Post on 18-Dec-2021
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)
1
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
2
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
3
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.
4
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
5
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
6
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
7
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
8
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.
1. GC Program
Column: Elite-5MS (5% Diphenyl/ 95% dimethyl poly siloxane), 30X0.25
mmX0.25μm
Equipment: GC Clarus 500 Perkin Elmer
Carrier Gas: 1ml per min, Split: 10:1
Detector: Mass detector Turbo gold-perkin Elimer
Software: Turbomass 5.2
Sample injected: 2μ l
Oven Temperature Programme-
1100 C-2 min hold
Up to 2000 C at the rate of 100 C/ min-No hold
Up to 2800C at the rate of 50C/ min-9 min hold
Injector temperature 2500 C
Total GC running time 36 min
2. MS Programme
Library used NIST Version-Year 2005
Inlet line temperature 2000C
Source temperature 2000C
Electron energy : 70 eV
Mass Scan (m/z) : 45-450
Solvent Delay : 0-2 min
Total MS running time : 36 min
9
PHARMACOLOGICAL EVALUATION
Approval of Experimental Work from IAEC/CPCSEA
The protocol of the animal experiments involved in this research work has been
approved by IAEC/CPCSEA constituted for this purposes.
Acute Toxicity Study
Acute oral toxicity refers to those adverse effects occurring following oral
administration of a single dose of a substance or multiple doses given within 24 hrs.
Acute toxic class method (OECD guidelines (2000), was followed to arrive at the
maximum safety dose of the drug extracts.
PRE CLINICAL EVALUATION IN ANIMAL MODELS
I HYPOGYCEMIC ACTIVITY
The present investigation is undertaken to the study the effect of Ethanolic Extract
from ripe fruits of Syzygium alternifolium on changes in Body weight, Plasma glucose,
and Hemoglobin and glycosylated hemoglobin and lipid profile.
II HEPATOPROTECTIVE ACTIVITY
In traditional medicines, various herbal preparations are being used for treating
liver disorders. In the absence of an effective treatment in modern medicine, efforts are
being made to find out suitable herbal drugs. In the line of previous work done in other
plants against paracetamol induced hepatotoxicity, the present study was taken up to
evaluate the effects of Ethanol Extract of Fruit Syzygium alternifolium against Ionized and
Rifampicin induced hepatotoxicity.
III ANTIOXIDANT ACTIVITY
Albino rats of wistar strains weighing between 180-220gm were produced from
animal experimental laboratory, and used throughout the study. They were housed in
micro nylon boxes in a control environment (temp 25+-20c) and 12 hrs dark\ light
cycle with standard laboratory diet and water ad libidum. The study was
conducted after obtaining institutional animal ethical committee clearance. As per
the standard practice, the rat were segregated based on their gender and
quarantined for 15 days before the commencement of the experiment. They were
fed on healthy diet and maintained in hygiene environment in our animal house.
10
Treatment protocol
The acclimatized animals were divided into 5 groups of each 6 animals,
designated as
Group 1: Served as normal control and receive normal diet and water.
Group 2: Toxic control received 400mg/kg IP D-galactosamine for 21 days
(400mg/kg)
Group 3: Standard control received 25mg/kg of silymarin orally for 21 Days
(25mg/kg).
Group 4: Served as a treatment control group and was administered Ethanolic
Extract of Syzygium Alternifolium at a dose of 200mg/kg through orally.
Group 5: Treatment control group and was Administered Ethanol Extract of
Syzygium alternifolium at a dose of 400mg/kg through orally.
METHODOLOGY
On day 22 24 hrs after Galactosamine administration, animals in all the groups
were humanely sacrificed using Ketamine HCL and 4ml of blood was withdrawn by
cardiac puncture and allowed to clot for 30 minutes at room temperature. The serum was
separated by using refrigerated centrifuge and used for the assay of marker enzymes
namely AST, ALT, ALP, TP, TB and LDH. The livers were dissected out immediately,
washed with ice-cold saline and 10% homogenates in phosphate buffer solution (PH 7.4)
were prepared. Liver homogenate was used for the assay of Lipid per oxidation (LPO)
while some fraction of homogenates were centrifuged at 7000rpm for 10 min at 40 C
using refrigerated centrifuge, and the supernatants were used for the assay of Superoxide
dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx). Some portion of liver
from each group was aseptically excused and stored in 10% formalin for
histopathological studies.
