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www.wjpps.com Vol 4, Issue 08, 2015.
1466
Darshan Babu N et al. World Journal of Pharmacy and Pharmaceutical Sciences
EXPERIMENTAL STUDY TO DETERMINE THE EFFECT OF C.M.I &
H.I. IN SELECTED DRUGS OF KUSHTAGHNA DASHEMANI IN
PARTHENIUM INDUCED ANIMAL MODEL
Darshan Babu N*1, Pampanna Gouda H
2, Umapati C. Baragi
3, Shrikanth P.H
4,
B. Ravishankar5
1Final year P.G. Scholar, Dept. of Samhita & Siddhanta, SDM College of Ayurveda, Rajiv
Gandhi University of Health Sciences, Udupi, Karnataka-574118, India.
2Dept. of Samhita & Siddhanta, SDM College of Ayurveda, Rajiv Gandhi University of
Health Sciences.Udupi, Karnataka, India.
3Dept. of Samhita & Siddhanta, SDM College of Ayurveda, Rajiv Gandhi University of
Health Sciences.Udupi, Karnataka, India.
4Dept. of Samhita & Siddhanta, SDM College of Ayurveda, Rajiv Gandhi University of
Health Sciences, Udupi, Karnataka, India.
5Director & Professor of Experimental Medicine S.D.M Centre for Research in Ayurveda &
Allied Sciences, Udupi, Karnataka, India.
ABSTRACT
Background: Integumentary system which includes skin, the largest
organ of the body, accounting for 16% to 20% of the total body
weight. The structure of human skin is complete, consisting of a
number of layers and tissue components with many important factors.
Though it is a protective barrier between the body and external
environment, it is easily exposed to infection. In environment we get a
noxious weed called Parthenium which is known for its Allergic
potentials like skin irritation. In classics, the description of tvak its
layers & disease pertaining to each layer has been explained. Kushta
(skin disorder) is a disease in which tvak (skin) is the vyakta sthana
(place) & shotha (swelling) is exhibited as a general symptom. In
classics it is explained that contact of poisonous herbs is a causative
factor for agantuja (exogenous) shotha. So experimental study was
carried out to evaluate the Kusthaghna property of kushtaghna
WWOORRLLDD JJOOUURRNNAALL OOFF PPHHAARRMMAACCYY AANNDD PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS
SSJJIIFF IImmppaacctt FFaaccttoorr 55..221100
VVoolluummee 44,, IIssssuuee 0088,, 11446666--11449944 RReesseeaarrcchh AArrttiiccllee IISSSSNN 2278 – 4357
Article Received on 15 June 2015,
Revised on 06 July 2015, Accepted on 26 July 2015
*Correspondence for
Author
Dr. Darshan Babu N
Final year P.G. Scholar,
Dept. of Samhita &
Siddhanta, SDM College
of Ayurveda, Rajiv
Gandhi University of
Health Sciences, Udupi,
Karnataka-574118, India.
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Darshan Babu N et al. World Journal of Pharmacy and Pharmaceutical Sciences
dashemani in parthenium induced dermatitis. Aims & Objectives: 1) To study the effect of
selected dravyas of Kushtaghna dashemani in parthenium induced dermatitis. 2) To assess
Humoral and Cell mediate immunity. Materials & Methods: Wistar strain albino rats of
either sex were selected, rats were randomly placed under 4 groups. Two control groups
divided into parthenium control & cyclophosphamide control & other two are test group i.e.
TED & 2 x TED dose of drugs were administered. Minimum of 6 rats were included in each
group. Conclusion: The study showed that the parthenium extract with adjuvant elicits
immunological oedema in pre-sensitized animals indicating CMI eliciting effect. Kushtaghna
Dashemani drugs showed the better activity in CMI and immunosuppressant activity in
humoral immunity.
KEYWORDS: Parthenium, Humoral & Cell Mediate Immunity, Kushtaghna Dashemani etc.
INTRODUCTION
Ayurveda, the science of life was revealed by the seers of India, thousands of years ago,
flourished as a comprehensive system of healthcare among the people. Base of this ancient
system of medicine can be traced in the Vedas (Atharva Veda) dated around 1200BC. This is
not only a science of life but it is the experience, observation & research of many great sages
in ancient period. Hence it is an external source of knowledge having multi -dimensional
textual work. Ayurveda is based on tridosha & pancha-mahabhuta siddhanta.[1] which are
the base for diagnosis and treatment aspects.
Like-wise tri-sutra i.e. hetu, linga & aushadha,[2] were given equal importance in the clinical
purview. In simple words hetu can be termed to be an aetiology of disease, linga as sign &
symptoms & aushadha is referred to be treatment.
Ayurvedic system of approach to diseased is entirely different from other system of medicine,
for both diagnosis & treatment; hetu or aetiology is considered first & given foremosst
importance in almost all diseases, then for other factors. During the 18 th & 19th century a
large number of cutaneous reactions due to contact with plants were documented. Parthenium
is a weed originally a resident of Mexico, introduced to India through wheat shipments from
USA in midst of 1950‟s & since then has spread far and wide throughout the India except
some mountainous and desert regions. In the context of contact dermatitis, Parthenium weed
tends to produce various patterns of hypersensitive reactions like allergic, irritant &
phototoxic dermatitis. Basic pathology behind any hypersensitive reaction will be marked
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changes in immunity level i.e. cell mediated immunity (CMI) & humoral immunity (HI). To
plan any type of treatments, where the basic pathology involves in immune level, it is
essential to review the drugs selected as a remedy acts at the level of above mentioned
immune level.
So study has been undertaken to review the effect C.M.I & H.I. in selected dravyas of
kushtaghna dashemani in parthenium induced animal model.
OBJECTIVE
1. To evaluate the efficacy of selected Kushtaghna Dashemani against Parthenium induced
dermatitis in experimental models.
2. To assess Cell mediated and Humoral immunity.
SKIN
Skin is the largest organ of the body. It is not uniformly thick. At some places it is thick and
at some places it is thin. The average thickness of the skin is about 1 to 2 mm. it, with all its
specialized derivatives, makes up what is called the integument (Latin: a covering) which
covers the entire surface of the human body.
Knowledge of its structure, physiology, chemistry and functions is essential to understand the
pathology of skin disorders and also a pre-requisite to understand the nature of the disease
and to plan the proper treatment. The skin covers the exterior of the body and is continuous
with the mucous membrane lining the body‟s orifices.
