TISSUE CULTURE AND THERAPEUTIC CAPABILITIES OF Syzygium alternifolium
(Wight) Walp. – AN EASTERN GHAT ENDANGERED ENDEMIC AT ALAGAR
HILLS OF MADURAI DISTRICT
B.Brief description
The floristic richness of the Indian forests supported the wide range of variations
in physiognomic features of the sub-continent remains an unceasing inspiration to any
inquisitive mind, lest not to a naturalist with a sense of appreciation and a probing
thought. The deeper cultural heritage of the land that gets groomed from the intelligent
understanding of people who have all along lived in accordance and harmony with their
land and natural elements speaks of the invaluable wisdom of natives. Indigenous
communities, who by intuition and practice have been at their best in controlling their
health, follow traditions which despite the lack of scientific validation have remained a
proven and settled practice in dealing with many common and acute ailments. Several
allopathic practitioners and proponents of modern medicine have come forward to adopt
and appreciate the wholesomeness of these practices although they call for a scrutiny of
the veracity of the claims made in folk medicine and native healing.
Among the ailments critical ailments where the traditional health care stands
indispensible practice in the Tamil heartland is the chronic cases of jaundice, diabetes,
bone and joint aliments and orthopedic inventions, let alone the treatments given for the
challenging cases of cancer, problems of gastrointestinal tracts, UTI and health issues
connected with reproduction, psychosomatic malfunctions, child, women and geriatric
issues. A number native, indigenous, endemic and exotic plant species have been
common and regular use among which the genus Syzygium of the family Mytraceae is one
that is popular, wide recommended and used, though less studied. With its 1200 species
that are pan global in distribution, the knowledge about the use of this genus is no new or
big news to communities that are familiar with its use. Ironically, but for the exception of
S cumini, scientific reporting has only been sparse.
This exercise on Syzygium alternifolium (Wight) Walp. a Eastern Ghat Taxon at
Alagar Hills of Madurai District seeks to address this issue. The notion that this species
can be used as medicinal in the treatment of diabetic, cancer and the fortification of liver
functions and potential to be used as an effective antioxidant, and that the field
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observation reporting the presence of this species recorded only once in the region in the
past presents the basic drive. The 19 individuals of the species are seen subdued due to
reckless tree felling activity, and intriguing anthropogenic and biotic pressures in the trail
that leads to Bison Valley naturally makes enough grounds for selecting S. alternifolium
as the choice and the subject of the study. That this specific taxon demands attention as an
ecological entity stems on the fact that as most other Indian species of Syzygium makes
Western Ghats abode, S. alternifolium finds the restricted region discontinuous mountain
range of Eastern Ghats as it original habitat, and by reasons hitherto unexplored have
somehow reached the floristically special Alagar Hills here at Tamilnadu.
The species known a Mogi, in the Middle Eastern Ghats where it is claimed to be
an endemic, is seen depleting in populations in its native site and has been reported
slipping into endangered list in the recently released red list is a botanists’ take on this
project. Eastern Ghat Hill tract which stretches as discontinuous hillocks and mountains
from Mahanadi basin of Orissa in north to Madurai district in Tamil Nadu at south is a
biogeographic zone that is quite unique in its contour and physiognomic features.
Notwithstanding to the exquisite floristic composition which arouses interest, the
overwhelming sensibilities of academics have not resulted in studies in terms attention
paid on Eastern Ghats. Somehow, said region stands neglected in comparison with its
western counterpart. Though Eastern Ghat Conservation Net Work had initiated some
studies to make some amends, Mogi, as S. alternifolium is called in its original habitat,
continues elude attention. That this species is spotted only once in past at Alagar Hills of
Madurai, interestingly a region with the overlaps of mountainous terrain of the Western
and Eastern Ghats of the Peninsular India is seen vulnerable makes room for this
investigation.