IV ANTI-CANCER ACTIVITY
Tumor being a mass of tissues which proliferate rapidly spreads throughout the
body and may eventually cause death of the host. Chemotherapy is an effective treatment
against various types of cancer either singly or in combination with surgery and/or
radiotherapy and in this an attempt is made to evaluate the effectiveness Ethanol Extract
of Fruit Syzygium Alternifolium against Dalton ascetic lymphoma.
11
Experimental
Selection Grouping and Acclimatization of Laboratory Animal
Technique for Inducing Tumor
EVALUATION OF ANTICANCER ACTIVITY
Induction of cancer using DLA cells
Treatment Protocol
Swiss Albino mice were divided in to five group of six each. All the animals in six
groups were injected with DLA cells (1 x 106 cells per mouse) intraperitonealy, and the
remaining one group is normal control group.
Group 1 serves as the normal control.
Group 2 Tumor Control. Group 1 and 2 receives normal diet and Water.
Group 3 Positive control, was treated with injection 5-fluoro uracil at 20mg/kg Body
weight by intra peritoneal injection.
Group 4 Treatment control group and was administered Ethanol Extract of Fruit
Syzygium alternifolium at a dose of 200mg/kg through orally.
Group 5 Treatment control group administered Ethanol Extract of Fruit Syzygium
alternifolium at a dose of 400mg/kg through orally.
Treatment
In this study, drug treatment was given after the 24 hrs of inoculation, once daily
for 14 days. On day 14, after the last dose, all mice from each group were sacrificed by
euthanasia. Blood was withdrawn from each mouse by retro orbital plexus bleeding and
the following parameters were checked.
1. Hematological parameters
a. WBC count
b. RBC count
c. Hb content
d. Platelet count
e. Packed cell volume
12
2. Serum enzyme and lipid profile
a. Total Cholesterol (TC)
b. Triglycerides (TG)
c. Aspartate amino Transferase (AST)
d. Alanine amino Transferase (ALT)
e. Alkaline Phosphatase (ALP)
3. Derived parameter
a. Body weight
b. Life span (%)
c. Cancer Cell Count
EVALUATION OF CLINICAL PARAMETERS
Cancer cell count
Hematological parameters
i. WBC count
ii. RBC count
iii. Platelet count
iv. Hemoglobin
v) Packed Cell Volume
SERUM ENZYME AND LIPID PROFILE
The serum was analyzed for the following parameters
1. Aspartate amino Transferase (AST)
2. Alanine amino Transferase (ALT)
3. Alkaline Phosphatase (ALP)
4. Total Cholesterol (TC)
5. Triglyceride (TG)
13
TOTAL CHOLESTEROL AND TRIGLYCERIDE (lipid profile)
Abnormal blood lipid profile has been associated with cancer. In Hodgkin
lymphoma, high cholesterol level and low triglyceride level has been reported and hence
the parameters are investigated in the study.
LIVER ENZYMES (AST, ALT, ALP).
Abnormal liver function seen in patient with Hodgkin lymphoma, that these liver
enzyme levels markedly increase in tumor bearing mice. ALP is an enzyme mainly
derived from the liver, bones and in lesser amount from intestines, placenta, kidneys and
leukocytes. An increase in ALP levels in the serum is frequently associated with the
variety of disease. ALP comprises a group of enzyme that catalyzes the phosphate esters
in an alkaline environment, generating an organic radical and inorganic phosphate.
Markedly elevated serum ALP, hyperalakline-phosphatasemia, is seen predominantly
with more specific disorders; including malignant biliary cirrhosis, hepatic lymphoma and
sarcoidosis. Therefore these said traits are investigated in the study.
DERIVED PARAMETERS
1. Body weight:
All the mice were weighed, from the beginning to 15th day of the study. Average
increase in body weight on the 15th day was determined.