FUNCTIONS OF SKIN
Skin acts as a protective covering for the body minimising loss of water from the body
tissues. The skin is a metabolically active organ with vital functions including the protection
and homeostatic of the body. However, it has many other important functions also.
i. Protection function
Skin forms the covering of all the organs of the body and protects these organs from the
following factors:
a) Bacteria and Toxic substances
b) Mechanical blow
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a) Protection from Bacteria & Toxic substance
Skin covers the organs of the body and protects the organs from having direct contact with
external environment. Thus, it prevents the bacterial infection.
Lysozyme secreted in skin destroys the bacteria. Keratinized stratum corneum of epidermis is
responsible for the protective function of skin. This layer also offers resistance against toxic
chemicals like acids and alkalis. If the skin is injured, infection occurs due to invasion of
bacteria from external environment.
During injury or skin infection, the keratinocytes secrete:
A. Cytokines like interleukins, α-tumor necrosis factor and γ-interferon, which play
important role in inflammation, immunological reactions, tissue repair & wound healing.
B. Antimicrobial peptides like β-defensins, which prevent invasion of microbes.
b) Protection from mechanical blow
Skin is not tightly placed over the underlying organs or tissues. It is somewhat loose and
moves over the underlying subcutaneous tissues. So, the mechanical impact of any blow to
the skin is not transmitted to the underlying tissues.
ii. Sensory function
Skin is considered as the largest sense organ in the body. It has many nerve endings, which
forms the specialized cutaneous receptors. These receptors are stimulated by sensations of
touch, pain, pressure or temperature sensation and convey these sensations to the brain via
afferent nerves. At the brain level, perception of different sensations occurs.
Apart from the varied functions that are enlisted above, the integumentary system separates
man‟s internal body systems from the direct external environmental interactions, thereby
providing the capacity to withstand the dry terrestrial habits. The success of this function is
remarkable in view of the fact that the epidermis is continuously exposed to both physical &
chemical insults. In fact, just as what cell wall and sub-cellular membrane do for an
individual cell, the epidermis does for the entire body. [3]
ALLERGY
An allergy is a hypersensitivity disorder of the immune system. Symptoms include red eyes,
itchiness and runny nose, eczema, hives or an asthma attack. Allergies can play a major role
in condition such as asthma & skin manifestations. In some people, severe allergies to
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environmental or dietary allergens or to medication may result in life threatening reactions
called anaphylaxis.
Allergic reactions occur when a person‟s immune system reacts to normally harmless
substances in the environment. A substance that causes a reaction is called an allergen. These
reactions are acquired, predictable and rapid. Allergy is one of four forms of hypersensitivity
and is formally called type I (or immediate) hypersensitivity. Allergic reactions are
distinctive because of excessive activation of certain white blood cells called mast cells and
basophils by a type of antibody called immunoglobulin E (IgE). This reaction results in an
inflammatory response which can range from uncomfortable to dangerous.
Cause
Risk factors for allergy can be placed in two general categories, namely host and
environmental factors. Host factors include heredity, sex, race and age with heredity being by
far the most significant. Four major environmental candidates are alterations in exposure to
infectious diseases during childhood, environmental pollution, allergen levels & dietary
changes.
Pathophysiology
This can be explained under 2 headings-
Acute response
In the early stages of allergy, a type I hypersensitivity reaction against an allergen
encountered for the first time & presented by a professional antigen-presenting cell causes a
response in a type of immune cell called a TH2 lymphocyte, which belongs to subset of T
cells that produce a cytokine called interleukin-4 (IL-4). These TH-2 cells interact with other
lymphocytes called B cells, whose role is production of antibodies. Some of them coupled
with signals provided by IL-4 & this interaction stimulates the B cell to begin production of a
large amount of a particular type of antibody known as IgE. Secreted IgE circulates in the
body and binds to an IgE-specific receptor (a kind of Fc receptor called FcεRI) on the surface
of other kinds of immune cells called mast cells & basophils, which are both involved in the
acute inflammatory response. The IgE-coated cells, at this stage, are sensitized to the
allergen.
If later exposure to the same allergen occurs, the allergen can bind to the IgE molecules held
on the surface of the mast cells or basophils. Cross linking of the IgE and Fc receptors occurs
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when more than one IgE-receptor complex interacts with the same allergenic molecule &
activates the sensitized cell. Activated mast cells and basophils undergo a process called
degranulation, during which they release histamine and other inflammatory chemical
mediators (cytokines, interleukins, leukotriene‟s and prostaglandins) from their granules into
the surrounding tissue causing several systemic effects such as, vasodilation, mucous
secretion, nerve stimulation & smooth muscle contraction. This results in itchiness and
anaphylaxis. Depending on the individual, allergen and mode of introduction, the symptoms
can be system-wide (classical anaphylaxis) or localized to particular body systems; asthma is
localized to the respiratory system & eczema is localized to the dermis.
Late-phase response
After the chemical mediators of the acute response subside, late-phase responses can often
occur. This is due to the migration of other leukocytes such as neutrophils, lymphocytes,
eosinophil & macrophages to the initial site. The reaction is usually seen 2-24 hours after the
original reaction. Cytokines from mast cells may play a role in the persistence of long-term
effects. Late-phase responses seen in asthma are slightly different from those seen in other
allergic responses, although they are still caused by release of mediators from eosinophil‟s
and are still dependent on activity of TH-2 cells.
Hypersensitivity Reactions
The immune system is an integral part of human protection against disease, but the normally
protective immune mechanisms can sometimes cause detrimental reactions in the host. Such
reactions are known as hypersensitivity reactions, and the study of these is termed
immunopathology. Hypersensitivity refers to excessive, undesirable (damaging, discomfort-
producing and sometimes fatal) reactions produced by the normal immune system.
Hypersensitivity reactions require a pre-sensitized (immune) state of the host.
Hypersensitivity reactions can be divided into four types: type I, type II, type III and type IV,
based on the mechanisms involved and time taken for the reaction. Frequently, a particular
clinical condition (disease) may involve more than one type of reactions.