C. Definition of the Problem
Reports are available on field, propagation and therapeutic evaluation of the S.
alternifolium is rare and this taxon has never been subjected to any academic study in this
region. Therefore, this present study on the field data and phenology of the smaller stand
of nineteen individual trees in Alagar hills is the first of its kind with only the exception
from the report made from the forest of Andhra Pradesh merits special attention. The
therapeutic and economic significance of this fragile small-made tree known only by
notion, but presumably sold in local market adds to woes of this species. The extremely
poor state to which the species is subjected due to the wagers of nature, plant being
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attacked and seen ravaged due the devouring by monkeys, animal predation, loss of seeds
due to pests and insects, and the problems pertaining to seed viability and germination
that limits the perpetuation of the species make this is investigation an impending
necessity.
As the difficulties faced by the species in completing the reproductive cycle,
makes the bid on protecting and rescuing the species through propagation through
conventional and tissue culture methods a need, efforts taken to evaluate the
phytochemical profile and the therapeutic efficacy for a proper scientific appraisal
intend fetching a technical data for an evaluation of economic potential, especially from
the folk medicine point of view that community sensitization and an awareness towards
the judicious use and conservation of this important species can generated from
rationalized stand point.
D. Scope of Research Work
Despite the claims on its medicinal and economic uses of the genus Syzygium, the
species of Syzygium alternifolium (Wight) Walp. (Myrtaceae) has not been investigated
frequently in great detail. It is reported that in recent years, the population size of this
taxon is declining due to habitat changes and felling of trees for trivial uses, and the
rampant devouring and collection of fruits. Besides anthropogenic pressures, the sparsely
distributed species is constrained with its discontinuous and erratic flowering and fruiting
and hence has only slim chances of repopulating itself in wild even at its natural habitats.
Keeping this in view and noting that only a tokenistic representation of the taxon
is available at Alagar Hills, field observations in the present study contemplate on
Scanning the distribution of the species along the slopes of the geomorphically
and floristically interesting Azhagar Koil Hills,
Finding reasons for the tokenistic presence of the otherwise endemic Middle
Eastern Ghats species in the present study site,
Exploring the biotic association of the trees that are seen as threats and support to
the survival individuals in field, and
Listing other disturbances caused to the specific site of distribution.
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Special efforts were taken to experiment the feasibility of
Propagating the species S. alternifolium via conventional nursery practices using
PGRs and other established and recommended procedures, both at field (in situ)
and departmental premises (ex situ), and
Reintroduce the cloned plants back in field at Alagar Hills.
As an adjunct to support conservation, in vitro trials were pursued by using certain
marked individuals among the nineteen trees as source plants from where explants were
collected for tissue culture investigations. While preliminary studies were under taken
To recover plant on one on one basis from somatic and embryonal explants,
Focused approaches were made to evaluate the feasibility establishing procedures
for micropropagation, and
The efficacy of inducing callus leading to redifferentiation and organogenesis was
also explored.
Biochemical and histochemical studies were initiated to probe the phytochemical
constitution of somatic and reproductive tissues.
Experiments were deigned to draw insights on therapeutic utility of the fruit extracts.
Antimicrobial assays were performed with certain common pathogenic microbes.
Preclinical investigations sought to establish the therapeutic significance forms
special feature of this study. The prophylactic and curative capabilities of aqueous
and solvent extracts of the fruits of S. alternifolium were assessed on certain
chosen ailments and disorders using animal models.
In specific terms, experiments on hypoglycemic activity, hepatoprotective ability,
anti-oxidant properties and anti-cancer effects formed a part of this study.
E. Methodology
The broad outline of the materials and methods followed in present investigation
is thematically listed as flows.
STUDY OUTLINE
This investigation on Syzygium alternifolium (Wight) Walp. pursued from the year
2015 onwards is a field cum lab study. As the nursery trials and tissue culture studies
were carried out in the American College, Madurai, field observations were made in
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Syzygium site located adjoining the temple premises of Alagar koil which is about 20 km
away north-east from the college campus. A scant population of 19 individuals of the
selected species located in the trail from Alagar koil to Natham was used as the principal
material for study. For want of expertise and overseeing of the animal ethics committee,
preclinical trials were conducted in KM college of Pharmacy situated in the outskirts of
Madurai.