2. Percentage increase in life span (ILS)
Life span was calculated by the following formulae
%ILS = 100x1groupcontrolofspanLife
grouptreatedofspanLife
All biochemical investigations were done by using COBAS MIRA PLUS-S Auto
analyzer from Roche Switzerland and Hematological test are carried out in COBAS
MICROS OT 18 from Roche. A Hi-Tech instrument MAX MAT used for an auto
analyzer for all biochemistry investigations in blood sample.
F. Original Contribution
This present investigation is the first attempt of its kind to generate information on
the distribution S alternifoilum, checks the feasibility of propagating species by
comparing extra vitrum and in vivo morphogenesis of the target species with an intent of
14
multiplying / cloning plants so as to assist reintroduction of plants in Syzygium site and
other selected location besides evolving a strategy of conservation and management of the
species. The thesis with an equal emphasis on asserting the curative abilities of the
ethanol based extractions of fruit extracts on
I HYPOGYCEMIC ACTIVITY
II HEPATOPROTECTIVE ACTIVITY
III ANTIOXIDANT ACTIVITY
IV ANTI-CANCER ACTIVITY
V ANTI-MICROBIAL ACTIVITY
The thesis dwells mainly on five chapters.
Chapter I: Introduction
This opening chapter offers an introduction to the genus Syzygium in general and
S. alternifolium in specific terms, presenting an idea on the floristic composition and
medicinal plant traditions of the Indian sub continent that a good lead is created to
introduce the different objectives of the experiment.
Chapter II: Review of literature.
The literature pertaining to the conservation efforts taken in the country under
different contexts, especially the extent of work pursed in the target species and related
genera in the various domains dealt in this study is epxaned with sufficient back ground
and literature support. Texts dealing with the propagation of trees, in vitro culture of
plants, and the photochemistry and solvent extraction of active ingredients are provided
along the pretexts of pre clinical studies carried out using Swiss albino rat models.
Broader procedures for ascertain the effects of plant extracts on Hypogylcemic activity,
Hepato protective activity, Antioxidant activity, Anti-Cancer activity and Anti-Microbial
Activity forms apart of this section.
Chapter III: Materials and methodology
The materials and methods in this study can be categorized into four sub heads
namely, (i) field investigations spotting the distribution, study of ecology and phenology
of S. alternifolium is done with an intent of determining the problems and prospects of the
species perpetuation in field, (ii) standardization of in vitro procedures of propagation
15
using embryos and embryonal explants, (iii) procedures followed for solvent extracts,
quantitative testing of phytochemicals, and (iv) the screening of the therapeutic activities
of the ethnolic fruit extracts on four different ailments and select micro organisms are
described.
Chapter IV: Results
Considering the paucity of data on the distribution of the S.alternifolium in the in
the southern eastern Ghats, in particular the Alagar Hills and the Madurai district the
exploratory visits and field estimation of the distribution the taxa undertaken are
described. These studies aimed at determining the problems faced by the species in
perpetuating itself in the conditions prevailing here naturally led to the trials on
conventional and tissue culture propagation that offered important leads for developing
procedure to be recommended for the propagation of Mogi. Besides presenting the
random observations an attempt has been made to propose a scheme for propagation of
the species is made that the fundamental aim of propagation and conservation of the
selected species is complied and tested.
Chapter V: Discussion
Observations made in the study and the experimental data gathered from different
trials are analyzed in the light of the literature available in this domain and suggestions
are made for follow-up investigations.
Chapter VI: Conclusion and summary
Finally the thesis is concludes by presenting an abstract report of the entire study
and highlights the novel findings and results.
G. Highlights, Conclusion and Scope
Leads from this study show that there is no dearth of flower and fruit production
even though the causality and morality rate in high at both instances. The higher
incidence of seed loss is due to pest infestation where the medium portions of the
cotyledons appear to be soft target and a preferred source of feed to the insects. Among
the abiotic stresses, moisture retention in embryos turns out a serious issue as the fruits
and seeds that have substantially lost water content could never be revived to be
rehydrated. This issue set aside, poor germinability is found mainly due to the seed borne
pest which totally eliminates the possibility of seed germination by any natural means.