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Table: 01 Showing the Comparison of Different Types of hypersensitivity reactions.
characteristics type-I
(anaphylactic)
type-II
(cytotoxic)
type-III
(immune
complex)
type-IV
(delayed type)
Antibody IgE IgG, IgM IgG, IgM None
Antigen Exogenous cell surface Soluble tissues & organs
Response time 15-30 minutes minutes-hours 3-8 hours 48-72 hours
Appearance Weal & flare lysis and necrosis
erythema and
edema,
necrosis
erythema and
induration
Histology Basophils and
eosinophil
antibody and
complement
complement
and
neutrophils
monocytes and
lymphocytes
Transferred
with Antibody Antibody Antibody T-cells
Examples
Allergic-
asthma,
Hay fever
Erythroblastosis
fetalis,
Goodpasture's
nephritis
SLE, farmer's
lung disease
tuberculin test,
poison ivy,
Parthenium,
granuloma
IMMUNE SYSTEM
The immune response of the human body against any non self are of two types: (a) innate (or
natural or non-specific) & (b) adaptive (or acquired or specific) (c) both these responses have
two components each viz. cellular & humoral. Innate immunity lacks specificity as there is no
involvement of memory cells. Acquired immunity on other hand is specifically adapted for
the inducing the pathogens & response improves with subsequent exposures to the same
pathogen due to the presence of memory cell line. In the innate cellular immunity there is
involvement of monocytes macrophage system, while in innate humoral immunity there is
activation of complement system. On the other hand the cellular component of acquired
immunity consists of T-lymphocytes while the humoral component of this immunity involves
the role of B lymphocytes. Normally in innate & acquired immune responses act in concerted
manner to contain or eradicate infection.
PARTHENIUM DERMATITIS
In India, Parthenium hysterophorus is the most notorious compositae weed known to
produce contact hypersensitivity. This plant is variously known as congress grass, carrot
weed, fever few, bastard fever few & white top.
Originally a resident of Mexico, this plant was introduced into India along with wheat
shipments from USA in the 1950‟s & since then has spread far & wide, covering almost the
whole of India except for mountainous and desert areas.
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The weed grows wildly on waste lands & along canals, railway tracks & roads. Though it
grows more profusely during the rainy season, the growth is almost perennial.
The first report of contact hypersensitivity to Parthenium hysterphorous in India was
recorded from Pune in 1966. Since then there have been many such reports from different
parts of India.
Parthenim hysterophorous has been found to contain parthenin as well as hymenin,
ambrosin & coron-opilin. The various dermatitis patterns described are airborne contact
dermatitis, atopic dermatitis, photo dermatitis, seborrhoeic dermatitis and even exfoliative
dermatitis. Eye lid involvement is quite common & some cases in the early phase of the
disease present only with eyelid dermatitis. In the initial stage, there is worsening of lesions
during the summer & monsoon with partial remission during the winters, but later on the
disease persists throughout the year with bouts of exacerbations.[4]
DRUG REVIEW
Kushtaghna dashemani
Drugs included under the kushtaghna dashemani are Khadira, Abhaya, Amalaka, Haridra,
Arushkara, Saptaparna, Aragvadha, Karavira, Vidanga and Jatipravala.[5] Out of these for
the experimental study purpose only 4 drugs have been selected i.e. Khadira (Acacia catechu
Willd), Aragvadha (Cassia fistula Linn), Vidanga (Embelia ribes Burm. F). & Haridra
(Curcuma longa Linn.)
MATERIALS AND METHODS
EXPERIMENTAL STUDY
The aim was to evaluate the effect of C.M.I & H.I. in selected dravyas of Kushtaghna
dashemani in parthenium induced animal models.
Experimental source
Wistar strain rats taken from the Animal House of S.D.M. Centre for Research in Ayurveda
& Allied Sciences, Udupi.
Test drugs
a) Whole Parthenium plant aqueous extract.
b) Kushtaghna Dashemani drugs like Aragvadha, Khadira, Vidanga & Haridra aqueous
extract.
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Method of collection of data
Wistar strain albino rats of either sex were selected from Animal House of S.D.M. Centre for
Research in Ayurveda & Allied Sciences, Udupi. Selected rats were randomly placed under 4
groups and in each group minimum of 6 rats were included.
Drug administration
Test drugs were administered for 7 & 14 days respectively for Cellular and Humoral
assessment.
Inclusion Criteria
1. Animals selected are adult albino rats having weight from 140-280g.
2. Healthy Wistar strain rats of either sex.
Exclusion Criteria
1. Wistar strain rats weighing less than 140g and above 280g.
2. Pregnant and diseased rats.
3. Rats used for and under trial of other experiments.
Dose Calculation
The dose of the formulations was calculated by AOT study.
Statistical analysis
The data obtained were analyzed using one way ANOVA followed by Dunnat multiple
comparison „t‟test for determining the level of significance of the observed effects, as post-
HOC test if „p‟ value of less than 0.05 was considered as statistically significant.
PHASE I
AOT STUDY I
Table: 02 Showing the details of AOT Study experimentation.
1 Animal species Rats
2 Strain Wistar albino
3 Source Animal house attached to SDM Research center,
SDM Ayurveda College Udyavara.
4 Selection
A total of 8 healthy either sex of body weight 160-
260g Rats were selected according to AOT
software.
5 Acclimatization
period
All the selected animals were kept Acclimatization
for 7 days before dosing.
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The group number, animal number and sex of the animal were identified with the help of
cage cards, as presented in the following table.
Table: 3: Showing the identification of animals, its desired dose (AOT), body weight &
calculated dose.
Sl.
no
Identification
of animals
Desired dose
(according to AOT)
Body weight
(grams )
Calculated dose
(ml)
1 Head 175mg/kg 275 0.8
2 Neck 550mg/kg 246 1.12
3 Back 175mg/kg 250 0.72
4 Base of the tail 550mg/kg 192 0.88
5 Tip of the tail 175mg/kg 240 0.7
6 Fore limb 550mg/kg 226 1.2
7 Hind limb 2000mg/kg 230 2.3
8 No mark 550mg/kg 225 1.03
Husbandry condition
1. Housing: Rats were housed in each cage of poly propylene with stainless steel top grill.
The dry paddy husk was used as bedding material and was changed every morning.
2. Environment: The animals were exposed to 12 hours light and 12 hours dark cycle with
the relative humidity of 50 to 70 % and the ambient temperature was 22 ± 030C degrees.
3. Diet: Sai Durga feed Bangalore rat pellet, was provided throughout the study period
except on previous night of dosing i.e. (over-night) fasting before dosing. The drinking
water was given ad-libitum in polypropylene bottles with stainless steel sipper tube.