FIELD INVESTIGATIONS focusses on
Distribution of the species
Study of Ecological factors
EXPERIMENTS ON PLANT PROPAGATION included attempts to
Study seed viability, seed germination and seedling establishment
Methods checking the feasibility of vegetative propagation
Plant Tissue Culture encompassing descriptions on
Source of Explants
Surface Sterilization
Explant Preparation
Culture Media
Inoculation and Subculture, and
Incubation
SPECIFIC EXPERIMENTS
Following the general procedures described above, parallel trails were made with
a preplan. Experiments were carried out individually to achieve three main objectives.
i. Effective establishment of culture to recover a plant each from every explant,
ii. De novo shoot multiplication leading to supernumerary shoot clusters, and the
iii. Opportunities vested with callus induction and regeneration was studied.
Cultures were examined on a regular basis and detailed time bound evaluations of
morphometric characters were made and suggested as the footnotes of the tables.
Parameters of assessment differed from experiment to experiment and specific
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information pertaining to the task is provided in the result section. Alongside these
attempts made to explore data that might assist the reintroduction of tissue cultured plants
in native sites, some efforts were taken to check the claims of therapeutic efficacy which
may augment bid to promote community participation in protecting the species pitching
on its utility value.
STUDIES ON THERAPEUTIC UTILITY
With the expertise from the pharmacognosy and pharmaceutics divisions of the
neighboring para-medical college under the approval of animal ethics committee, studies
preclinical trials were initiated. Pharmacological potential of S.alternifolium was
evaluated with the procedures outline below. Solvent extracts were performed using
Analar grade chemicals procured form standard companies such as Merck, Lobo and
Ranbaxy laboratories. Medium requirements for anti microbial assays were met Hi media
and the test organism of anti bacterial studies and test animals used as animal models,
and samples such as cell lines for anti-cancer studies and animals for preclinical
evaluations were supplied by reputed research institutes and professional labs.
Preparation of crude extracts
Preparation of fruit extracts by hot continuous percolation method
The ethanol extract of Syzygium alternifolium was subjected to qualitative tests
for identification of various plant constituents.
Qualitative Phytochemical Analysis
The Ethanol extract of Syzygium alternifolium was subjected to specific tests for
identification of constituents. Identification of the various phytochemical constituents was
carried out adopting the procedures available in Trease and Evans, 1983 and
Krishnamurthy (1988). Qualitative tests were carried out in accordance with the general
plan of the experiment.
1) Detection of Carbohydrates
A. Molisch’s Test
B. Fehling’s Test
C. Benedict’s Test
D. Barford’s Test
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E. Test for Starch
2) Tests for Gums and Mucilage
3) Test for Proteins and Amino Acids
A. Ninhydrin Test
B. Biuret Test
C. Millon’s Test of Cole’s Mercuric Nitrite Test.
D. Xanthoprotein Test
4) Test for Fixed Oils and Fats
A. Spot Test
B. Saponification Test
5) Test for Alkaloids
A. Mayer’s Test
B. Dragendorff’s Test
C. Wagner’s Test
D. Hager’s Test
6) Tests for Glycosides
A. Legal’s Test
B. Baljet’s Test
C. Borntrager’s Test
D. Test for Deoxy Sugar (Keller-Killani Test)
7) Test for Phytosterols
A. Libermann-Burchard’s Test
B. Salkowski’s test
8) Test for Flavanoids
A. Ferric chloride Tests
B. Shinoda’s test
C. Flavones
D. Reaction with alkali and acid
9) Test for Tannins
A. Ferric chloride test
B. Reaction with lead acetate
C. Gelatin Test
10) Test for Saponins
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Analysis of Ethanol Extract of Syzygium alternifolium by Gas Chromatography-
Mass Spectrometry (GC-MS)
Analysis of Sample
The given extracts were analyzed in GC-MS for different components.