16
In vitro experiments pursued in this study presented interesting possibilities and
inevitable challenges. During initial trials, somatic explants and leaves posed problems of
metabolite exudation and phenolic oxidation and hence higher percentage of culture loss
due the browning of medium has to be contained. Though direct explanation of embryos
from immature and nearly mature fruits offered bypass, stages closer to maturing of fruits
and seeds alone evinced competency to produce plants. With not even remote chances of
recovering plants from seeds and embryos picked from ripe fruits shed tree, even one on
one plant recovery from each seed/ embryo is considered advantageous in this endemic
and endangered tree species.
Primary shoot axes raised from plumular part of the growing axis evinced a higher
propensity for axillary branching and de novo production of shoots that supernumerary
shoot buds providing for cloning of shoots can be achieved by sequential sub culture
following cloning schedule developed highlighted in the study.
An interesting outcome from this study relates to the feasibility of raising plants
through alternate mode of plant formation. It is observed that the cotyledon and the
cotyledon derived callus tissue emerging from the median part the adaxial surface of
embryos presented a higher probability for propagation. Plant emergence can be noticed
at the said region at higher frequencies both with and without the formation of
intermediary callus tissue.
This region vulnerable to destroyed by pest in the dehisced fruits dropped on
ground at natural conditions turned out to be a promising investment for tissue culture.
Embryos and embryonal explants prepared from stage III embryos through
microdissection from the prematurely picked fruits of the marked source plants in Alagar
Hills site at stages preceding shedding (fruits appearing deep pink to red color with faint
streaks of purple at the pericarp surface) were amenable for regeneration.
Micro surgically prepared explant and thin layer sliced segments of embryonal
explants involving this region alone was seen to offer to formation of organogenetically
competent callus. As the nature, color and texture of callus emerging from the excised cut
surface and the de differentiation of the adaxial surface and the abaxial epidermis surfaces
differed in their proliferation and redifferentiation capabilities, a scheme for orgongenesis
besides the earlier one suggested for de novo multiplication of the preexisting embryonal
bud is developed.
17
That the scope of variations and the genetic integrity relies on extent of callus
multiplication and the potential regeneration gets delicately poised and adjusted to the
profitability of the callus tissue will have be seen as both an opportunity and an
impediment which calls for skilled and tactful manipulation of the in vitro environment.
Though further studies are suggested to develop protocols from the two suggested
proposal made by the consolidation of results observed in the various experiments of this
study, this part of the thesis presents the possibility of judiciously manipulating the spatial
and temporal variables for effective tissue culture intervention the conservation and
propagation of the valuable endangered and threatened taxon reported to be an endemic
of mid eastern ghats surviving here far ways from its original home.
Even though the observation on the metabolite exudation is left beyond the scope
of this study for an exclusive follow-up investigation, the interest evinced by the natives
and ethnic community to use Syzygium as a valuable and effective therapeutic pressed for
the need to evaluate its medicinal properties. The field observation that despite its smaller
size and the limited presence of edible portions of the fruits, S. alternifolium is seen
devoured by primates and sold in traditional herbal market substantiated an evaluation of
its therapeutic competency.
Considering the fact that the ethnolic extracts of S alternifolium fruits are abound
an array of secondary plant chemicals as seen in solvent extraction and preliminary
phytochemical tests kept the option of pursuing further investigation on hypoglycemic
activity, hepatoprotective activity, anti-oxidant activity and anti cancer activity important
to be validated in preclinical study with animal models.
The broader metabolite profile with positive testing for presence of steroids,
terpenoids, carotenoids, flavanoids, alkaloids, tannins and glycoside in the qualitative
tests and GC based profiling ethanol fractions from the fruit extracts clearly illustrate the
presence of compounds that might be positively prophylactic and curative in their action
against the four common and important ailments.