Preparation of Test formulation for administration
1. Test drug : Parthenium whole plant aqueous extract
2. Vehicle : Gum acacia
3. Dose preparation : The test drug was made in to fine suspension in vehicle
with suitable concentration. All the animals were dosed
constant dose volume (1 ml/ 100g body weight)
175mg/kg, 550mg/kg, 2000mg/kg
4. Schedule : Single dose per animal
a) Administration : The test formulation was administered through intra-
peritoneal at different dose levels to respective animal
through sterile disposable syringe.
6 Numbering and
Identification
The animal was marked with saturated Picric acid
solution in water for proper Identification. The
marking within the cages is as follows.
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b) Dose fixation : According to the AOT Software.
C) Route : Intra-peritoneal
d) Dose : 175mg/kg, 550mg/kg, 2000mg/kg test substance
e) Dose volume : 1ml/100g animal
OBSERVATION
Examination of Physical and Behavioral changes
The animal was observed continuously for 4 hours after the dosing. The careful cage side
observation was done without disturbing the animal attention and at the end of the every hour
the animal was individually exposed to open arena for recording the behavioral changes like
increased or decreased motor activity , convulsions, straub‟s reaction, muscle spasm,
catatonia, spasticity, ophisthotonus , hyperesthesia , muscle relaxation, anesthesia, arching
and rolling, lacrimation, salivation , diarrhea, writhing , mode of respiration, changes in skin
color etc. exitus, CNS depression – hypo activity, passivity, relaxation, ataxia, narcosis, etc.
Mortality
All the animals were observed at ½, 1, 2, 3, 4, 24 h, 48 h after dosing and there after daily
once for mortality during the entire period of the study (i.e.14 days).
RESULTS
Experimental evaluation of Parthenium aqueous extract for acute toxicity
1) As per the guidelines 175 mg/kg of Parthenium aqueous extract was administered for the
rat weighing about 275g i.e. 0.8 ml of solution.
Observation: The animal looked active in each hour.
2) As there was no mortality in previous dosed rat, for the next rat 550 mg/kg of Parthenium
aqueous extract was given as per the protocol and verified with AOT software. The
weight of that rat was 246g i.e., 1.12 ml of solution was administered.
Observation: Jerk movements were present at 30 minutes & rat died after 30 minutes of
drug administration.
3) As there was mortality in previous dosed rat, once again rat was dosed with 175mg/kg of
Parthenium aqueous extract was administered for the rat weighed about 250g. i.e., 0.72ml
of solution.
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Observation: Straub‟s reaction present at 1 to 4 hours, irritability present at 1 to 48hours,
rearing present at 1 to 48hours and jerk movements were present at 1 to 4 hours of drug
administration.
4) As there was no mortality in previous dosed rat, for the next rat 550 mg/kg of Parthenium
aqueous extract was given as per the protocol and verified with AOT software. The
weight of that rat was 192g i.e., 0.88 ml of solution was administered.
Observation: Rearing and jerk movements were present in 1 hour of drug administration.
Rat died after 60 minutes after drug administration.
5) As there was mortality in previous dosed rat, once again rat was dosed with 175mg/kg of
Parthenium aqueous extract was administered for the rat weighed about 240g. i.e., 0.7 ml
of solution.
Observation: Straub‟s reaction present at 3rd to 4th hour, Irritability, rearing and jerk
movements were present in 1-4 hour of drug administration.
6) As there was no mortality in previous dosed rat, for the next rat 550 mg/kg of Parthenium
aqueous extract was given as per the protocol and verified with AOT software. The
weight of that rat was 226g i.e., 1.2 ml of solution was administered.
Observation: The animal looked normal with little sign of toxicity. The increased motor
activity and auditory response were observed after 1-48 hour of drug administration.
Rearing present in 1-4 hours & Straub‟s reaction was present 1-48 hour of drug
administration.
7) As there was no mortality in previous dosed rat, once again rat was dosed with
2000mg/kg - the rat weighed about 230g. i.e., 2.23ml of solution was administered.
Observation: Rat was active, Straub‟s reaction & convulsion was observed in 30
minutes. Rat died after 30 minute of drug administration.
8) As there was mortality in previous dosed rat, once again rat was dosed with 550 mg/kg of
Parthenium aqueous extract was given as per the protocol and verified with AOT
software. The weight of that rat was 225g i.e., 1.03 ml of solution was administered.
Observation: Bleeding seen in lacrimal sac & hyperpigmentation in injected site was
noticed, Rat was active in 1 – 48 hours, increased motor activity seen 1- 48 hours,
Straub‟s reaction present in 2-24 hours, increased auditory response seen in 3rd - 48th
hour, rearing seen in 1-4 hours & rat died after 4 hours of drug administration.
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The data was fed into the AOT software to obtain the LD 50 value with confidence limits.
The LD 50 value was found to be 550mg/kg with a confidence limit of 196.4 to
884mg/kg. The AOT software generated data sheet can be seen further.
AOT STUDY-II
Table: 4 Showing the group number, animal number and sex of the animal were
identified with the help of cage cards, as presented in the following table.
Sl.
no
Identification
of animals
Desired dose
(according to AOT)
Body weight
(grams )
Calculated dose
(ml)
1 Head 175mg/kg 180 0.52
2 Neck 550mg/kg 141 0.41
3 Back 2000mg/kg 161 1.7
4 Base of the tail 2000mg/kg 166 1.77
5 No mark 2000mg/kg 160 1.6
Preparation of Test formulation for administration
1. Test drug : Drugs of Kushtaghna Dashaemani i.e. Aragvadha,
Khadira, Vidanga & Haridra aqueous extract
2. Vehicle : Gum acacia
3. Dose preparation : The test formulation was made in to fine suspension in
vehicle with suitable concentration. All the animals
were dosed constant dose volume (1 ml/ 100g body
weight) 175mg/kg, 550mg/kg, 2000mg/kg
4. Schedule : Single dose per animal
a) Administration : The test formulation was administered through oral route
at different dose levels to respective animal through oral
feeding needle sleeved on to disposable syringe
b) Dose fixation : According to the AOT Software.
c) Route : Oral
d) Dose : 175mg/kg, 550mg/kg, 2000mg/kg test substance
e) Dose volume : 1ml/100g animal
RESULTS
Physical & behavioral examination
There were no physical & behavioral changes except mild increase in motor activity and
rearing activity seen in 2 rats in the group 2000mg/kg in all the treated animals one day one at
1,2,3,4 hours intervals after dosing and there after once daily for 14 consecutive days. Thus
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data obtained from the study on single dose administration of Kushtaghna dashemani drugs
oral administration up to 14 days of observation period does not result in any physical and
behavioral changes.
