experimental study to determine the effect of c.m.i & …

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.


Health Sciences.Udupi, Karnataka, India.


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

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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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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.




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.




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 (…)


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)


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 (%)


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)


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)


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 (%)


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 (%)


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


Table: 14. Showing the effect of Kushtaghna dashemani on Red cell distribution width

Standard deviation

Group RDWSD (fl)


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


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



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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


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 ----


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 ----


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.

REFERENCES

1. Charaka. Atreyabhadrakaapiya adyaya. In: vaidya Yadavji Trikamji Acharya (eds)

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.

experimental study to determine the effect of c.m.i & …

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