The positive effects of the extracts in controlling the hyperglycemic conditions
imposed by the i.p. administered Alloxan monohydrate in direct and comparable
proportions with the standard drug Glipizide in terms of the check in loss of body weight
and raise in blood sugar ably substantiated by the investigations on hematological
18
parameters and serum enzymes provides ample scope for recommending the use of
botanical in treatment of diabetics.
In a similar vein, liver toxicity registering manifold increase in the level of AST,
ALT and ALP and biliproteins and the marginal climb-up of LDH being the considered
key parameters in this study accompanying a fall of total protein content in the blood
samples of the diseased animals offering a reference showed that the ethanol extracts of
S. alternifolium are potential enough to exert a convincing and an equivalent effect much
like the standard hepatoprotective drug, Silymarin
Data procured in favor of the use of fruit extract on trials focusing on oxidative
stress showed that the anti-stress action can be deduced from the various biochemical
parameters and enzyme mediated functions assayed in D-Gal induced animal models.
Anticancer investigations performed with DLA inflicted cancer growth in swiss
albino mice also offered a convincing proof to the favorable effects evoked by the ethanol
fractions of fruit extracts of S. alternifolium. The visible and direct evidence gained in
terms of the check in rise of WBC and the ability to ensure RBC count in the treated
animals coincided with the impacts hemoglobin level and the curbs put on the increases in
platelets and PCV reveals the feasibility of carrying forward further studies.
The promising indications seen in pre clinical trials with animals models on anti-
diabetic effects, hepato protective functions anti- cancer abilities and anti-oxidant activity
and anti-microbial proprieties of the fruit extracts S alternifolium provides a great deal of
support to affirm the folk notion that the chosen taxon of Syzygium can be a multi
potent raw and crude drug. Since S. alternifolium appears to be a useful botanical of great
diversified ability to alleviate or ameliorate an array diseases, it can be easily be
recommended for next level follow-up evaluation via pharmacognosic and
pharmacological characterization.
19
H. Articles Published/Presented in National or International Conferences:
LIST OF ORAL PRESENTATION
Dorai Pandian and Sangeli Mohan, 2011 Ecological Monitoring: Relating Human
Activities and Ecohealth of Nagamalai, Foothill Springs Madurai, Tamil Nadu,
India International Wetlands Conference Organized by University of Florida at
USA.
Mohan, S., Shanmugam, S., Eswaran, V.M. and Abraham, G.C..2013 Medicinal plants
from Yanaimalai Hillock in Madurai District of Tamil Nadu – a Preliminary study
in the National Seminar on Discourses on Past, Present and Future Scenario on
Medicinal Plant Conservation in India organized by PG & Reasearch Department
of Botany, Saraswathi Narayanan College, Madurai, Tamilnadu, India.
Mohan, S., Chidhambaranathan .V and Abraham, G.C. 2015 Phytochemical
Charecterization and Vegetative Propagation of Syzygium rama – varmae (Bourd.)
Chithra, National Conference on Recent Advances in Industrial Biotehnological
Skills Development, organized by Department of Botany, Thiagarajar College,
Madurai, Tamilnadu, India.
Mohan, S., Chidhambaranathan, V. and Abraham, G.C. 2015 Phytochemical
Charecterization and Vegetative Propagation of Syzygium rama – varmae (Bourd.)
Chithra, National Conference on Recent Advances in Industrial Biotehnological
Skills Development,held at Department of Botany, Thiagarajar College, Madurai,
Tamilnadu, India.
Mohan, S., Chidhambaranathan .V. Abraham.G.C. and Grace Lydial pushpalatha,G.2015
Cursory search on Antioxident properties and Therapeutic efficacy of Syzygium
gambleanum for Bioprospecting towards stepping up Peoples Initiatives in
Conservation”. National Conference on Recent Advances in Industrial
Biotehnological Skills Development organized by Department of Botany,
Thiagarajar College, Madurai, Tamilnadu, India.