Mortality
All the animals belonging to the treated group survived throughout the 14 days observation
period after dosing.
The data was fed into the AOT software to obtain the LD50 value with confidence limits.
The LD 50 is greater than 2000mg/kg.
The AOT software generated data sheet can be seen further.
PHASE II
A) i) Drug Used
c) Whole Parthenium plant aqueous extract.
d) Kushtaghna Dashemani drugs like Aragvadha, Khadira, Vidanga & Haridra aqueous
extract.
ii) Chemicals Used
a) Potash Alum
b) Normal Saline
c) Cyclophosphamide
d) 10% Sodium Carbonate
e) 30% SRBC solution (Sheep blood would be collected from city slaughter house in a
sterilized bottle.)
iii) Equipment’s & glass ware to be used: Glass tubes, Glass beakers, PH Meter, Syringes
etc.
Dose for Rats
1) Parthenium whole plant aqueous extract: From AOT study
LD50 = 550mg/kg, then 1/5th of LD50 is 110mg/kg
1/10th of LD50 is 55mg/kg.
2) Kushtagna Dashemani drugs aqueous extract
Drugs like Aragvadha, Khadira, Vidanga & Haridra made into aqueous extract form. By
AOT study dose has been calculated like-
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As LD50 is more than 2000 mg/kg, so 1/10th of LD50 is 200mg/kg
200mg/kg (TED) & 400mg/kg (TEDx2)
B) Route of Drug Administration
The test drug Parthenium was administered according to the body weight of the animals by
intra-peritoneal route & SRBC by subcutaneous route with the help of a sterile injection
syringe & Kushtaghna Dashemani drugs was administered according to the body weight of
the animals by oral route with the help of an oral feeding tube attached to injection syringe.
C) Animals
Wistar strain albino rats of either sex weighing between 140-280g were used for
experimental study with the following conditions.
Rats were used for experimental study with the following conditions.
The animals were obtained from animal house attached to the Pharmacology Laboratory
S.D.M Centre for Research in Ayurveda and Allied Sciences. ETHICS NO: SDM-
CAU/IAEC/13-14-08.
They were exposed to natural day and night cycles with ideal laboratory condition in
terms of ambient temperature, humidity.
They were fed with pellets from “Sai Durga Feeds”, Bengaluru and water ad libitum.
Inclusion Criteria
1. Animals selected are adult albino rats having weight from 140-280g.
2. Healthy Wistar strain rats of either sex.
Exclusion Criteria
1. Wistar strain rats weighing less than 140g and above 280g.
2. Pregnant and diseased rats.
3. Rats used for and under trial of other experiments.
STOCK SOLUTION PREPARATION
a) The aqueous extract of Parthenium should be stored in a container and kept in desiccator
for future usage. Dose of Parthenium which is set by AOT study is referred as a standard
marker and to assess the cell mediated and humoral immunity required dose should be
taken i.e. 1/10th of LD50 dose and mixed with 10ml of distilled water.
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b) Two grams of individual drug extract weighed individually and mixed well using mortar
& pestle and after thorough mixing, drugs were stored in a container and kept in
desiccator. Later for dosage purpose daily 200mg of Kushtaghna Dashemani drugs taken
and 10ml of Distilled water with 50 mg of Gum acacia used for test 1 group & 400mg
of Kushtaghna Dashemani drugs taken and 10ml of Distilled water with 50 mg of Gum
acacia used for test 2 group.
Drug administration
Test drugs were administered for 7 & 14 days respectively for cellular and humoral
assessment in the morning session between 9-10 AM orally & on experiment day
subcutaneous and intra peritoneal injection was carried out in the morning session between 9-
10 AM.
D) Grouping
I ASSESMENT OF CELL MEDIATED IMMUNITY:
The rats were grouped into different groups randomly irrespective of sexes and each group
comprised of six animals.
Group 1: Water control + Parthenium.
Group 2: Cyclophophamide control
Group 3: Kushtaghna Dashemani solution (TED)
Group 4: Kushtaghna Dashemani solution (TEDx2)
The rats were sensitized subcutaneously (1ml/100g body weight) on first day & seventh day
of drug administration with the following solution:
i. Parthenium Solution – 1ml
ii. Normal Saline – 4ml
iii. Potash Alum-1ml
PH of the above reagent (i.e. potash alum adjuvant) was maintained between 5.6-6.8 using
10% sodium carbonate.
On Seventh day initial paw volume of left hind paw were noted and 0.1 ml of (Parthenium
Solution – 1ml, Normal Saline – 4ml, Potash Alum-1ml) were injected into plantar
aponeurosis of left hind paw, volume of immunological edema thus produced was measured
by volume displacement method. After 24 hours & 48 hours with plethysmograph.
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Percentage increase in paw volume, which is the induced edema formation over initial value,
was calculated. The values from control group were compared with the values from the test
drug administered groups to assess the cell mediated immunity response of the drug.
II ASSESMENT OF HUMORAL IMMUNITY
ANIMAL GROUPING
The selected animals were grouped into different groups randomly irrespective of sexes and
each group comprised of six animals.
Group 1: Water control (SRBC Control)
Group 2: Cyclophosphamide control
Group 3: Kushtaghna Dashemani solution (TED)
Group 4: Kushtaghna Dashemani solution (TEDx2)
ASSESSMENT CRITERIA
The test drug vehicle was administered for 10 consecutive days. On third day of test drug
administration the animal was first sensitized with parthenium solution, for this sensitizing it
was injected intra peritoneal in the dose of 0.055mg/g of body weight to the rats. Then again
sensitizing has done with 30% SRBC by injecting subcutaneously in the dose of 0.5ml/100g
of body weight to the rats. On the 10th day blood was collected by supra-orbital puncture with
the help of micro capillary tubes under mild ether anesthesia for estimation of hematological
parameters followed by sacrifice with over dose of ether anesthesia. The abdomen was
opened through midline incision for dissecting out the important organs as mentioned below
and lymph node was removed and weighed. Blood was collected in the sterile test tube.
