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.

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

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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.

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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.

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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

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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)

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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

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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

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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.

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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.

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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

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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.