Mohan. S and Abraham, G.C 2019 Ethinic interests and ecological threats and challenges
at the Alagarkovil hills in Madurai District in the International Eco-Conference on
Greening the Globe for Sustainable Living organized by CSI Synod Centre,
Chennai, Tamil Nadu, India
20
Ebenezar Immanuel.J, Mohan. S and Eswaran. V.M. 2019 Presented the Oral entitled on
Pseudomonas fluorescens, a Compatible Bacteria Strain against the Prevailling
Diseases in the Agro Ecosystem in the International Eco-Conference on Greening
the Globe for Sustainable Living organized by CSI Synod Centre, Chennai, Tamil
Nadu, India
Mohan. S, Shanmugam, S., Esvaran, V.M., and Abraham, G.C 2008 Syzygium
alternifolium (Wight) Walp., a rare endemic of the Middle Southern Eastern Ghats
finds a habitat at Alagar Hills of Madurai District in Tamilnadu in the
International Conference on Innovative Techniques in Agricultural and Biological
Science For Sustainable Development organized by PG Departments Botany &
Zoology, Sri Vidya Mandir College, Uthangarai, Krishnagiri, Tamil Nadu, India
LIST OF POSTER PRESENTATION
Mohan, S., Ramesh, S. M., and Abraham, G. C. 2013 Cd-Cr (VI) Influenced changes in
Morphogenetic Competency of a Commercial Legume, Butter Bean (Phaseolus
lunatus) in the National Conference on Frontier Vistas in Modern Biotechnology
(FVMBT-2013) organized by Department of Immunology & Microbiology, The
American College, Madurai, Tamil Nadu, India
Mohan, S., Shanmugam, S., Esvaran, V.M. and Abraham, G.C., 2019 Insights on the
efficacy of survival and reproductive effectiveness in Syzygium alternifolium
(Wight) Walp.,a rare endangered Eastern Ghat inhabitant spotted at Alagar Hills,
Madurai in the Mega Science Congress – MSC 2K19 organized by PRIST
University, Madurai Campus, Arasanoor, Sivagangai, Tamil Nadu, India
Mohan. S., Nisha, N., Ebenezr Immanuel, J. and Abraham, G.C 2016 In-vitro technique
for recovering plants from the medicinal ornamental Bauhinia purpurea L. in the
International Conference on Frontier in Life Science organized by Department of
Botany, ST. Joseph’s Colege, Trichy, Tamil Nadu, India
21
LIST OF PAPER PUBLICATION
Shanmugam, S., P. Selvakumar, S. Mohan, G. Dinesh kumar and K. Rajendran, 2010
Angiospermic Flora of Pachalur Hills in Dindigul District of Tamilnadu, India –
A Checklist. Journal of Phytological Research, 23(2): 223 – 238.
Dhanasekaran, M., Mohan, S., Abraham. G.C. 2014 Investigation of Micropropagation
on Kigelia pinnata D.C. International Journal of Pharmaceutical, Chemical and
Biological Sciences 4(2), 226-228.
Dhanasekaran, M., Mohan, S., Abraham,G.C. 2014, Preliminary Phytochemical and
Histochemical Investigation on Kigelia pinnata D.C. International Journal of
Pharma Sciences and Research (IJPSR), Vol (5) 413- 419.
Dhanasekaran, M., Mohan. S, Abraham.G.C. 2014 Evaluation of Pharmacological
potential on Kigelia pinnata D.C. International Journal of Pharma Sciences and
Research (IJPSR) Vol (5) 489 – 494.
Mohan, S., Chidambaranathan.N., Grace Lydial Pushpalatha, G. Dhanasekaran. M, and
Abraham.G.C., Phytochemical Profile and Acute Toxicity Studies of Ethanolic
Extract of Syzygium alternifolium (Wight) Walp International Research Journal of
Pharmacy Int. Res. J.Pharm Pharm. 2019;10(11):38 – 43.
Mohan, S., Chidambaranathan.N., Grace Lydial Pushpalatha, G., Dhanasekaran, M., and
Abraham, G.C. 2020 Anti - Hyperglycemic Effects of Ethanolic extract of
Syzygium alternifolium (Wight) Walp. in Alloxan Induced Diabetes Rat Model,
International Research Journal of Pharmacy Int. Res. J.Pharm.;11(2):67-71.