Serum was separated from it the component would be inactivated by incubating for 30 min at
56 degree C temperature in a serological water bath.
The micro titer plate was filled with 0.1ml sterile normal saline and serial 2 fold dilution of
0.1ml of the serum in sterile saline solution was made in the micro titer plate. 0.1ml of thrice
saline washed 30% SRBC was added to each well of the tray. Blood from the same animal or
sheep was used for both sensitization and to determine anti body titer. The trays were covered
and placed in refrigerator overnight. Antibody titer (haemaglutinaization titer) was noted on
next day. The titter was converted to log 2 values for easy comparison. Spleen was dissected
out from the sacrificed animal and their weight was recorded. Tissues including lymph node
were transferred to 10 % formaldehyde solution and later on processed for histological
studies.
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TESTS PERFORMED
Hematological Examination
The following parameters measured using automatic cell counter where applicable –
1. Hemoglobin
2. RBC count
3. Mean corpuscular volume
4. Mean corpuscular hemoglobin
5. MCHC
6. Platelet count
7. RDW
8. Packed
9. cell volume
10. IgE
OBSERVATION AND RESULTS
Humoral immunity
Table: 5. Showing the effect of Kushtaghna dashemani on Antibody Titer:
The data shows there was decrease in anti-body titer (log 2 value) in cyclophosphamide
group TED & TEDx2 groups when compared to the SRBC control group, the observed
decrease was found to be statistically very significant.
Table: 6 Showing the effect of Kushtaghna dashemani on Hemoglobin level:
Group Hb% (Gm%)
MEAN ± SEM % change
Control 16.13 ± 0.49 ----
Test 1 14.45 ± 0.66 10.41↓
Test 2 13.22 ± 0.71* 18.04↓
Data: MEAN ± SEM, * P<0.05
The data shows there was decrease in haemoglobin level in TED group when compared to the
control group & the observed decrease found to be statistically non-significant.
The data shows there was decrease in haemoglobin level in TEDx2 group when compared to
the control group the observed decrease found to be statistically significant.
Group Log 2 (…)
MEAN ± SEM % change
SRBC control 3.46 ± 0.30 ----
Cyclophosphamide 1.52 ± 0.50** 56.06↓
Test 1 1.56 ± 0.29** 54.91↓
Test 2 1.32 ± 0.12** 61.84↓
Data: MEAN ± SEM, **P<0.01
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Table: 7. Showing the effect of Kushtaghna dashemani on Total WBC Count:
Group TC (Cells/Cu mm)
MEAN ± SEM % change
Control 11383.33 ± 954.78 ----
Test 1 12233.33 ± 1311.7 7.46↑
Test 2 10740 ± 975.50 5.65↓
Data: MEAN ± SEM
The data shows there was increase in Total Count Level in TED group when compared to the
control group the observed increase was found to be statistically non-significant.
The data shows there was decrease in Total Count Level in TEDx2 group when compared to
the control group the observed decrease was found to be statistically non-significant.
Table: 8. Showing the effect of Kushtaghna dashemani on Red blood cell count:
Group
RBC (Millions/cu
mm)
MEAN ± SEM
% change
Control 7.79 ± 0.28 ----
Test 1 6.76 ± 0.44 13.22↓
Test 2 6.29 ± 0.41* 18.98↓
Data: MEAN ± SEM, * P<0.05
The data shows there was decrease in Red blood cell count in TED group when compared to
the control group & the observed decrease found to be statistically non-significant.
The data shows there was decrease in Red blood cell count in TEDx2 group when compared
to the control group & the observed decrease found to be statistically significant.
Table: 9. Showing the effect of Kushtaghna dashemani on Packed Cell Volume:
Group PCV (%)
MEAN ± SEM % change
Control 46.11 ± 1.45 ----
Test 1 40.8 ± 1.76 11.51↓
Test 2 38.52 ± 1.43** 16.46↓
Data: MEAN ± SEM, ** P<0.01
The data shows there was decrease in packed cell volume in TED group when compared to
the control group & the observed decrease found to be statistically non-significant.
The data shows there was decrease in packed cell volume in TEDx2 group when compared to
the control group & the observed decrease found to be statistically very significant.
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Table: 10. Showing the effect of Kushtaghna dashemani on Mean cell volume:
Group MCV (fl)
MEAN ± SEM % change
Control 59.33 ± 0.97 ----
Test 1 16.98 ± 2.11 71.38↓
Test 2 61.99 ± 2.70 4.48↑
Data: MEAN ± SEM
The data shows there was decrease in Mean cell volume in TED group when compared to the
control group & the observed decrease was found to be statistically non-significant.
The data shows there was increase in Mean cell volume in TEDx2 group when compared to
the control group & the observed increase was found to be statistically non-significant.
Table: 11. Showing the effect of Kushtaghna dashemani on Mean cell hemoglobin level
Group MCH (Picogram)
MEAN ± SEM % change
Control 20.7 ± 0.31 ----
Test 1 21.5 ± 0.69 3.86↑
Test 2 21.08 ± 0.63 122.58↑
Data: MEAN ± SEM
The data shows there was increase in Mean cell haemoglobin level in TED group when
compared to the control group & the observed increase was found to be statistically non-
significant.
The data shows there was increase in Mean cell haemoglobin level in TEDx2 group when
compared to the control group & the observed increase was found to be statistically non-
significant.
Table: 12. Showing the ffect of Kushtaghna dashemani on Mean cell hemoglobin
Concentration
Group MCHC (%)
MEAN ± SEM % change
Control 34.95 ± 0.23 ----
Test 1 35.33 ± 0.13 1.08↑
Test 2 34.2 ± 0.60 2.14↓
Data: MEAN ± SEM
The data shows there was increase in Mean cell haemoglobin Concentration in TED group
when compared to the control group & the observed increase was found to be statistically
non-significant.
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The data shows there was decrease in Mean cell haemoglobin Concentration in TEDx2 group
when compared to the control group & the observed decrease was found to be statistically
non-significant.
Table: 13. Showing the effect of Kushtaghna dashemani on Red cell distribution width
co-efficient variation
Group RDWCV (%)
MEAN ± SEM % change
Control 14.76 ± 0.24 ----
Test 1 15.4 ± 0.80 4.33↑
Test 2 16.44 ± 0.85 11.38↑
Data: MEAN ± SEM
The data shows there was increase in Red cell distribution width co-efficient variation in
TED group when compared to the control group & the observed increase was found to be
statistically non-significant.
The data shows there was increase in Red cell distribution width co-efficient variation in
TEDx2 group when compared to the control group & the observed decrease was found to be
statistically non-significant.
Table: 14. Showing the effect of Kushtaghna dashemani on Red cell distribution width
Standard deviation
Group RDWSD (fl)
MEAN ± SEM % change
Control 30.6 ± 0.72 ----
Test 1 32.71 ± 1.94 6.89↑
Test 2 35.06 ± 2.93 14.83↑
Data: MEAN ± SEM
The data shows there was increase in Red cell distribution width Standard deviation in TED
group when compared to the control group & the observed increase was found to be
statistically non-significant.
The data shows there was increase in Red cell distribution width Standard deviation in
TEDx2 group when compared to the control group & the observed decrease was found to be
statistically non-significant.
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Table: 15. Showing the effect of Kushtaghna dashemani on Platelet Count:
Group
PLATELET
(Lakhs/Cu mm)
MEAN ± SEM
% change
Control 6.88 ± 0.19 ----
Test 1 7.22 ± 0.61 4.94↑
Test 2 6.65 ± 0.42 3.34↓
Data: MEAN ± SEM
The data shows there was increase in Platelet count in TED group when compared to the
control group & the observed increase was found to be statistically non-significant.
The data shows there was decrease in Platelet count in TEDx2 group when compared to the
control group & the observed decrease was found to be statistically non-significant.
Table: 16. Showing the effect of Kushtaghna dashemani on IgE
Group 1 2 3 % change
Control 12.5 2.5 2.5 5.84
Test 1 3.51 2.8 2.6 2.94↓
Test 2 7.8 2.6 2.5 4.3↑
The data shows there was no significant change in the IgE.
Table: 17. Showing the effect of Kushtaghna dashemani on Spleen weight
Group
Spleen weight
(g)
MEAN ± SEM
% change
Normal Control 0.82 ± 0.13 ----
SRBC control 1.11 ± 0.26 35.36↑
Cyclophosphamide 0.86 ± 0.01 22.52↓
Test 1 0.88 ± 0.10 20.72↓
Test 2 1.16 ± 0.16 4.50↑
Data: MEAN ± SEM
The data shows there was increase in Spleen weight in SRBC group, when compared to the
control group, the observed increase was found to be statistically non-significant.
The data shows there was decrease in Spleen weight in Cyclophosphamide & TED group
when compared to the SRBC control group, the observed decrease was found to be
statistically non-significant.
The data shows there was increase in Spleen weight in TEDx2 group when compared to the
SRBC control group, the observed increase was found to be statistically non-significant.
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Cell-mediated immunity
Table: 18: Showing the effect of Kushtaghna Dashemani on cell-mediated immunity in
24 & 48 hours
Group Initial
MEAN ± SEM
24 hr
MEAN ± SEM
48 hr
MEAN ± SEM
Parthenium control 0.72 ± 0.03 1.10 ± 0.04** 1 ± 0.06**
Cyclophosphamide 1.02 ± 0.06 1.34 ± 0.10** 1.21 ± 0.07*
Test 01 0.77 ± 0.03 1.13 ± 0.08** 1.05 ± 0.05**
Test 02 0.82 ± 0.01 1.13 ± 0.05** 0.94 ± 0.02**
Data: MEAN ± SEM ** P<0.01
Table: 19. Showing the effect of Kushtaghna dashemani in cell mediated immunity: (%
change in 24hrs)
Group
% change in
24hrs
MEAN ± SEM
% change
Parthenium control 54.40 ± 4.23 ----
Cyclophosphamide 31.94 ± 9.72 41.28↓
Test 01 54.34 ± 7.35 0.11↓
Test 02 38.02 ± 3.34 30.11↓
Data: MEAN ± SEM
The data shows there was decrease in Percentage change in paw oedema in 24 hours in
cyclophosphamide group TED & TEDx2 groups when compared to the Parthenium control
group, the observed decrease was found to be statistically non-significant.
Table: 20. Showing the effect of Kushtaghna dashemani in cell mediated immunity (%
change in 48hrs)
Group
% change in
48hrs
MEAN ± SEM
% change
Parthenium control 38.64 ± 4.74 ----
Cyclophosphamide 26.05 ± 13.45 32.58↓
Test 01 42.28 ± 7.57 9.42↑
Test 02 15.27 ± 2.47 60.48↓
Data: MEAN ± SEM
The data shows there was decrease in Percentage change in paw oedema in 48 hours in
cyclophosphamide group, TEDx2 group when compared to the Parthenium control group, the
observed decrease was found to be statistically non-significant.
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The data shows there was increase in Percentage change in 48 hours in TED group when
compared to the Parthenium control group, the observed increase was found to be statistically
non-significant.
Histopathological observations
Lymph node
Examination of lymph node sections obtained from different groups was carried out under
trinocular microscope at different magnifications. Sections from control group exhibited
normal cytoarchitecture. In SRBC treated group increased cellularity was observed but no
follicle formation could be noticed. In SRBC and TED administered group increased
cellularity was noted but once again no nodular or follicle formation could be observed as
happens in sensitized animals. In SRBC and 2 X TED administered group also increased
cellularity was noted but without nodular formation. (Details as in Plate 01)
Spleen
Examination of sections of spleens from different groups was carried out under different
magnifications. Normal control group sections exhibited normal cytoarchitecture with red
pulp predominating over white pulp proportion. In SRBC sensitized rats increase in the white
pulp proportion was observed in comparison to control group sections in two rats. The rest
did not exhibit any remarkable change. In SRBC and TED injected rats no remarkable
changes could be observed. In SRBC and 2 X TED treated rats increased proportion of white
pulp was observed in 4 rats while normal cytoarchitecture was observed in the remaining two
rats. (Details as in Plate 02).
PLATE-01
Photo 1.2- Lymph node Control Photo 1.1- Lymph node Control
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PLATE-02
Photo 1.3- Lymph node test Photo 1.4- Lymph node test
Photo 1.5- Lymph node test
Photo 1.6- Lymph node test
Photo 2.1- Spleen Control Photo 2.2- Spleen Control
Photo 2.3- Spleen Test
Photo 2.4- Spleen Test
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DISCUSSION
The present study had objective to detect the cell mediated & humoral immunity in
parthenium induced animal model.
Immunology is one which covers the study of all aspects of immune system. It deals with the
physiological functioning of the immune system in states of both healthy & diseased.
Malfunctions of the immune system may produce varied immunological disorders like
hypersensitivity, immune deficiency disorders etc.
Haematological parameter like Hb showed non-significant decrease in test 1 group &
significant decrease in test 2 group. Total count showed non-significant increase in test 1
group & non-significant decrease in test 2 group. RBC & PCV showed non-significant
decrease in test 1 group & significant decrease in test 2 group. MCV showed non-significant
decrease in test 1 group & non-significant increase in test 2 group, MCH showed non-
significant increase in both the groups, MCHC shows non-significant increase in test 1 group
& non-significant decrease in test 2 group. RDWCV & RDWSD shows non-significant
increase in both the groups. Platelet showed non-significant increase in test 1 group & non-
significant decrease in test 2 group.
From the results projected above it can be seen that cell mediated immunity group shows
non-significant decrease at 24th hour in both the test group & on 48th hour there is non-
significant decrease in control group, non-significant increase in test 1 group & non-
significant decrease in test 2 group were observed. Humoral immunity group shows
significant decrease in both control & test group.
Photo 2.5- Spleen Test Photo 2.6- Spleen Test
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Cell-mediated immunity animal model
It is a well-known fact that to assess effect of test drug on cell mediated immunity egg
albumin paw edema in pre-sensitized animals is used as a model. In this case egg albumin or
similar antigenic agent is injected with adjuvant containing potassium alum. To ascertain
whether such a CMI is elicited by parthenium suspension was injected in to the plantar
aponeurosis of animals pre-sensitized with parthenium-potassium alum suspension. The
injection of parthenium suspension elicited significant edema indicating that it has the
potential to elicit cell mediated immunity (CMI). This was though non-significant was
suppressed by cyclophosphamide. TED dose of test formulation had only a marginal effect
whereas TED x 2 dose produced more than 60% inhibition of 48 hour immunological
oedema. Thus the study meets both the requirements mentioned in the objective. The first one
clearly shows that parthenium is allergogenic and produced CMI- mediated pedal oedema
like egg albumin. This may be the reason for the strong contact dermatitis many a times
associated with exposure to parthenium. Secondly kusthaghna dashameni gana drugs were
moderately effective like cyclophosphamide in suppressing this CMI eliciting effect. Further
refinement of the formulation may provide better effect. This clearly proves that this
Kushtaghna dashemani gana has good potential in the treatment of allergic dermatitis.
Analysis of the data shows in the control group weak suppression of immunological oedema
at 24th and 48th hour after injection of the paw edema eliciting agent was observed. In test
drug (Kushtaghna dashemani) administered group significant decrease was observed i.e.
suppression of paw oedema was observed in 24th hour and stimulation in test 1 group and
suppression in test 2 group of paw oedema was observed in 48th post injection. This indicates
the effect and test drug formulation (kushtaghna dashemani drugs) possess very good
immunological suppression effect and slight stimulation which is of higher magnitude. The
immunological oedema represents expression of cell mediated immunity hence based on the
results obtained it can be inferred that Kushtaghna dashemani dravyas has cell mediated
immunity suppression effect. It is pertinent to recollect here that most of the contact
dermatitis type of allergy is due to CMI its suppression by the test drug group may provide
evidence for their efficacy against it.
Effect on anti-body formation
Haemagglutination antibody titer is a primary parameter for studying the humoral response.
Antigen and antibody reaction results in agglutination. Antibody molecules secreted by
plasma cells mediate the humoral immune response.
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The test formulation shows significant decrease against SRBC in sensitized animals. This
indicates at the dose level employed Kushtaghna dashemani has immuno suppressant effect
on TH-2 mediated pathway of immune reaction. It is common observation that only cytotoxic
agents suppress both anti-bodies mediated & cell mediated immunity while specific
immunomodulators generally suppress either of the above reaction. Thus it can be suggested
that the test drug has cell mediated immunity & humoral immunity suppression.
Histological examination
Histological examination of lymph nodes cytoarchitecture did not reveal any significant
changes after SRBC injection & test drug administration. In Spleen SRBC sensitized rats
increase in the white pulp proportion was observed in comparison to control group sections.
In TED group no remarkable changes could be observed and in 2 TED group significant
increase in white pulp proportion of spleen was observed after SRBC injection. This is
indicative of stimulation of the splenic activity & is as expected after injection of antigen.
CONCLUSION
Analysis of the results clearly indicates that parthenium extract with adjuvant elicits
immunological oedema in pre-sensitized animals indicating CMI eliciting effect. This effect
was moderately suppressed by both cyclophosphamide and TED x 2 dose of test formulation.
In anti-body formation test both cyclophosphamide and test drug formulations at both the
doses studied suppressed anti-body formation against SRBC in parthenium extract injected
rats. Further the injected site in control animals exhibited moderate to marked erythema and
oedema which was very much reduced in reference standard and test formulation
administered groups. No remarkable effect could be observed on the haematological
parameters except for mild anaemia like condition. Spleen and lymph node exhibited
increased cellularity but nodular formation was not observed. This may be due to antigen
induced stimulation of the organs which was not modified to significant extent by the test
formulation. It can be concluded that better activity of Kushtaghna Dashemani drugs was
seen in CMI and Immunosuppressant activity in humoral immunity.
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Charaka Samhita. Varanasi: Chaukambha Sanskrit Sansthan; 2013; 138.
2. Charaka. Dheerganjeevithiya adyaya. In: vaidya Yadavji Trikamji Acharya (eds) Charaka
Samhita. Varanasi: Chaukambha Sanskrit Sansthan; 2013; 7.
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3. K. Sembulingam, Prema Sembulingam. Essential of Medical Physiology. 6th Ed., New
Delhi; Jaypee Brothers medical Publishers (p) Ltd., 2013; 351-355.
4. R.G. Valia and Ameet R Valia, IADVL Textbook & atlas of dermatology, second edition
ed. Mumbai: Bhalani publishing house; 2003; 470-471.
5. Charaka. Shadvirechanashatiya adyaya. In: vaidya Yadavji Trikamji Acharya (eds)
Charaka Samhita. Varanasi: Chaukambha Sanskrit Sansthan; 2013; 33.
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